Caldimonas thermodepolymerans Sugar Preference in Polyhydroxyalkanoates Production from Lignocellulose

Caldimonas thermodepolymerans Sugar Preference in Polyhydroxyalkanoates Production from Lignocellulose | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Caldimonas thermodepolymerans Sugar Preference in Polyhydroxyalkanoates Production from Lignocellulose Xenie Hajkova, Anastasia Grybchuk-Ieremenko, Pavel Dvorak, Iva Buchtikova, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7619151/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Caldimonas thermodepolymerans DSM 15344, a moderately thermophilic bacterium, has emerged as a promising candidate for next-generation industrial biotechnology (NGIB) due to its ability to utilize lignocellulose-derived sugars for polyhydroxyalkanoate (PHA) production. This study assesses its metabolic potential by evaluating the utilization of various plant-derived sugars and their mixtures, with a focus on xylose, glucose, and cellobiose. The results indicate that C. thermodepolymerans exhibits a strong preference for xylose over glucose but demonstrates even greater efficiency in metabolizing cellobiose. However, extracellular hydrolysis of cellobiose leads to glucose accumulation, which constrains overall productivity. Our findings suggest that the primary limitation in glucose metabolism is inefficient glucose transport rather than intracellular catabolism. To address this bottleneck, the glf glucose facilitator from the mesophilic bacterium Zymomonas mobilis was introduced into C. thermodepolymerans , enhancing its glucose utilization capacity. The engineered strain (Caldi_GLF3) exhibited significantly improved PHA productivity, particularly when cultivated on sugar mixtures containing cellobiose. Despite being grown at suboptimal temperatures due to the thermal instability of glf from Z. mobilis , Caldi_GLF3 outperformed the wild-type strain, achieving notably high PHA yields, especially in the presence of cellobiose. These findings highlight the critical role of glucose transport in the metabolism of C. thermodepolymerans and suggest that targeted engineering can further enhance its biotechnological potential. This study establishes C. thermodepolymerans as a promising thermophilic chassis for PHA production from lignocellulosic sugars, contributing to sustainable biopolymer synthesis. Biotechnology and Bioengineering Caldimonas thermodepolymerans polyhydroxyalkanoates thermophiles sugar metabolism glucose transporters lignocelluloses Figures Figure 1 Figure 2 Figure 3 Highlights • DSM 15344 produces PHA from lignocellulose-derived sugars • Xylose and cellobiose are preferred substrates, while glucose is poorly utilized • Deficient glucose transporter in DSM 15344 restored by gene 1. Introduction Modern human society faces a myriad of challenges, and among the most pivotal, unavoidable, and formidable is the ongoing shift of the chemical industry from reliance on fossil resources to renewable alternatives. Microbial biotechnologies stand poised to play an important role in facilitating this transition. Microorganisms exhibit the remarkable ability to utilize various renewable resources and transform them into a diverse array of valuable products, encompassing fuels, chemicals, and materials (Jerome et al. 2022 ; Gawel et al. 2019 ). Microbial biotechnologies can also contribute to solution of “plastic crisis” (Borrelle et al. 2020 ) by providing alternatives to petrochemical polymers. Polyhydroxyalkanoates (PHAs) represent microbial polyesters that various prokaryotes accumulate as storage and stress robustness enhancing metabolites (Obruca et al. 2022 ). As eco-friendly alternatives, PHAs surpass synthetic polymers in sustainability and environmental impact (Fu et al. 2023 ; Rajvanshi et al. 2023 ). Nevertheless, traditional microbial technologies exhibit several weaknesses which are manifested in high investment and operational costs for these processes. In response to these challenges, Chen and Jiang ( 2018 ) has recently introduced the concept of Next-Generation Industrial Biotechnology (NGIB). This innovative approach relies on employing extremophiles as chassis for the bioproduction. The inherent resilience of extremophiles to extreme conditions endows these processes with natural robustness against microbial contamination (Yu et al. 2019 ). Apart from cultivation, the extremophilic nature of employed microorganisms provides benefits in up-stream processing, for instance, salt tolerance of some extremophilic microorganisms allows the simple integration of acidic/alkaline hydrolysis step in the processing of complex substrates such as lignocelluloses (Kucera et al. 2018 ). Furthermore, it may also facilitate streamlined downstream processing as demonstrated in the isolation of intracellular products from hypotonic lysis-sensitive cells (Novackova et al. 2022 ). To establish the NGIB process, identifying the optimal extremophilic microbial host is paramount. The desirable microorganism should exhibit resilience, stability, and the ability to efficiently utilize cost-effective renewable resources such as lignocelluloses. A high-level understanding of its genetic and metabolic features is essential for further improvement of the bacterium employing synthetic biology tools and approaches. Currently, within the family of halophiles, the moderately halophilic bacterium Halomonas bluephagenesis stands out as a promising candidate for manufacturing not only PHAs but other high-value products as well (Park et al. 2023 ; Zhang et al. 2023 ). However, the utilization of thermophiles—microorganisms adapted to high temperatures—offers a set of advantages by introducing extreme conditions solely through elevated temperature. Unlike with halophiles, challenges such as salt-related costs and corrosion are absent. Contrary to common misconceptions about the energy demands of thermophilic biotechnologies, well-isolated bioreactors require relatively low energy for heating to moderate thermophilic temperatures. Additionally, thermophilic cultivations can function as "self-heating systems" due to the heat energy generated by microbial metabolism and dissipated through stirring, particularly in industrial-scale cultivations with high cell densities. Furthermore, thermophilic processes exhibit high energy efficiency, as only minimal energy-demanding cooling efforts are necessary (Turner et al. 2007 ; Ibrahim et al. 2010). Caldimonas thermodepolymerans DSM 15344 (formerly known as Schlegelella thermodepolymerans ) is a gram-negative, moderately thermophilic, aerobic bacterium, emerging as a promising candidate for a thermophilic chassis in NGIB processes. This bacterium was initially isolated in by Elbana et al. (2003) due to its notable ability to degrade various polyesters, as reflected in its taxonomic name. Despite this capability being investigated in subsequent studies (Romen et al. 2004 ; Elbanna et al. 2004 ), the bacterium received limited attention for an extended period. However, our recent findings demonstrated that C. thermodepolymerans not only degrades PHAs, but it can also produce them in high quantities, and it shows preference for xylose. These characteristics are not only intriguing from a fundamental perspective but also position C. thermodepolymerans as a robust candidate for NGIB processes and industrial PHA production, especially from diverse lignocellulose-based resources (Kourilova et al. 2020 ). The genomic features and some of the metabolic characteristics of this bacterium have been recently elucidated (Musilova et al. 2021 ; 2023 ). The bacterium was evaluated as a potent candidate for PHA production from xylose-rich hemicellulose-based resources and demonstrated its proficiency in xylose metabolism and tolerance to lignocellulose relevant microbial inhibitors such as phenolic compounds or derivates of furfural (Kourilova et al. 2021 ). In a study, Zhou et al. ( 2023 ) delved into cultivation parameters influencing PHA synthesis in C. thermodepolymerans and employed proteomics to unravel metabolic pathways for PHA synthesis from xylose in this bacterium. Recently, C. thermodepolymerans demonstrated the ability to achieve high cell densities and elevated PHA productivity on xylose in laboratory bioreactors using a fed-batch cultivation strategy under nitrogen-limited conditions (Jang et al. 2025 ). Capability of C. thermodepolymerans with respect to direct utilization of xylan was also reported (Zhou et al. 2025 ), C. thermodepolymerans was also utilized for PHA biosynthesis from wine-lees, side products of prosecco wine production (Caminiti et al. 2025 ). In the immediate past, initial genome-editing toolkit was developed for C. thermodepolymerans , potentially opening numerous opportunities for its utilization as a thermophilic chassis for bioproduction (Grybchuk-Ieremeko et al. 2025). A detailed understanding of metabolic features is a crucial step in the development of extremophilic chassis. Musilova et al. ( 2023 ) proposed a comprehensive scheme of the central carbohydrate metabolism of three related Caldimonas strains, including C. thermodepolymerans DSM 15344. However, the range of plant-derived sugars—both monomers and oligomers—and their mixtures that C. thermodepolymerans can metabolize and efficiently convert into PHA has not yet been thoroughly investigated. Furthermore, other critical metabolic aspects of C. thermodepolymerans , such as the kinetics of sugar utilization and potential bottlenecks in the efficient processing of certain sugars, remain to be elucidated. A deeper understanding of these fundamental characteristics could be crucial in establishing C. thermodepolymerans as a promising thermophilic chassis in the NGIB framework. The objective of this study was to assess various plant-derived sugars and their mixtures as potential substrates for PHA production by C. thermodepolymerans . We investigated the utilization kinetics of xylose, glucose, cellobiose, and their combinations, alongside studying microbial culture growth and PHA biosynthesis parameters. Additionally, we addressed the deficiency in glucose metabolism within C. thermodepolymerans by introducing a high-capacity glucose transporter from Zymomonas mobilis . Our findings suggest that the limitation in glucose metabolism in C. thermodepolymerans is primarily attributed to glucose transportation into bacterial cells rather than a lack of metabolic capacity for glucose utilization. These results underscore the significant potential of C. thermodepolymerans as a microbial platform for PHA production within the framework of NGIB. Furthermore, our study demonstrates that its biotechnological potential can be further enhanced through metabolic engineering. 2. Materials and Methods 2.1 Strains and cultivation media The microorganism used in this study is a bacterial strain Caldimonas thermodepolymerans DSM 15344 from DSMZ-German Collection of Microorganisms and Cell Cultures. Bacterial suspensions were stored in cryotubes with 10% glycerol as cryoprotectant in a deep freezer at -80°C. Bacteria were cultivated in two types of media. First, nutrient-rich complex medium Nutrient Broth w/w 1% Peptone (HiMedia, India) was used – 50 mL in 100 mL Erlenmeyer flasks. The basic mineral salt medium (MSM) with the following composition was used as production medium - Na 2 HPO 4 · 12 H 2 O (9.0 g/L), KH 2 PO 4 (1.5 g/L), NH 4 Cl (1.0 g/L), MgSO 4 · 7 H 2 O (0.2 g/L), CaCl 2 · 2 H 2 O (0.02 g/L), Fe (III) NH 4 citrate (0.0012 g/L), yeast extract (0.5 g/L), 1 mL/L of microelements solution (EDTA (50.0 g/L), FeCl 3 · 6 H 2 O (13.8 g/L), ZnCl 2 (0.84 g/L), CuCl 2 · 2 H 2 O (0.13 g/L), CoCl 2 · 6 H 2 O (0.1 g/L), MnCl 2 · 6 H 2 O (0.016 g/L), H 3 BO 3 (0.1 g/L), dissolved in distilled water) and the specific type and concentration of the carbon source (usually 20 g/L). 2.2 Integration of the glucose transporter into C. thermodepolymerans genome The glf gene from Zymomonas mobilis (NCBI gene ID: 79904430), which codes for the glucose facilitator protein, was integrated into the chromosome of Caldimonas strain DSM 15344. The mini-Tn5 transposon vector pBAMD1-6_ glf constructed previously (Bujdoš et al. 2023 ) was electroporated into Caldimonas competent cells. To prepare electrocompetent bacterial cells, the night culture of Caldimonas was inoculated into 100 mL of fresh LB medium (Serva) to the OD 600 of 0.05 and shaken at 220 rpm at 37°C. Upon reaching an OD 600 of ~ 0.5, the bacterial culture was cooled on ice and washed three times with 10% glycerol in milli-Q water. Cells were then resuspended in 1.5 mL of ice-cold 10% glycerol, aliquoted, and frozen at -60°C (Grybchuk-Ieremenko et al. 2025 ). Ten electroporations with pBAMD1-6_ glf vector were conducted in parallel. For this purpose, 100 µL of cells were thawed on ice, and 100 ng of plasmid DNA was added. Electroporation was performed using a Biorad Gene Pulser Xcell (2,500 V, 25 µF, 200 Ω) with a 2 mm gap cuvette. Immediately following the pulse, 0.9 mL of prewarmed (37°C) LB was added to the cells, which were then recovered and shaken at 220 rpm at 37°C for 2 h. Afterward, bacterial cultures were transferred into 10 mL of LB (in 50 mL Erlenmeyer flasks) supplied with 10 µg/mL gentamicin and cultured at 42°C, 220 rpm for 24 h. Subsequently, isolates were passaged twice each 24 h in 10 mL of MSM medium with 5 g/L glucose and 10 µg/mL gentamicin at 42°C, 220 rpm. Then the ODs were measured and the isolate that showed the fastest growth on glucose was chosen for further examination. It was spread for single colonies on MSM agar plates supplied with 5 g/L glucose and 10 µg/mL gentamicin and incubated at 42°C. The streaking on fresh MSM plates with glucose was repeated several times to ensure the stability of the integration. The growth of all obtained mutants was tested in the presence of 5 g/L glucose also at Caldimonas' optimal temperature of 50°C. Forty single colonies in two replicates obtained from preselection in liquid and solid media were tested for the growth in 600 µL of MSM with 2 g/L glucose in a 48-well plate in Infinite® 200 PRO plate reader (Tecan). The isolate Cald_GLF3, which showed repeatedly the fastest growth at 42°C, was taken for further investigations and its growth was compared to the wild type in the 48-well plate assay under the conditions described above. To identify the locus of the glf integration in the chromosome, arbitrary PCR with standard primers as described by Martínez-García et al. ( 2014 ) was used. 2.3 Screening of lignocellulose-derived sugar utilization potential of C. thermodepolymerans Screening for carbohydrates occurring in lignocellulosic matrices was performed in 12-well plates for suspension culture. The prepared plates were incubated in the thermoshaker Biosan PST-60HL-4 at 50°C with constant shaking (280 rpm). The volume of MSM including bacterial culture (5 vol. %) was 2 mL per well in biological triplicates. The carbohydrates tested were xylose, xylobiose, glucose, cellobiose, cellotriose, maltose and maltotriose. For control, cultivation without carbohydrate source was also performed. Due to the high purchase cost of some of the tested carbohydrates, specifically cellotriose, maltotriose, and xylobiose, the substrate concentration was adjusted to 10 g/L. As a consequence of the lower substrate concentration, the cultivation time was reduced to 48 hours. After this period the samples for biomass and PHA analysis were collected. 2.3 Kinetics of utilization of xylose, glucose, and cellobiose and their mixtures The ability and efficiency of utilizing various carbohydrates (xylose, glucose, cellobiose) and their combinations for growth and PHAs production were monitored using submerged cultivations in Erlenmeyer flasks in biological triplicates, with sampling every 12 hours. The medium composition and cultivation protocol were based on section 2.1 . The inoculation phase lasted 20 hours, after which 5 vol. % of the culture was transferred to the production medium (100 mL in 250 mL Erlenmeyer flasks) containing—in addition to MSM—20 g/L of a carbon source. The tested carbohydrates were added in equal proportions. In the case of two carbohydrates, each was present at 10 g/L. When xylose, glucose, and cellobiose were combined, the concentration of each carbohydrate was 6 g/L. Cultivation in MSM was terminated after 72 hours. Yield coefficients Y X/S , Y P/S were calculated as the ratio of the biomass or PHA concentration after 72 hours to the amount of substrate consumed during the same period. Maximal volumetric biomass and PHA productivity, and maximum substrate utilization rates were obtained by fitting linear segment of the growth curve graph in MS Excel with a straight line. The resulting values correspond to the slopes of the linear regressions. 2.4 Analytical methods After the cultivations were completed, samples were taken to obtain biomass and supernatants for further analyses. Dry biomass was determined gravimetrically. From each cultivation, performed in biological triplicates, 10 mL of culture was collected. The sample was then centrifuged, washed with distilled water, and centrifuged again. The resulting pellet was dried to a constant weight and subsequently weighed. The content of PHA in dried biomass was analysed by gas chromatography with a flame ionization detector, following the method described by Obruca et al. ( 2013 ). PHA content was determined as methyl esters of 3-hydroxy acids, formed by methanolysis of the intracellular polymer. Approximately 8–11 mg of dry biomass was reacted with 1 mL of chloroform and 0.8 mL of 15% sulfuric acid in methanol containing 5 mg/mL of benzoic acid as an internal standard. The reaction was carried out at 94°C for 3 hours. After completion, 0.5 mL of 0.05 M NaOH was added, and the mixture was vigorously shaken. Following phase separation, 0.05 mL of the organic phase was mixed with 0.9 mL of isopropyl alcohol. The resulting methyl esters were analysed using a Trace GC Ultra (Thermo Fisher Scientific) equipped with a Stabilwax column (30 m × 0.32 mm × 0.5 µm) and a flame ionization detector. Calibration curve was prepared using commercially available polymer standard. The supernatants obtained during sampling were used for the analysis of carbohydrates present in the cultivation medium. Prior to measurement by high-performance liquid chromatography (HPLC) with a refractive index detector, the samples were filtered through a nylon membrane filter with a pore size of 0.45 µm. The analysis was performed using a Shimadzu LC-10AD HPLC system. For isocratic separation, a Water Carbohydrate Analysis column (3.9 × 300 mm) was used, with a mobile phase consisting of acetonitrile and water in a ratio of 80:20. Carbohydrate concentrations were calculated from peak areas using calibration curves for xylose, glucose and cellobiose. 3. Results and discussion 3.1 Utilization of lignocellulose-relevant sugars by C. thermodepolymerans Plant biomass holds promise as a resource serving as a viable alternative to fossil resources. Therefore, we screened the capacity of the C. thermodepolymerans to utilize various plant-relevant sugars including disaccharides and trisaccharides, the results are shown in the Table 1 . Consistent with prior findings (Kourilova et al. 2020 ; Musilova et al. 2023 ), the bacterium exhibited a pronounced preference for xylose among the tested monosaccharides, surpassing its affinity for other sugars, including glucose. This holds significant importance as xylose stands out as the most abundant pentose and, after glucose, the second most prevalent monosaccharide in lignocellulosic biomass. The efficient conversion of xylose into valuable chemicals, fuels, and materials emerges as a critical task for establishing a sustainable bioeconomy (Narisetty et al. 2022 ). In this light, due to its high affinity to xylose, C. thermodepolymerans appears to be a distinctive bacterium, presenting a promising candidate as an NGIB chassis for valorising xylose-rich resources. Nevertheless, our results indicate that the bacterium is unable to cleave glycosidic bonds between xyloses in xylobiose since we observed only negligible growth of the culture on this disaccharide (Table 1 ). A distinct scenario arises when examining the metabolism of glucose in C. thermodepolymerans . The wild-type strain of this bacterium exhibits limited efficiency in metabolizing the monosaccharide. Surprisingly, the bacterium demonstrates remarkable proficiency in utilizing glucose oligomers linked by β-1,4-glycosidic bonds, such as cellobiose and cellotriose. Strikingly, the growth and synthesis of polyhydroxyalkanoates (PHA) on these glucose oligosaccharides, particularly cellotriose, surpass even that observed on the preferred substrate—xylose. This may reflect a potential energetic advantage of transporting longer oligosaccharides, such as cellotriose, compared to shorter ones like cellobiose or monomeric glucose. If sugars are actively transported through one of the present ABC transporters and intracellular cleavage of cellooligosaccharides occurs, only one ATP molecule is required per cellotriose, yielding three glucose molecules. In contrast, cellobiose yields only two glucose molecules for the same energy cost (Dvorak and de Lorenzo 2018; Parisutham et al. 2017 ). In contrast, the presence of α-1,4-glycosidic bonds, as found in maltose or maltotriose, impedes the metabolization of these glucose oligosaccharides by C. thermodepolymerans . The inability to cleave α-1,4-glycosidic bonds renders the wild-type strain of C. thermodepolymerans unsuitable as a PHA producer from starch and similar saccharides. However, it is worth noting that other members of the Caldimonas genus, such as Caldimonas taiwanensis (Chen et al. 2005 ; Sheu et al. 2009 ), possess amylolytic activity. Therefore, they could serve as suitable sources for genes that could enhance the biotechnological potential of C. thermodepolymerans through metabolic engineering techniques. Table 1 Screening of catabolic potential of C. thermodepolymerans with respect to selected plant-derived sugars , cultivations were carried out in 12-well plates within 48 h time interval at 50°C with initial sugar concentration 10 g/L. OD 630 nm CDW [g/L] PHA [g/L] Xylose 6.443 ± 1.025 2.933 ± 0.283 0.773 ± 0.019 Xylobiose 0.583 ± 0.039 n.a.* n.a. Glucose 0.406 ± 0.160 n.a. n.a. Cellobiose 9.460 ± 0.608 1.300 ± 0.071 0.279 ± 0.008 Cellotriose 10.595 ± 0.246 3.767 ± 0.071 0.978 ± 0.022 Maltose 0.387 ± 0.104 n.a. n.a. Maltotriose 0.645 ± 0.010 n.a. n.a. No sugar 0.386 ± 0.034 n.a. n.a. *n.a. – not analysed (lack of biomass) 3.2 Insight into the kinetics of utilization of xylose, glucose and cellobiose To gain deeper insights into the sugar preference and metabolism of C. thermodepolymerans , we conducted a study on the kinetics of culture growth, PHA accumulation, and sugar consumption. The organism was cultivated on xylose, glucose, cellobiose, and various combinations of these sugars. In all cases of experimental set in Erlenmeyer flasks, the initial sugar concentration was set at 20 g/L. The results of these experiments are demonstrated in Table 2 and in Fig. 1 . When evaluating growth on the monosaccharides xylose and glucose, as expected, xylose proved to be a more suitable carbon source for the cultivation of C. thermodepolymerans . The final CDW value, PHA titer, and yield coefficients were significantly higher on xylose compared to glucose. Growth on glucose was associated with a prolonged lag phase (36 h), which led to lower overall efficiency in terms of PHA production. However, once the culture overcame the lag phase, the kinetic parameters—including maximal volumetric biomass or PHA productivity, and sugar consumption rate (see Table 2 )—observed on glucose were comparable to those obtained on xylose. This observation suggests that the culture requires an adaptation period to glucose, likely involving the activation of specific metabolic processes that enable efficient glucose utilization. Interestingly, among the sugars tested in flask experiment, the disaccharide cellobiose was found to be the most preferred. It supported the highest CDW and PHA titers. The culture also exhibited the greatest biomass and PHA productivity, and the most efficient sugar consumption. This observation aligns with the previously discussed findings that the metabolism of oligosaccharides is often more efficient than that of monosaccharides (Dvorak and de Lorenzo 2018). However, even the utilization of cellobiose revealed some problematic aspects. During cultivation, glucose was detected in the culture medium, suggesting that a portion of cellobiose underwent hydrolysis. Two potential mechanisms may explain this phenomenon. The first hypothesis is that cellobiose is transported into the cells, hydrolysed to glucose intracellularly, and subsequently excreted before its phosphorylation by glucokinase Glk. However, this scenario is very unlikely, as cells rarely excrete valuable metabolites like hexoses and phosphorylation is a rapid step. A more plausible explanation is that C. thermodepolymerans possesses enzymatic machinery capable of both intracellular and extracellular cleavage of cellobiose. The majority of cellobiose is transported into bacterial cells and metabolized intracellularly, while a significant portion is cleaved extracellularly or in the periplasm. For instance, approximately 38% of cellobiose consumed during the first 12 hours of cultivation was extracellularly converted into glucose, and 23% was similarly converted at 36 hours. Genome analysis of C. thermodepolymerans identified a gene encoding β-glucosidase, which may be responsible for cellobiose hydrolysis (gene locus ID IS481_00935) (Musilova et al. 2023 ). Notably, a signal sequence specific to the twin-arginine translocation (Tat) pathway was identified at the 5′ end of the β-glucosidase gene. Since the Tat pathway is known to translocate fully folded and active proteins across the cytoplasmic membrane (Berks et al. 2003 ), it is hypothesized that enzyme hydrolyses cellobiose during its passage from the cell, in the cytoplasm, periplasm, and the surrounding medium. Nevertheless, the extracellular cleavage of cellobiose into glucose appears to be a disadvantage for efficient cellobiose utilization. Due to the low efficiency of glucose metabolism in C. thermodepolymerans , extracellularly cleaved glucose accumulates in the medium, with residual concentrations reaching 4.22 g/L after 72 hours of cultivation. This inefficiency suggests that the overall utilization of cellobiose could be significantly improved if extracellular cleavage was minimized or even eliminated. On the other hand, the ability of C. thermodepolymerans to efficiently utilize cellobiose suggests that in this bacterium the challenges associated with glucose assimilation are more likely related to deficiencies in glucose transport into the cell rather than limitations in its intracellular catabolic pathways. 3.3 Growth, PHA production and sugars utilization kinetics on sugar mixtures In addition to single sugars, we also conducted a study on binary and ternary mixtures of glucose, xylose, and cellobiose. All the tested sugars were applied at equal concentrations, with an initial total sugar concentration of 20 g/L. The results are presented in Fig. 1 and Table 2 . The utilization of sugar mixtures, particularly binary sugar mixtures, generally resulted in lower culture growth efficiency and, most notably, reduced PHA accumulation compared to cultivation on single sugars, such as xylose or cellobiose. The cultivations using sugar mixtures yielded lower PHA titers and a reduced PHA content in bacterial biomass (see Fig. 1 h and 1 i). When glucose was combined with xylose, as expected, xylose was consumed at a significantly higher rate and was completely depleted during cultivation. In contrast, glucose was utilized at a considerably lower rate, with approximately 50% of its initial concentration remaining unconsumed. This clearly demonstrates the preferential uptake of xylose over glucose by C. thermodepolymerans . However, this preference is most likely not driven by a carbon catabolite repression mechanism (Görke and Stülke 2008 ), as no diauxia was observed and both substrates were co-consumed throughout the entire cultivation. Surprisingly, even the combination of xylose and cellobiose—two sugars that are efficiently utilized when supplied individually—resulted in a low Y P/S coefficient and reduced PHA titers. During cultivation, the consumption rate of cellobiose (0.253 g/(L·h)) was nearly twice that of xylose (0.129 g/(L·h)). However, a significant portion of the consumed cellobiose underwent extracellular cleavage. For instance, after 12 hours of cultivation, nearly all the utilized cellobiose had been converted into glucose, and by 24 hours, the cellobiose-to-glucose conversion rate reached 46%. Notably, the resulting glucose remained unutilized in the culture medium. This indicates that the extracellular cleavage of cellobiose—particularly pronounced in the presence of xylose—negatively impacted the efficiency of PHA production by C. thermodepolymerans when cultivated on a xylose-cellobiose mixture. Furthermore, cultivation on a glucose-cellobiose mixture resulted in the lowest PHA yield and titer among all tested cultures. Once again, cellobiose was consumed at a very high rate (0.475 g/(L·h), the highest observed in this study). However, due to partial extracellular cleavage of cellobiose, an increase in glucose concentration was observed in the cultivation medium during the initial 24 hours. As cellobiose was completely depleted during the initial 24 h, the microbial culture subsequently began utilizing the accumulated glucose. Nevertheless, as previously noted, the overall productivity of the system remained very low. Last but not least, the cells were also cultivated in a ternary mixture containing all three sugars—glucose, xylose, and cellobiose. Compared to binary mixtures, the PHA yields, PHA titers, and PHA content in biomass were significantly higher and nearly comparable to those obtained with single xylose or cellobiose as the carbon source. Once again, cellobiose was consumed at a higher rate than xylose, and its metabolism was associated with glucose formation. This effect was particularly evident between 12 and 36 hours of cultivation when an increase in glucose concentration was detected. By 48 hours, both xylose and cellobiose were fully utilized, and even the residual glucose concentration remained very low (only about 1.14 g/L) compared to other cultivation scenarios tested in this experiment. Generally, it is evident that the biotechnological potential of C. thermodepolymerans in metabolizing lignocellulose-derived sugars is significantly constrained by its inefficient glucose utilization and the partial extracellular hydrolysis of cellobiose into glucose. Both of these phenomena have a substantial negative impact on the productivity of the microbial culture, particularly in binary mixtures of the investigated sugars. However, as mentioned above, since the bacterial culture efficiently utilizes cellobiose, we hypothesize that the issue with glucose utilization is primarily due to inefficient glucose transport into the bacterial cell, rather than a deficiency in the metabolic pathways required for glucose catabolism. Therefore, equipping C. thermodepolymerans with a highly efficient glucose transporter could potentially resolve both issues—it would enhance glucose uptake and utilization, thereby mitigating also the negative effects associated with partial extracellular hydrolysis of cellobiose into glucose. Table 2 Kinetic parameters and yield coefficients describing the growth of C. thermodepolymerans on xylose, glucose, cellobiose and their binary and ternary mixtures. Cultivations were carried out in flasks in MSM mineral medium at 50°C under constant shaking at 180 rpm. The initial concentrations of the tested sugars were set at 20 g/L. In the case of binary and ternary mixtures, all sugars were applied in equal amounts. Substrate Y X/S Y P/S Maximal volumetric biomass productivity [g/(Lh)] Maximal volumetric PHA productivity [g/(Lh)] Maximal sugar utilization rate [g/(Lh)] Xylose Glucose Cellobiose Xyl 0.30 0.21 0.082 0.066 0.269 n.a. n.a. Glu 0.26 0.17 *0.084 0.068 n.a. 0.252 n.a. Cel 0.44 0.31 0.140 0.074 n.a. n.a. **0.432 Xyl + Glu 0.35 0.20 0.060 0.040 0.152 0.090 n.a. Glu + Cel 0.24 0.04 0.111 0.060 n.a. 0.213 0.475 Xyl + Cel 0.25 0.09 0.089 0.025 0.129 n.a. 0.253 Xyl + Glu + Cel 0.36 0.26 0.129 0.096 0.107 0.145 0.132 * Lag-phase of 36 h in case of glucose **Conversion of cellobiose into glucose – from 38% (12 h of cultivation) to 23% (36 h of cultivation) Y X/S , Y P/S – yield coefficient; biomass (X), respectively product (P) yield on substrate (g/g) 3.4 Integration of exogenous glucose transporter from Zymomonas mobilis into genome of C. thermodepolymerans To test the hypothesis on the glucose transport bottleneck, we endowed C. thermodepolymerans with well-characterized glucose facilitator Glf from Zymomonas mobilis (Parker et al. 1995 ). The glf gene with consensus Shine-Dalgarno sequence AGGAGG was randomly integrated into the Caldimonas chromosome via the mini-Tn5 transposon vector pBAMD1-6 (Martínez-García et al. 2014 ) allowing for a chromosome positioning effect and selection of transformants with balanced glf expression and reduced metabolic burden from exogenous part (Demko et al. 2019 ). The introduction of the vector into the cells was achieved through electroporation, followed by a selection in minimal medium with glucose at 42°C. We did not observe any growth of transformants at 50°C most probably due to the lack of thermal stability of Glf, which originates from a mesophilic bacterium (Parker et al. 1995 ). However, the selection at 42°C resulted in visible growth of the library of transformants in liquid medium with glucose. Individual clones growing fast on glucose were isolated on agar plates. Forty isolated clones were tested for growth on glucose as a sole carbon and energy source in a 48-well plate format and on agar plates (Fig. 2 ). The successful integration of a suitable glucose transporter, which enabled C. thermodepolymerans to grow on this sugar, clearly supports our hypothesis that the bacterium's inability to metabolize glucose is due to a deficiency in its transport rather than a limitation in its catabolism. Cald_GLF3 with confirmed glf integration in the intergenic region between the GMC family oxidoreductase (locus tag IS481_17915) and the hypothetical protein (locus tag IS481_17920) in chromosome emerged as the most promising candidate for further characterization (Fig. 2 ). Further, we tested the PHA production performance of the Caldi_GLF3 strain and compared it to the wild type when cultivated on xylose and glucose. The results are presented in Fig. 3 . It should be noted that this experiment was conducted at 42°C due to the thermal instability of the Glf transporter. However, this temperature is suboptimal for the cultivation of C. thermodepolymerans , which explains the lower PHA, and biomass yields compared to those observed in the wild type cultivated at 50°C (see Fig. 1 and Table 2 ). Interestingly, the growth and PHA production capacity of Caldi_GLF3 on xylose remained unchanged compared to the wild type. However, as expected, the introduction of the Glf transporter significantly improved growth on glucose. While the wild type exhibited only minimal growth on glucose at 42°C, the Caldi_GLF3 strain demonstrated robust growth without a noticeable lag phase. Moreover, the PHA titer obtained on glucose was comparable to that observed for both the wild type and Caldi_GLF3 on xylose. Motivated by the successful PHA production of the Caldi_GLF3 strain on glucose, we decided to further investigate its sugar-utilization capacity. Therefore, similar to the wild-type strain (Table 2 , Fig. 1 ), we cultivated Caldi_GLF3 in the presence of xylose, glucose, cellobiose, as well as their binary and ternary mixtures. Again, the cultivation of Caldi_GLF3 was carried out at 42°C. In addition to measuring CDW and PHA yields, we also recorded the concentrations of residual sugars, and the results are summarized in Table 3 . As previously mentioned, the suboptimal cultivation temperature resulted in lower CDW values for Caldi_GLF3 cultures grown on glucose and xylose compared to the wild-type strain cultivated at 50°C. Interestingly, we observed relatively high amounts of residual monosaccharides after cultivation, which can likely be attributed to less efficient metabolism at a lower temperature. However, the results for cellobiose were surprisingly different. The Caldi_GLF3 strain was able to almost completely utilize cellobiose, achieving the highest CDW and PHA titers observed in our flask cultivations so far—11.5 g/L and 9.6 g/L, respectively. These findings suggest that equipping the strain with a potent glucose transporter effectively resolved limitations associated with cellobiose metabolism, particularly the extracellular cleavage of cellobiose into glucose which was most likely quickly and efficiently transferred to the cells and metabolized. However, we must acknowledge that the exceptionally high productivity of Caldi_GLF3 on cellobiose exceeded our expectations, and we currently lack a definitive explanation for this remarkable result. We hypothesize that the introduction of a new glucose transporter may have also enhanced the activity or expression of the native cellobiose transporter. This potential crosstalk between sugar transport systems could have resulted in highly efficient sugar uptake and subsequent metabolism. Alternatively, it can be speculated that the Glf facilitator of Z. mobilis may exhibit broader substrate specificity (especially at elevated temperatures), potentially facilitating the transport of cellobiose as well. This speculation aligns with the general understanding that some sugar transporters, particularly those in fungi and bacteria, can transport a variety of sugars, including disaccharides, due to their low substrate specificity (Barbi et al. 2021 ). Undoubtedly, the observed improvement in cellobiose utilization by Caldi_GLF3 is a fascinating phenomenon that deserves further investigation. In the case of saccharide mixtures, the introduction of glf from Z. mobilis into the genome of C. thermodepolymerans had a notably positive effect, especially in the presence of cellobiose (Table 3 ). Despite being cultivated at a suboptimal temperature, the Caldi_GLF3 strain demonstrated higher PHA productivity than the wild-type strain (Table 2 ). This effect was particularly evident in the binary mixture of xylose and cellobiose, which resulted in exceptionally high CDW and PHA titers (7.66 g/L and 5.61 g/L, respectively). Similarly, the ternary mixture of the tested saccharides yielded promising results. Conversely, the mixture of glucose and cellobiose was the least efficient. Notably, a significant portion of cellobiose (92% of the initial amount) remained unconsumed in the cultivation medium. Interestingly, this combination was also the least productive for the wild-type strain (Table 2 ). However, in this case, the Caldi_GLF3 strain exhibited substantially higher PHA productivity (1.83 g/L) compared to the wild-type strain (0.64 g/L), highlighting the potential of engineered C. thermodepolymerans for PHA production from plant-derived sugars. Results from cultivations with glucose and xylose, or mixtures of glucose, xylose, and cellobiose, indicate a reversed sugar preference in the Cald_GLF3 strain compared to the wild type. This intriguing phenomenon suggests that activated glucose transport may trigger previously dormant innate carbon catabolite repression mechanism(s), leading to a bacterial preference for glucose over xylose (Görke and Stülke 2008 ). This offers an attractive opportunity for biotechnologists to employ either xylose- or glucose-preferring Caldimonas strains—or to combine them in a synthetic co-culture—depending on the substrate composition and the specific bioprocess scenario (Studer and Troiano 2025 ). Taken together, these results confirm our hypothesis and show that C. thermodepolymerans DSM 15344 can use glucose monomer as the sole carbon and energy source for growth and PHB biosynthesis once the transport bottleneck is removed, e.g. by implantation of an exogenous glucose facilitator. It should be noted that this is not yet an ideal solution as the temperature optimum of the glucose transporter used does not match the growth optimum of C. thermodepolymerans . This temperature incompatibility can be overcome by protein engineering and stabilization of the Glf transporter (Dvorak et al. 2017 ; Musil et al. 2023 ) or by searching for a suitable thermostable homologue in databases (Hon et al., 2020 ). Alternatively, faster growth of DSM 15344 on glucose can be achieved by laboratory evolution (Espeso et al. 2020 ). We plan to investigate these options in our further work. Table 3 Growth, PHA production and sugar consumption of Cald_GLF3 strain cultivated on xylose, glucose, cellobiose and their binary and ternary mixtures. Cultivations were carried out in biological triplicates in flasks with MSM mineral medium at 42°C under constant shaking at 180 rpm, 72 hours. The initial concentrations of the tested sugars were set at 20 g/L. In the case of binary and ternary mixtures, all sugars were applied in equal amounts. CDW [g/L] PHB g/L [g/L] PHB % [g/L] Residual saccharide [g/L] Yield coefficients Xyl Glu Cel Y X/S Y P/S Xyl 4.04 ± 0.12 1.99 ± 0.08 49.33 ± 2.07 12.79 ± 2.35 n.d. n.d. 0.56 0.28 Glu 2.65 ± 0.09 1.85 ± 0.07 69.84 ± 2.52 n.d. 15.92 ± 1.85 n.d. 0.65 0.45 Cel 11.49 ± 0.22 9.62 ± 0.33 83.74 ± 2.40 n.d. 0.82 ± 0.07 n.d. 0.60 0.50 Xyl + glu 3.14 ± 0.17 2.30 ± 0.14 73.17 ± 1.52 10.95 ± 0.75 3.17 ± 0.98 n.d. 0.53 0.39 Glu + cel 2.70 ± 0.42 1.83 ± 0.31 67.66 ± 2.54 n.d. 6.57 ± 0.40 9.20 ± 0.31 0.64 0.43 Xyl + cel 7.66 ± 0.40 5.61 ± 0.27 73.33 ± 1.69 0.25 ± 0.01 0.75 ± 0.01 5.25 ± 0.72 0.56 0.41 Xyl + glu + cel 5.52 ± 0.19 3.83 ± 0.22 69.40 ± 2.30 4.18 ± 1.05 1.70 ± 0.92 0.20 ± 0.34 0.41 0.28 4. Conclusion C. thermodepolymerans DSM 15344 exhibits a distinctive utilization profile for lignocellulose-derived sugars. Despite its limited native capacity for glucose assimilation, primarily due to a transport bottleneck, the bacterium shows remarkable potential for PHA production from selected plant-relevant carbohydrates. The observed extracellular cleavage of cellobiose into glucose, coupled with inefficient glucose uptake, significantly constrains bioprocess efficiency, particularly in sugar mixtures. Introducing of the glf gene from Z. mobilis successfully removed this transport limitation, enabling robust glucose metabolism and markedly improving cellobiose utilization. However, due to the mesophilic origin of Glf, the engineered strain operates optimally only at 42°C, below the natural growth optimum of C. thermodepolymerans . Future work should therefore focus on stabilizing the Glf transporter to withstand higher cultivation temperatures or on identifying alternative thermostable glucose transporters, allowing growth at 50°C. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7619151","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":515227562,"identity":"500c531e-cc6d-4e23-b0e4-21bbd91dd3b7","order_by":0,"name":"Xenie Hajkova","email":"","orcid":"","institution":"Faculty of Chemistry, Brno University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Xenie","middleName":"","lastName":"Hajkova","suffix":""},{"id":515227563,"identity":"98b7430d-6c33-42c1-87a6-37cde374b3d9","order_by":1,"name":"Anastasia Grybchuk-Ieremenko","email":"","orcid":"","institution":"Faculty of Science, Masaryk University","correspondingAuthor":false,"prefix":"","firstName":"Anastasia","middleName":"","lastName":"Grybchuk-Ieremenko","suffix":""},{"id":515227564,"identity":"2c4a472b-fb08-4523-aeca-a58fbbe76ff7","order_by":2,"name":"Pavel Dvorak","email":"","orcid":"","institution":"Faculty of Science, Masaryk University","correspondingAuthor":false,"prefix":"","firstName":"Pavel","middleName":"","lastName":"Dvorak","suffix":""},{"id":515227565,"identity":"70de4439-ddf3-455b-8874-c4d33720cbc1","order_by":3,"name":"Iva Buchtikova","email":"","orcid":"","institution":"Faculty of Chemistry, Brno University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Iva","middleName":"","lastName":"Buchtikova","suffix":""},{"id":515227566,"identity":"fc86ea54-57f2-42c0-94e9-98c961a5ad3e","order_by":4,"name":"Vojtech Cerny","email":"","orcid":"","institution":"Faculty of Chemistry, Brno University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Vojtech","middleName":"","lastName":"Cerny","suffix":""},{"id":515227567,"identity":"9d141710-057f-4dbe-ba5b-5fa7943afad1","order_by":5,"name":"Viktorie Chvatalova","email":"","orcid":"","institution":"Faculty of Chemistry, Brno University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Viktorie","middleName":"","lastName":"Chvatalova","suffix":""},{"id":515227568,"identity":"8851b5e0-3370-4be1-9b4c-2449c89ff7a9","order_by":6,"name":"Stanislav Obruca","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuElEQVRIiWNgGAWjYDACCTBpww9lEK8lTbIBzEggXsthErTwS/c+k/hRc16Cf3YDmzTvDwZ5+QYCWiTnHDeT7Dl2W0LizgE2aZ4EBsNGQloMbqSxSTOw3a4zkEhgNgZqSWAm5DCIln/nJOBa2IjSwth2AKSF8TFICw8hLZJzjjFb9vYlS0jcSGx8OCdNwnAGIS380m2MN358s5Pgn5F84MAbGxvCIYYEGEFqiU8Do2AUjIJRMArwAACjOTKdQgZhdgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-9270-195X","institution":"Faculty of Chemistry, Brno University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Stanislav","middleName":"","lastName":"Obruca","suffix":""}],"badges":[],"createdAt":"2025-09-15 10:02:48","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-7619151/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7619151/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91460015,"identity":"8409e9e4-4d40-473f-b381-3aea7f6a624f","added_by":"auto","created_at":"2025-09-16 16:57:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":241656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eGrowth of C. thermodepolymerans\u003c/strong\u003e\u003c/em\u003e \u003cem\u003e\u003cstrong\u003eon xylose, glucose, cellobiose and their binary and ternary mixtures. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eCultivations were carried out in flasks in MSM mineral medium at 50 °C under constant shaking at 180 rpm in biological triplicates. The initial concentrations of the tested sugars were set at 20 g/L. In the case of binary and ternary mixtures, all sugars were applied in equal amounts. The samples were taken every 12 h and analysed for CDW, PHA and residual sugar contents.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7619151/v1/0fc04b9a622455a3bb289d63.png"},{"id":91460017,"identity":"4b447062-9215-49d9-a459-6fe03c664447","added_by":"auto","created_at":"2025-09-16 16:57:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":44278,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eGrowth of Caldimonas thermodepolymerans clones isolated after the integration of the glf glucose facilitator gene into the chromosome on glucose A.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003eGrowth of C. thermodepolymerans isolates with integrated glf and wild-type control (red line) in MSM minimal medium with 2 g/L glucose and 10 µg/mL gentamicin in 48-well plate at 42 °C. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eB.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e C. thermodepolymerans wild type and isolated clones 1-22 with integrated glf grown on MSM plates supplied with 5 g/L glucose and 10 µg/mL gentamicin at 42 °C.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7619151/v1/d33b24f3aae62b1b4dcbffdc.png"},{"id":91460018,"identity":"2f6cead1-4fdd-4d1b-80fe-8a256199ed35","added_by":"auto","created_at":"2025-09-16 16:57:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":23724,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eGrowth and PHA production capacity of C. thermodepolymerans – wild type and strain Cald_GLF3 harbouring the glucose transporter from Z. mobilis - on xylose and glucose. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eCultivations were carried out in biological triplicates in flasks with MSM mineral medium at 42 °C (a suboptimal temperature for C. thermodepolymerans) due to the temperature instability of the Glf transporter. The initial sugar concentration was set at 20 g/L, and cultivations were performed under constant shaking (180 rpm) for 72 hours.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7619151/v1/d4b4ab32e2b2538d356fa4df.png"},{"id":91461217,"identity":"6a998d48-1b52-47c6-b9e7-abd09e30ad26","added_by":"auto","created_at":"2025-09-16 17:13:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1710400,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7619151/v1/ab92d12c-e31e-4ab0-971d-47202fbd87d9.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCaldimonas thermodepolymerans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Sugar Preference in Polyhydroxyalkanoates Production from Lignocellulose\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Highlights","content":"\u003cp\u003e\u0026bull; DSM 15344 produces PHA from lignocellulose-derived sugars\u003c/p\u003e\u003cp\u003e\u0026bull; Xylose and cellobiose are preferred substrates, while glucose is poorly utilized\u003c/p\u003e\u003cp\u003e\u0026bull; Deficient glucose transporter in DSM 15344 restored by gene\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eModern human society faces a myriad of challenges, and among the most pivotal, unavoidable, and formidable is the ongoing shift of the chemical industry from reliance on fossil resources to renewable alternatives. Microbial biotechnologies stand poised to play an important role in facilitating this transition. Microorganisms exhibit the remarkable ability to utilize various renewable resources and transform them into a diverse array of valuable products, encompassing fuels, chemicals, and materials (Jerome et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Gawel et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMicrobial biotechnologies can also contribute to solution of \u0026ldquo;plastic crisis\u0026rdquo; (Borrelle et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) by providing alternatives to petrochemical polymers. Polyhydroxyalkanoates (PHAs) represent microbial polyesters that various prokaryotes accumulate as storage and stress robustness enhancing metabolites (Obruca et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). As eco-friendly alternatives, PHAs surpass synthetic polymers in sustainability and environmental impact (Fu et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Rajvanshi et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eNevertheless, traditional microbial technologies exhibit several weaknesses which are manifested in high investment and operational costs for these processes. In response to these challenges, Chen and Jiang (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) has recently introduced the concept of Next-Generation Industrial Biotechnology (NGIB). This innovative approach relies on employing extremophiles as chassis for the bioproduction. The inherent resilience of extremophiles to extreme conditions endows these processes with natural robustness against microbial contamination (Yu et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Apart from cultivation, the extremophilic nature of employed microorganisms provides benefits in up-stream processing, for instance, salt tolerance of some extremophilic microorganisms allows the simple integration of acidic/alkaline hydrolysis step in the processing of complex substrates such as lignocelluloses (Kucera et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Furthermore, it may also facilitate streamlined downstream processing as demonstrated in the isolation of intracellular products from hypotonic lysis-sensitive cells (Novackova et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo establish the NGIB process, identifying the optimal extremophilic microbial host is paramount. The desirable microorganism should exhibit resilience, stability, and the ability to efficiently utilize cost-effective renewable resources such as lignocelluloses. A high-level understanding of its genetic and metabolic features is essential for further improvement of the bacterium employing synthetic biology tools and approaches. Currently, within the family of halophiles, the moderately halophilic bacterium \u003cem\u003eHalomonas bluephagenesis\u003c/em\u003e stands out as a promising candidate for manufacturing not only PHAs but other high-value products as well (Park et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHowever, the utilization of thermophiles\u0026mdash;microorganisms adapted to high temperatures\u0026mdash;offers a set of advantages by introducing extreme conditions solely through elevated temperature. Unlike with halophiles, challenges such as salt-related costs and corrosion are absent. Contrary to common misconceptions about the energy demands of thermophilic biotechnologies, well-isolated bioreactors require relatively low energy for heating to moderate thermophilic temperatures. Additionally, thermophilic cultivations can function as \"self-heating systems\" due to the heat energy generated by microbial metabolism and dissipated through stirring, particularly in industrial-scale cultivations with high cell densities. Furthermore, thermophilic processes exhibit high energy efficiency, as only minimal energy-demanding cooling efforts are necessary (Turner et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Ibrahim et al. 2010).\u003c/p\u003e\u003cp\u003e\u003cem\u003eCaldimonas thermodepolymerans\u003c/em\u003e DSM 15344 (formerly known as \u003cem\u003eSchlegelella thermodepolymerans\u003c/em\u003e) is a gram-negative, moderately thermophilic, aerobic bacterium, emerging as a promising candidate for a thermophilic chassis in NGIB processes. This bacterium was initially isolated in by Elbana et al. (2003) due to its notable ability to degrade various polyesters, as reflected in its taxonomic name. Despite this capability being investigated in subsequent studies (Romen et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Elbanna et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), the bacterium received limited attention for an extended period. However, our recent findings demonstrated that \u003cem\u003eC. thermodepolymerans\u003c/em\u003e not only degrades PHAs, but it can also produce them in high quantities, and it shows preference for xylose. These characteristics are not only intriguing from a fundamental perspective but also position \u003cem\u003eC. thermodepolymerans\u003c/em\u003e as a robust candidate for NGIB processes and industrial PHA production, especially from diverse lignocellulose-based resources (Kourilova et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The genomic features and some of the metabolic characteristics of this bacterium have been recently elucidated (Musilova et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The bacterium was evaluated as a potent candidate for PHA production from xylose-rich hemicellulose-based resources and demonstrated its proficiency in xylose metabolism and tolerance to lignocellulose relevant microbial inhibitors such as phenolic compounds or derivates of furfural (Kourilova et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In a study, Zhou et al. (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) delved into cultivation parameters influencing PHA synthesis in \u003cem\u003eC. thermodepolymerans\u003c/em\u003e and employed proteomics to unravel metabolic pathways for PHA synthesis from xylose in this bacterium. Recently, \u003cem\u003eC. thermodepolymerans\u003c/em\u003e demonstrated the ability to achieve high cell densities and elevated PHA productivity on xylose in laboratory bioreactors using a fed-batch cultivation strategy under nitrogen-limited conditions (Jang et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Capability of \u003cem\u003eC. thermodepolymerans\u003c/em\u003e with respect to direct utilization of xylan was also reported (Zhou et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), C. \u003cem\u003ethermodepolymerans\u003c/em\u003e was also utilized for PHA biosynthesis from wine-lees, side products of prosecco wine production (Caminiti et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In the immediate past, initial genome-editing toolkit was developed for \u003cem\u003eC. thermodepolymerans\u003c/em\u003e, potentially opening numerous opportunities for its utilization as a thermophilic chassis for bioproduction (Grybchuk-Ieremeko et al. 2025).\u003c/p\u003e\u003cp\u003eA detailed understanding of metabolic features is a crucial step in the development of extremophilic chassis. Musilova et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) proposed a comprehensive scheme of the central carbohydrate metabolism of three related \u003cem\u003eCaldimonas\u003c/em\u003e strains, including \u003cem\u003eC. thermodepolymerans\u003c/em\u003e DSM 15344. However, the range of plant-derived sugars\u0026mdash;both monomers and oligomers\u0026mdash;and their mixtures that \u003cem\u003eC. thermodepolymerans\u003c/em\u003e can metabolize and efficiently convert into PHA has not yet been thoroughly investigated. Furthermore, other critical metabolic aspects of \u003cem\u003eC. thermodepolymerans\u003c/em\u003e, such as the kinetics of sugar utilization and potential bottlenecks in the efficient processing of certain sugars, remain to be elucidated. A deeper understanding of these fundamental characteristics could be crucial in establishing \u003cem\u003eC. thermodepolymerans\u003c/em\u003e as a promising thermophilic chassis in the NGIB framework.\u003c/p\u003e\u003cp\u003eThe objective of this study was to assess various plant-derived sugars and their mixtures as potential substrates for PHA production by \u003cem\u003eC. thermodepolymerans\u003c/em\u003e. We investigated the utilization kinetics of xylose, glucose, cellobiose, and their combinations, alongside studying microbial culture growth and PHA biosynthesis parameters. Additionally, we addressed the deficiency in glucose metabolism within \u003cem\u003eC. thermodepolymerans\u003c/em\u003e by introducing a high-capacity glucose transporter from \u003cem\u003eZymomonas mobilis\u003c/em\u003e. Our findings suggest that the limitation in glucose metabolism in \u003cem\u003eC. thermodepolymerans\u003c/em\u003e is primarily attributed to glucose transportation into bacterial cells rather than a lack of metabolic capacity for glucose utilization. These results underscore the significant potential of \u003cem\u003eC. thermodepolymerans\u003c/em\u003e as a microbial platform for PHA production within the framework of NGIB. Furthermore, our study demonstrates that its biotechnological potential can be further enhanced through metabolic engineering.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Strains and cultivation media\u003c/h2\u003e\u003cp\u003eThe microorganism used in this study is a bacterial strain \u003cem\u003eCaldimonas thermodepolymerans\u003c/em\u003e DSM 15344 from DSMZ-German Collection of Microorganisms and Cell Cultures. Bacterial suspensions were stored in cryotubes with 10% glycerol as cryoprotectant in a deep freezer at -80\u0026deg;C. Bacteria were cultivated in two types of media. First, nutrient-rich complex medium Nutrient Broth w/w 1% Peptone (HiMedia, India) was used \u0026ndash; 50 mL in 100 mL Erlenmeyer flasks. The basic mineral salt medium (MSM) with the following composition was used as production medium - Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e \u0026middot; 12 H\u003csub\u003e2\u003c/sub\u003eO (9.0 g/L), KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (1.5 g/L), NH\u003csub\u003e4\u003c/sub\u003eCl (1.0 g/L), MgSO\u003csub\u003e4\u003c/sub\u003e \u0026middot; 7 H\u003csub\u003e2\u003c/sub\u003eO (0.2 g/L), CaCl\u003csub\u003e2\u003c/sub\u003e \u0026middot; 2 H\u003csub\u003e2\u003c/sub\u003eO (0.02 g/L), Fe\u003csup\u003e(III)\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003ecitrate (0.0012 g/L), yeast extract (0.5 g/L), 1 mL/L of microelements solution (EDTA (50.0 g/L), FeCl\u003csub\u003e3\u003c/sub\u003e \u0026middot; 6 H\u003csub\u003e2\u003c/sub\u003eO (13.8 g/L), ZnCl\u003csub\u003e2\u003c/sub\u003e (0.84 g/L), CuCl\u003csub\u003e2\u003c/sub\u003e \u0026middot; 2 H\u003csub\u003e2\u003c/sub\u003eO (0.13 g/L), CoCl\u003csub\u003e2\u003c/sub\u003e \u0026middot; 6 H\u003csub\u003e2\u003c/sub\u003eO (0.1 g/L), MnCl\u003csub\u003e2\u003c/sub\u003e \u0026middot; 6 H\u003csub\u003e2\u003c/sub\u003eO (0.016 g/L), H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e (0.1 g/L), dissolved in distilled water) and the specific type and concentration of the carbon source (usually 20 g/L).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Integration of the glucose transporter into C. thermodepolymerans genome\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe \u003cem\u003eglf\u003c/em\u003e gene from \u003cem\u003eZymomonas mobilis\u003c/em\u003e (NCBI gene ID: 79904430), which codes for the glucose facilitator protein, was integrated into the chromosome of \u003cem\u003eCaldimonas\u003c/em\u003e strain DSM 15344. The mini-Tn5 transposon vector pBAMD1-6_\u003cem\u003eglf\u003c/em\u003e constructed previously (Bujdoš et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) was electroporated into \u003cem\u003eCaldimonas\u003c/em\u003e competent cells.\u003c/p\u003e\u003cp\u003eTo prepare electrocompetent bacterial cells, the night culture of \u003cem\u003eCaldimonas\u003c/em\u003e was inoculated into 100 mL of fresh LB medium (Serva) to the OD\u003csub\u003e600\u003c/sub\u003e of 0.05 and shaken at 220 rpm at 37\u0026deg;C. Upon reaching an OD\u003csub\u003e600\u003c/sub\u003e of ~\u0026thinsp;0.5, the bacterial culture was cooled on ice and washed three times with 10% glycerol in milli-Q water. Cells were then resuspended in 1.5 mL of ice-cold 10% glycerol, aliquoted, and frozen at -60\u0026deg;C (Grybchuk-Ieremenko et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Ten electroporations with pBAMD1-6_\u003cem\u003eglf\u003c/em\u003e vector were conducted in parallel. For this purpose, 100 \u0026micro;L of cells were thawed on ice, and 100 ng of plasmid DNA was added. Electroporation was performed using a Biorad Gene Pulser Xcell (2,500 V, 25 \u0026micro;F, 200 Ω) with a 2 mm gap cuvette. Immediately following the pulse, 0.9 mL of prewarmed (37\u0026deg;C) LB was added to the cells, which were then recovered and shaken at 220 rpm at 37\u0026deg;C for 2 h. Afterward, bacterial cultures were transferred into 10 mL of LB (in 50 mL Erlenmeyer flasks) supplied with 10 \u0026micro;g/mL gentamicin and cultured at 42\u0026deg;C, 220 rpm for 24 h. Subsequently, isolates were passaged twice each 24 h in 10 mL of MSM medium with 5 g/L glucose and 10 \u0026micro;g/mL gentamicin at 42\u0026deg;C, 220 rpm. Then the ODs were measured and the isolate that showed the fastest growth on glucose was chosen for further examination. It was spread for single colonies on MSM agar plates supplied with 5 g/L glucose and 10 \u0026micro;g/mL gentamicin and incubated at 42\u0026deg;C. The streaking on fresh MSM plates with glucose was repeated several times to ensure the stability of the integration. The growth of all obtained mutants was tested in the presence of 5 g/L glucose also at \u003cem\u003eCaldimonas'\u003c/em\u003e optimal temperature of 50\u0026deg;C. Forty single colonies in two replicates obtained from preselection in liquid and solid media were tested for the growth in 600 \u0026micro;L of MSM with 2 g/L glucose in a 48-well plate in Infinite\u0026reg; 200 PRO plate reader (Tecan). The isolate Cald_GLF3, which showed repeatedly the fastest growth at 42\u0026deg;C, was taken for further investigations and its growth was compared to the wild type in the 48-well plate assay under the conditions described above. To identify the locus of the \u003cem\u003eglf\u003c/em\u003e integration in the chromosome, arbitrary PCR with standard primers as described by Mart\u0026iacute;nez-Garc\u0026iacute;a et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) was used.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Screening of lignocellulose-derived sugar utilization potential of C. thermodepolymerans\u003c/h2\u003e\u003cp\u003eScreening for carbohydrates occurring in lignocellulosic matrices was performed in 12-well plates for suspension culture. The prepared plates were incubated in the thermoshaker Biosan PST-60HL-4 at 50\u0026deg;C with constant shaking (280 rpm). The volume of MSM including bacterial culture (5 vol. %) was 2 mL per well in biological triplicates. The carbohydrates tested were xylose, xylobiose, glucose, cellobiose, cellotriose, maltose and maltotriose. For control, cultivation without carbohydrate source was also performed. Due to the high purchase cost of some of the tested carbohydrates, specifically cellotriose, maltotriose, and xylobiose, the substrate concentration was adjusted to 10 g/L. As a consequence of the lower substrate concentration, the cultivation time was reduced to 48 hours. After this period the samples for biomass and PHA analysis were collected.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Kinetics of utilization of xylose, glucose, and cellobiose and their mixtures\u003c/h2\u003e\u003cp\u003eThe ability and efficiency of utilizing various carbohydrates (xylose, glucose, cellobiose) and their combinations for growth and PHAs production were monitored using submerged cultivations in Erlenmeyer flasks in biological triplicates, with sampling every 12 hours. The medium composition and cultivation protocol were based on section \u003cspan refid=\"Sec3\" class=\"InternalRef\"\u003e2.1\u003c/span\u003e. The inoculation phase lasted 20 hours, after which 5 vol. % of the culture was transferred to the production medium (100 mL in 250 mL Erlenmeyer flasks) containing\u0026mdash;in addition to MSM\u0026mdash;20 g/L of a carbon source. The tested carbohydrates were added in equal proportions. In the case of two carbohydrates, each was present at 10 g/L. When xylose, glucose, and cellobiose were combined, the concentration of each carbohydrate was 6 g/L. Cultivation in MSM was terminated after 72 hours. Yield coefficients Y\u003csub\u003eX/S\u003c/sub\u003e, Y\u003csub\u003eP/S\u003c/sub\u003e were calculated as the ratio of the biomass or PHA concentration after 72 hours to the amount of substrate consumed during the same period. Maximal volumetric biomass and PHA productivity, and maximum substrate utilization rates were obtained by fitting linear segment of the growth curve graph in MS Excel with a straight line. The resulting values correspond to the slopes of the linear regressions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Analytical methods\u003c/h2\u003e\u003cp\u003eAfter the cultivations were completed, samples were taken to obtain biomass and supernatants for further analyses. Dry biomass was determined gravimetrically. From each cultivation, performed in biological triplicates, 10 mL of culture was collected. The sample was then centrifuged, washed with distilled water, and centrifuged again. The resulting pellet was dried to a constant weight and subsequently weighed.\u003c/p\u003e\u003cp\u003eThe content of PHA in dried biomass was analysed by gas chromatography with a flame ionization detector, following the method described by Obruca et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). PHA content was determined as methyl esters of 3-hydroxy acids, formed by methanolysis of the intracellular polymer. Approximately 8\u0026ndash;11 mg of dry biomass was reacted with 1 mL of chloroform and 0.8 mL of 15% sulfuric acid in methanol containing 5 mg/mL of benzoic acid as an internal standard. The reaction was carried out at 94\u0026deg;C for 3 hours. After completion, 0.5 mL of 0.05 M NaOH was added, and the mixture was vigorously shaken. Following phase separation, 0.05 mL of the organic phase was mixed with 0.9 mL of isopropyl alcohol. The resulting methyl esters were analysed using a Trace GC Ultra (Thermo Fisher Scientific) equipped with a Stabilwax column (30 m \u0026times; 0.32 mm \u0026times; 0.5 \u0026micro;m) and a flame ionization detector. Calibration curve was prepared using commercially available polymer standard.\u003c/p\u003e\u003cp\u003eThe supernatants obtained during sampling were used for the analysis of carbohydrates present in the cultivation medium. Prior to measurement by high-performance liquid chromatography (HPLC) with a refractive index detector, the samples were filtered through a nylon membrane filter with a pore size of 0.45 \u0026micro;m. The analysis was performed using a Shimadzu LC-10AD HPLC system. For isocratic separation, a Water Carbohydrate Analysis column (3.9 \u0026times; 300 mm) was used, with a mobile phase consisting of acetonitrile and water in a ratio of 80:20. Carbohydrate concentrations were calculated from peak areas using calibration curves for xylose, glucose and cellobiose.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Utilization of lignocellulose-relevant sugars by C. thermodepolymerans\u003c/h2\u003e\u003cp\u003ePlant biomass holds promise as a resource serving as a viable alternative to fossil resources. Therefore, we screened the capacity of the \u003cem\u003eC. thermodepolymerans\u003c/em\u003e to utilize various plant-relevant sugars including disaccharides and trisaccharides, the results are shown in the Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Consistent with prior findings (Kourilova et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Musilova et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), the bacterium exhibited a pronounced preference for xylose among the tested monosaccharides, surpassing its affinity for other sugars, including glucose. This holds significant importance as xylose stands out as the most abundant pentose and, after glucose, the second most prevalent monosaccharide in lignocellulosic biomass. The efficient conversion of xylose into valuable chemicals, fuels, and materials emerges as a critical task for establishing a sustainable bioeconomy (Narisetty et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In this light, due to its high affinity to xylose, \u003cem\u003eC. thermodepolymerans\u003c/em\u003e appears to be a distinctive bacterium, presenting a promising candidate as an NGIB chassis for valorising xylose-rich resources. Nevertheless, our results indicate that the bacterium is unable to cleave glycosidic bonds between xyloses in xylobiose since we observed only negligible growth of the culture on this disaccharide (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eA distinct scenario arises when examining the metabolism of glucose in \u003cem\u003eC. thermodepolymerans\u003c/em\u003e. The wild-type strain of this bacterium exhibits limited efficiency in metabolizing the monosaccharide. Surprisingly, the bacterium demonstrates remarkable proficiency in utilizing glucose oligomers linked by β-1,4-glycosidic bonds, such as cellobiose and cellotriose. Strikingly, the growth and synthesis of polyhydroxyalkanoates (PHA) on these glucose oligosaccharides, particularly cellotriose, surpass even that observed on the preferred substrate\u0026mdash;xylose. This may reflect a potential energetic advantage of transporting longer oligosaccharides, such as cellotriose, compared to shorter ones like cellobiose or monomeric glucose. If sugars are actively transported through one of the present ABC transporters and intracellular cleavage of cellooligosaccharides occurs, only one ATP molecule is required per cellotriose, yielding three glucose molecules. In contrast, cellobiose yields only two glucose molecules for the same energy cost (Dvorak and de Lorenzo 2018; Parisutham et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn contrast, the presence of α-1,4-glycosidic bonds, as found in maltose or maltotriose, impedes the metabolization of these glucose oligosaccharides by \u003cem\u003eC. thermodepolymerans\u003c/em\u003e. The inability to cleave α-1,4-glycosidic bonds renders the wild-type strain of \u003cem\u003eC. thermodepolymerans\u003c/em\u003e unsuitable as a PHA producer from starch and similar saccharides. However, it is worth noting that other members of the \u003cem\u003eCaldimonas\u003c/em\u003e genus, such as \u003cem\u003eCaldimonas taiwanensis\u003c/em\u003e (Chen et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Sheu et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), possess amylolytic activity. Therefore, they could serve as suitable sources for genes that could enhance the biotechnological potential of \u003cem\u003eC. thermodepolymerans\u003c/em\u003e through metabolic engineering techniques.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eScreening of catabolic potential of C. thermodepolymerans with respect to selected plant-derived sugars\u003c/b\u003e, \u003cem\u003ecultivations were carried out in 12-well plates within 48 h time interval at 50\u0026deg;C with initial sugar concentration 10 g/L.\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOD 630 nm\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCDW [g/L]\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePHA [g/L]\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eXylose\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e6.443\u0026thinsp;\u0026plusmn;\u0026thinsp;1.025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.933\u0026thinsp;\u0026plusmn;\u0026thinsp;0.283\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.773\u0026thinsp;\u0026plusmn;\u0026thinsp;0.019\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eXylobiose\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.583\u0026thinsp;\u0026plusmn;\u0026thinsp;0.039\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003en.a.*\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003en.a.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGlucose\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.406\u0026thinsp;\u0026plusmn;\u0026thinsp;0.160\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003en.a.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003en.a.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCellobiose\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e9.460\u0026thinsp;\u0026plusmn;\u0026thinsp;0.608\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.300\u0026thinsp;\u0026plusmn;\u0026thinsp;0.071\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.279\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCellotriose\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e10.595\u0026thinsp;\u0026plusmn;\u0026thinsp;0.246\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.767\u0026thinsp;\u0026plusmn;\u0026thinsp;0.071\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.978\u0026thinsp;\u0026plusmn;\u0026thinsp;0.022\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMaltose\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.387\u0026thinsp;\u0026plusmn;\u0026thinsp;0.104\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003en.a.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003en.a.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMaltotriose\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.645\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003en.a.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003en.a.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNo sugar\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.386\u0026thinsp;\u0026plusmn;\u0026thinsp;0.034\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003en.a.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003en.a.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003cem\u003e*n.a. \u0026ndash; not analysed (lack of biomass)\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Insight into the kinetics of utilization of xylose, glucose and cellobiose\u003c/h2\u003e\u003cp\u003eTo gain deeper insights into the sugar preference and metabolism of \u003cem\u003eC. thermodepolymerans\u003c/em\u003e, we conducted a study on the kinetics of culture growth, PHA accumulation, and sugar consumption. The organism was cultivated on xylose, glucose, cellobiose, and various combinations of these sugars. In all cases of experimental set in Erlenmeyer flasks, the initial sugar concentration was set at 20 g/L. The results of these experiments are demonstrated in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eWhen evaluating growth on the monosaccharides xylose and glucose, as expected, xylose proved to be a more suitable carbon source for the cultivation of \u003cem\u003eC. thermodepolymerans\u003c/em\u003e. The final CDW value, PHA titer, and yield coefficients were significantly higher on xylose compared to glucose. Growth on glucose was associated with a prolonged lag phase (36 h), which led to lower overall efficiency in terms of PHA production. However, once the culture overcame the lag phase, the kinetic parameters\u0026mdash;including maximal volumetric biomass or PHA productivity, and sugar consumption rate (see Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e)\u0026mdash;observed on glucose were comparable to those obtained on xylose. This observation suggests that the culture requires an adaptation period to glucose, likely involving the activation of specific metabolic processes that enable efficient glucose utilization.\u003c/p\u003e\u003cp\u003eInterestingly, among the sugars tested in flask experiment, the disaccharide cellobiose was found to be the most preferred. It supported the highest CDW and PHA titers. The culture also exhibited the greatest biomass and PHA productivity, and the most efficient sugar consumption. This observation aligns with the previously discussed findings that the metabolism of oligosaccharides is often more efficient than that of monosaccharides (Dvorak and de Lorenzo 2018).\u003c/p\u003e\u003cp\u003eHowever, even the utilization of cellobiose revealed some problematic aspects. During cultivation, glucose was detected in the culture medium, suggesting that a portion of cellobiose underwent hydrolysis. Two potential mechanisms may explain this phenomenon. The first hypothesis is that cellobiose is transported into the cells, hydrolysed to glucose intracellularly, and subsequently excreted before its phosphorylation by glucokinase Glk. However, this scenario is very unlikely, as cells rarely excrete valuable metabolites like hexoses and phosphorylation is a rapid step. A more plausible explanation is that \u003cem\u003eC. thermodepolymerans\u003c/em\u003e possesses enzymatic machinery capable of both intracellular and extracellular cleavage of cellobiose. The majority of cellobiose is transported into bacterial cells and metabolized intracellularly, while a significant portion is cleaved extracellularly or in the periplasm. For instance, approximately 38% of cellobiose consumed during the first 12 hours of cultivation was extracellularly converted into glucose, and 23% was similarly converted at 36 hours. Genome analysis of \u003cem\u003eC. thermodepolymerans\u003c/em\u003e identified a gene encoding β-glucosidase, which may be responsible for cellobiose hydrolysis (gene locus ID IS481_00935) (Musilova et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Notably, a signal sequence specific to the twin-arginine translocation (Tat) pathway was identified at the 5\u0026prime; end of the β-glucosidase gene. Since the Tat pathway is known to translocate fully folded and active proteins across the cytoplasmic membrane (Berks et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), it is hypothesized that enzyme hydrolyses cellobiose during its passage from the cell, in the cytoplasm, periplasm, and the surrounding medium.\u003c/p\u003e\u003cp\u003eNevertheless, the extracellular cleavage of cellobiose into glucose appears to be a disadvantage for efficient cellobiose utilization. Due to the low efficiency of glucose metabolism in \u003cem\u003eC. thermodepolymerans\u003c/em\u003e, extracellularly cleaved glucose accumulates in the medium, with residual concentrations reaching 4.22 g/L after 72 hours of cultivation. This inefficiency suggests that the overall utilization of cellobiose could be significantly improved if extracellular cleavage was minimized or even eliminated.\u003c/p\u003e\u003cp\u003eOn the other hand, the ability of \u003cem\u003eC. thermodepolymerans\u003c/em\u003e to efficiently utilize cellobiose suggests that in this bacterium the challenges associated with glucose assimilation are more likely related to deficiencies in glucose transport into the cell rather than limitations in its intracellular catabolic pathways.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Growth, PHA production and sugars utilization kinetics on sugar mixtures\u003c/h2\u003e\u003cp\u003eIn addition to single sugars, we also conducted a study on binary and ternary mixtures of glucose, xylose, and cellobiose. All the tested sugars were applied at equal concentrations, with an initial total sugar concentration of 20 g/L. The results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eThe utilization of sugar mixtures, particularly binary sugar mixtures, generally resulted in lower culture growth efficiency and, most notably, reduced PHA accumulation compared to cultivation on single sugars, such as xylose or cellobiose. The cultivations using sugar mixtures yielded lower PHA titers and a reduced PHA content in bacterial biomass (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei).\u003c/p\u003e\u003cp\u003eWhen glucose was combined with xylose, as expected, xylose was consumed at a significantly higher rate and was completely depleted during cultivation. In contrast, glucose was utilized at a considerably lower rate, with approximately 50% of its initial concentration remaining unconsumed. This clearly demonstrates the preferential uptake of xylose over glucose by \u003cem\u003eC. thermodepolymerans\u003c/em\u003e. However, this preference is most likely not driven by a carbon catabolite repression mechanism (G\u0026ouml;rke and St\u0026uuml;lke \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), as no diauxia was observed and both substrates were co-consumed throughout the entire cultivation.\u003c/p\u003e\u003cp\u003eSurprisingly, even the combination of xylose and cellobiose\u0026mdash;two sugars that are efficiently utilized when supplied individually\u0026mdash;resulted in a low Y\u003csub\u003eP/S\u003c/sub\u003e coefficient and reduced PHA titers. During cultivation, the consumption rate of cellobiose (0.253 g/(L\u0026middot;h)) was nearly twice that of xylose (0.129 g/(L\u0026middot;h)). However, a significant portion of the consumed cellobiose underwent extracellular cleavage. For instance, after 12 hours of cultivation, nearly all the utilized cellobiose had been converted into glucose, and by 24 hours, the cellobiose-to-glucose conversion rate reached 46%. Notably, the resulting glucose remained unutilized in the culture medium. This indicates that the extracellular cleavage of cellobiose\u0026mdash;particularly pronounced in the presence of xylose\u0026mdash;negatively impacted the efficiency of PHA production by \u003cem\u003eC. thermodepolymerans\u003c/em\u003e when cultivated on a xylose-cellobiose mixture.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurthermore, cultivation on a glucose-cellobiose mixture resulted in the lowest PHA yield and titer among all tested cultures. Once again, cellobiose was consumed at a very high rate (0.475 g/(L\u0026middot;h), the highest observed in this study). However, due to partial extracellular cleavage of cellobiose, an increase in glucose concentration was observed in the cultivation medium during the initial 24 hours. As cellobiose was completely depleted during the initial 24 h, the microbial culture subsequently began utilizing the accumulated glucose. Nevertheless, as previously noted, the overall productivity of the system remained very low.\u003c/p\u003e\u003cp\u003eLast but not least, the cells were also cultivated in a ternary mixture containing all three sugars\u0026mdash;glucose, xylose, and cellobiose. Compared to binary mixtures, the PHA yields, PHA titers, and PHA content in biomass were significantly higher and nearly comparable to those obtained with single xylose or cellobiose as the carbon source. Once again, cellobiose was consumed at a higher rate than xylose, and its metabolism was associated with glucose formation. This effect was particularly evident between 12 and 36 hours of cultivation when an increase in glucose concentration was detected. By 48 hours, both xylose and cellobiose were fully utilized, and even the residual glucose concentration remained very low (only about 1.14 g/L) compared to other cultivation scenarios tested in this experiment.\u003c/p\u003e\u003cp\u003eGenerally, it is evident that the biotechnological potential of \u003cem\u003eC. thermodepolymerans\u003c/em\u003e in metabolizing lignocellulose-derived sugars is significantly constrained by its inefficient glucose utilization and the partial extracellular hydrolysis of cellobiose into glucose. Both of these phenomena have a substantial negative impact on the productivity of the microbial culture, particularly in binary mixtures of the investigated sugars. However, as mentioned above, since the bacterial culture efficiently utilizes cellobiose, we hypothesize that the issue with glucose utilization is primarily due to inefficient glucose transport into the bacterial cell, rather than a deficiency in the metabolic pathways required for glucose catabolism. Therefore, equipping \u003cem\u003eC. thermodepolymerans\u003c/em\u003e with a highly efficient glucose transporter could potentially resolve both issues\u0026mdash;it would enhance glucose uptake and utilization, thereby mitigating also the negative effects associated with partial extracellular hydrolysis of cellobiose into glucose.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eKinetic parameters and yield coefficients describing the growth of C. thermodepolymerans on xylose, glucose, cellobiose and their binary and ternary mixtures.\u003c/b\u003e \u003cem\u003eCultivations were carried out in flasks in MSM mineral medium at 50\u0026deg;C under constant shaking at 180 rpm. The initial concentrations of the tested sugars were set at 20 g/L. In the case of binary and ternary mixtures, all sugars were applied in equal amounts.\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSubstrate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eY\u003csub\u003eX/S\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eY\u003csub\u003eP/S\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMaximal volumetric biomass productivity [g/(Lh)]\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMaximal volumetric PHA productivity [g/(Lh)]\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e\u003cp\u003eMaximal sugar utilization rate [g/(Lh)]\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eXylose\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eGlucose\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eCellobiose\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eXyl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.082\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.066\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.269\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003en.a.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003en.a.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlu\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e*0.084\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.068\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003en.a.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.252\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003en.a.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCel\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.140\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.074\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003en.a.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003en.a.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e**0.432\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eXyl\u0026thinsp;+\u0026thinsp;Glu\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.060\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.040\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.152\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.090\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003en.a.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlu\u0026thinsp;+\u0026thinsp;Cel\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.111\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.060\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003en.a.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.213\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.475\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eXyl\u0026thinsp;+\u0026thinsp;Cel\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.089\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003en.a.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.253\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eXyl\u0026thinsp;+\u0026thinsp;Glu\u0026thinsp;+\u0026thinsp;Cel\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.096\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.107\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.145\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.132\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003e* Lag-phase of 36 h in case of glucose\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003e**Conversion of cellobiose into glucose \u0026ndash; from 38% (12 h of cultivation) to 23% (36 h of cultivation)\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003eY\u003csub\u003eX/S\u003c/sub\u003e, Y\u003csub\u003eP/S\u003c/sub\u003e \u0026ndash; yield coefficient; biomass (X), respectively product (P) yield on substrate (g/g)\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Integration of exogenous glucose transporter from Zymomonas mobilis into genome of C. thermodepolymerans\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTo test the hypothesis on the glucose transport bottleneck, we endowed \u003cem\u003eC. thermodepolymerans\u003c/em\u003e with well-characterized glucose facilitator Glf from \u003cem\u003eZymomonas mobilis\u003c/em\u003e (Parker et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). The \u003cem\u003eglf\u003c/em\u003e gene with consensus Shine-Dalgarno sequence AGGAGG was randomly integrated into the \u003cem\u003eCaldimonas\u003c/em\u003e chromosome via the mini-Tn5 transposon vector pBAMD1-6 (Mart\u0026iacute;nez-Garc\u0026iacute;a et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) allowing for a chromosome positioning effect and selection of transformants with balanced \u003cem\u003eglf\u003c/em\u003e expression and reduced metabolic burden from exogenous part (Demko et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The introduction of the vector into the cells was achieved through electroporation, followed by a selection in minimal medium with glucose at 42\u0026deg;C. We did not observe any growth of transformants at 50\u0026deg;C most probably due to the lack of thermal stability of Glf, which originates from a mesophilic bacterium (Parker et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). However, the selection at 42\u0026deg;C resulted in visible growth of the library of transformants in liquid medium with glucose. Individual clones growing fast on glucose were isolated on agar plates. Forty isolated clones were tested for growth on glucose as a sole carbon and energy source in a 48-well plate format and on agar plates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The successful integration of a suitable glucose transporter, which enabled \u003cem\u003eC. thermodepolymerans\u003c/em\u003e to grow on this sugar, clearly supports our hypothesis that the bacterium's inability to metabolize glucose is due to a deficiency in its transport rather than a limitation in its catabolism. Cald_GLF3 with confirmed \u003cem\u003eglf\u003c/em\u003e integration in the intergenic region between the GMC family oxidoreductase (locus tag IS481_17915) and the hypothetical protein (locus tag IS481_17920) in chromosome emerged as the most promising candidate for further characterization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurther, we tested the PHA production performance of the Caldi_GLF3 strain and compared it to the wild type when cultivated on xylose and glucose. The results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. It should be noted that this experiment was conducted at 42\u0026deg;C due to the thermal instability of the Glf transporter. However, this temperature is suboptimal for the cultivation of \u003cem\u003eC. thermodepolymerans\u003c/em\u003e, which explains the lower PHA, and biomass yields compared to those observed in the wild type cultivated at 50\u0026deg;C (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Interestingly, the growth and PHA production capacity of Caldi_GLF3 on xylose remained unchanged compared to the wild type. However, as expected, the introduction of the Glf transporter significantly improved growth on glucose. While the wild type exhibited only minimal growth on glucose at 42\u0026deg;C, the Caldi_GLF3 strain demonstrated robust growth without a noticeable lag phase. Moreover, the PHA titer obtained on glucose was comparable to that observed for both the wild type and Caldi_GLF3 on xylose.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMotivated by the successful PHA production of the Caldi_GLF3 strain on glucose, we decided to further investigate its sugar-utilization capacity. Therefore, similar to the wild-type strain (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), we cultivated Caldi_GLF3 in the presence of xylose, glucose, cellobiose, as well as their binary and ternary mixtures.\u003c/p\u003e\u003cp\u003eAgain, the cultivation of Caldi_GLF3 was carried out at 42\u0026deg;C. In addition to measuring CDW and PHA yields, we also recorded the concentrations of residual sugars, and the results are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. As previously mentioned, the suboptimal cultivation temperature resulted in lower CDW values for Caldi_GLF3 cultures grown on glucose and xylose compared to the wild-type strain cultivated at 50\u0026deg;C. Interestingly, we observed relatively high amounts of residual monosaccharides after cultivation, which can likely be attributed to less efficient metabolism at a lower temperature.\u003c/p\u003e\u003cp\u003eHowever, the results for cellobiose were surprisingly different. The Caldi_GLF3 strain was able to almost completely utilize cellobiose, achieving the highest CDW and PHA titers observed in our flask cultivations so far\u0026mdash;11.5 g/L and 9.6 g/L, respectively. These findings suggest that equipping the strain with a potent glucose transporter effectively resolved limitations associated with cellobiose metabolism, particularly the extracellular cleavage of cellobiose into glucose which was most likely quickly and efficiently transferred to the cells and metabolized. However, we must acknowledge that the exceptionally high productivity of Caldi_GLF3 on cellobiose exceeded our expectations, and we currently lack a definitive explanation for this remarkable result. We hypothesize that the introduction of a new glucose transporter may have also enhanced the activity or expression of the native cellobiose transporter. This potential crosstalk between sugar transport systems could have resulted in highly efficient sugar uptake and subsequent metabolism. Alternatively, it can be speculated that the Glf facilitator of \u003cem\u003eZ. mobilis\u003c/em\u003e may exhibit broader substrate specificity (especially at elevated temperatures), potentially facilitating the transport of cellobiose as well. This speculation aligns with the general understanding that some sugar transporters, particularly those in fungi and bacteria, can transport a variety of sugars, including disaccharides, due to their low substrate specificity (Barbi et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Undoubtedly, the observed improvement in cellobiose utilization by Caldi_GLF3 is a fascinating phenomenon that deserves further investigation.\u003c/p\u003e\u003cp\u003eIn the case of saccharide mixtures, the introduction of \u003cem\u003eglf\u003c/em\u003e from \u003cem\u003eZ. mobilis\u003c/em\u003e into the genome of \u003cem\u003eC. thermodepolymerans\u003c/em\u003e had a notably positive effect, especially in the presence of cellobiose (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Despite being cultivated at a suboptimal temperature, the Caldi_GLF3 strain demonstrated higher PHA productivity than the wild-type strain (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This effect was particularly evident in the binary mixture of xylose and cellobiose, which resulted in exceptionally high CDW and PHA titers (7.66 g/L and 5.61 g/L, respectively). Similarly, the ternary mixture of the tested saccharides yielded promising results. Conversely, the mixture of glucose and cellobiose was the least efficient. Notably, a significant portion of cellobiose (92% of the initial amount) remained unconsumed in the cultivation medium. Interestingly, this combination was also the least productive for the wild-type strain (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, in this case, the \u003cem\u003eCaldi_GLF3\u003c/em\u003e strain exhibited substantially higher PHA productivity (1.83 g/L) compared to the wild-type strain (0.64 g/L), highlighting the potential of engineered \u003cem\u003eC. thermodepolymerans\u003c/em\u003e for PHA production from plant-derived sugars. Results from cultivations with glucose and xylose, or mixtures of glucose, xylose, and cellobiose, indicate a reversed sugar preference in the Cald_GLF3 strain compared to the wild type. This intriguing phenomenon suggests that activated glucose transport may trigger previously dormant innate carbon catabolite repression mechanism(s), leading to a bacterial preference for glucose over xylose (G\u0026ouml;rke and St\u0026uuml;lke \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). This offers an attractive opportunity for biotechnologists to employ either xylose- or glucose-preferring \u003cem\u003eCaldimonas\u003c/em\u003e strains\u0026mdash;or to combine them in a synthetic co-culture\u0026mdash;depending on the substrate composition and the specific bioprocess scenario (Studer and Troiano \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTaken together, these results confirm our hypothesis and show that \u003cem\u003eC. thermodepolymerans\u003c/em\u003e DSM 15344 can use glucose monomer as the sole carbon and energy source for growth and PHB biosynthesis once the transport bottleneck is removed, e.g. by implantation of an exogenous glucose facilitator. It should be noted that this is not yet an ideal solution as the temperature optimum of the glucose transporter used does not match the growth optimum of \u003cem\u003eC. thermodepolymerans\u003c/em\u003e. This temperature incompatibility can be overcome by protein engineering and stabilization of the Glf transporter (Dvorak et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Musil et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) or by searching for a suitable thermostable homologue in databases (Hon et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Alternatively, faster growth of DSM 15344 on glucose can be achieved by laboratory evolution (Espeso et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). We plan to investigate these options in our further work.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eGrowth, PHA production and sugar consumption of Cald_GLF3 strain cultivated on xylose, glucose, cellobiose and their binary and ternary mixtures.\u003c/b\u003e \u003cem\u003eCultivations were carried out in biological triplicates in flasks with MSM mineral medium at 42\u0026deg;C under constant shaking at 180 rpm, 72 hours. The initial concentrations of the tested sugars were set at 20 g/L. In the case of binary and ternary mixtures, all sugars were applied in equal amounts.\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCDW\u003c/p\u003e\u003cp\u003e[g/L]\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePHB g/L\u003c/p\u003e\u003cp\u003e[g/L]\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePHB %\u003c/p\u003e\u003cp\u003e[g/L]\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e\u003cp\u003eResidual saccharide [g/L]\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003eYield coefficients\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eXyl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eGlu\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCel\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eY\u003csub\u003eX/S\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eY\u003csub\u003eP/S\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eXyl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e4.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e49.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e12.79\u0026thinsp;\u0026plusmn;\u0026thinsp;2.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003en.d.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003en.d.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlu\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e2.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e69.84\u0026thinsp;\u0026plusmn;\u0026thinsp;2.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003en.d.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e15.92\u0026thinsp;\u0026plusmn;\u0026thinsp;1.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003en.d.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.45\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCel\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e11.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e9.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e83.74\u0026thinsp;\u0026plusmn;\u0026thinsp;2.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003en.d.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003en.d.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eXyl\u0026thinsp;+\u0026thinsp;glu\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e3.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e2.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e73.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003en.d.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.39\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlu\u0026thinsp;+\u0026thinsp;cel\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e2.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e67.66\u0026thinsp;\u0026plusmn;\u0026thinsp;2.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003en.d.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e9.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.43\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eXyl\u0026thinsp;+\u0026thinsp;cel\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e7.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e5.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e73.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.41\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eXyl\u0026thinsp;+\u0026thinsp;glu\u0026thinsp;+\u0026thinsp;cel\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e5.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e3.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e69.40\u0026thinsp;\u0026plusmn;\u0026thinsp;2.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.18\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003e\u003cem\u003eC. thermodepolymerans\u003c/em\u003e DSM 15344 exhibits a distinctive utilization profile for lignocellulose-derived sugars. Despite its limited native capacity for glucose assimilation, primarily due to a transport bottleneck, the bacterium shows remarkable potential for PHA production from selected plant-relevant carbohydrates. The observed extracellular cleavage of cellobiose into glucose, coupled with inefficient glucose uptake, significantly constrains bioprocess efficiency, particularly in sugar mixtures. Introducing of the \u003cem\u003eglf\u003c/em\u003e gene from \u003cem\u003eZ. mobilis\u003c/em\u003e successfully removed this transport limitation, enabling robust glucose metabolism and markedly improving cellobiose utilization. However, due to the mesophilic origin of Glf, the engineered strain operates optimally only at 42\u0026deg;C, below the natural growth optimum of \u003cem\u003eC. thermodepolymerans\u003c/em\u003e. Future work should therefore focus on stabilizing the Glf transporter to withstand higher cultivation temperatures or on identifying alternative thermostable glucose transporters, allowing growth at 50\u0026deg;C. These improvements, alongside broader transporter engineering and adaptive laboratory evolution, could unlock the full potential of \u003cem\u003eC. thermodepolymerans\u003c/em\u003e as an NGIB chassis for efficient lignocellulose valorisation into sustainable bioplastics.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgments:\u003c/h2\u003e\u003cp\u003eThis study was supported by grant project GACR GA22-10845S.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBarbi F, Vallon L, Guerrero-Gal\u0026aacute;n C, Zimmermann SD, Melayah D, Abrouk D, Dor\u0026eacute; J, Lemaire M, Fraissinet-Tachet L, Luis P, Marmeisse R (2021) Datamining and functional environmental genomics reassess the phylogenetics and functional diversity of fungal monosaccharide transporters. 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RSC Sustain 3:1685. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/d5su00040h\u003c/span\u003e\u003cspan address=\"10.1039/d5su00040h\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Czech Science Foundation","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Caldimonas thermodepolymerans, polyhydroxyalkanoates, thermophiles, sugar metabolism, glucose transporters, lignocelluloses","lastPublishedDoi":"10.21203/rs.3.rs-7619151/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7619151/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eCaldimonas thermodepolymerans\u003c/em\u003e DSM 15344, a moderately thermophilic bacterium, has emerged as a promising candidate for next-generation industrial biotechnology (NGIB) due to its ability to utilize lignocellulose-derived sugars for polyhydroxyalkanoate (PHA) production. This study assesses its metabolic potential by evaluating the utilization of various plant-derived sugars and their mixtures, with a focus on xylose, glucose, and cellobiose. The results indicate that \u003cem\u003eC. thermodepolymerans\u003c/em\u003e exhibits a strong preference for xylose over glucose but demonstrates even greater efficiency in metabolizing cellobiose. However, extracellular hydrolysis of cellobiose leads to glucose accumulation, which constrains overall productivity. Our findings suggest that the primary limitation in glucose metabolism is inefficient glucose transport rather than intracellular catabolism. To address this bottleneck, the \u003cem\u003eglf\u003c/em\u003e glucose facilitator from the mesophilic bacterium \u003cem\u003eZymomonas mobilis\u003c/em\u003e was introduced into \u003cem\u003eC. thermodepolymerans\u003c/em\u003e, enhancing its glucose utilization capacity. The engineered strain (Caldi_GLF3) exhibited significantly improved PHA productivity, particularly when cultivated on sugar mixtures containing cellobiose. Despite being grown at suboptimal temperatures due to the thermal instability of \u003cem\u003eglf\u003c/em\u003e from \u003cem\u003eZ. mobilis\u003c/em\u003e, Caldi_GLF3 outperformed the wild-type strain, achieving notably high PHA yields, especially in the presence of cellobiose. These findings highlight the critical role of glucose transport in the metabolism of \u003cem\u003eC. thermodepolymerans\u003c/em\u003e and suggest that targeted engineering can further enhance its biotechnological potential. This study establishes \u003cem\u003eC. thermodepolymerans\u003c/em\u003e as a promising thermophilic chassis for PHA production from lignocellulosic sugars, contributing to sustainable biopolymer synthesis.\u003c/p\u003e","manuscriptTitle":"Caldimonas thermodepolymerans Sugar Preference in Polyhydroxyalkanoates Production from Lignocellulose","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-16 16:57:28","doi":"10.21203/rs.3.rs-7619151/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0c84f057-e8d9-44c9-933a-25d705c33449","owner":[],"postedDate":"September 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54726528,"name":"Biotechnology and Bioengineering"}],"tags":[],"updatedAt":"2025-09-16T16:57:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-16 16:57:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7619151","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7619151","identity":"rs-7619151","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}