Engineering a sustainable system for pectin-based lipid metabolism through modular co-utilization of two Kluyveromyces marxianus strains

Engineering a sustainable system for pectin-based lipid metabolism through modular co-utilization of two Kluyveromyces marxianus strains | 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 Engineering a sustainable system for pectin-based lipid metabolism through modular co-utilization of two Kluyveromyces marxianus strains Bofan Yu, He Qiao, Xuye Lang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6193521/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Aug, 2025 Read the published version in Bioresources and Bioprocessing → Version 1 posted 5 You are reading this latest preprint version Abstract Pectin bioconversion from renewable feedstocks represents an appealing and sustainable production route. However, the microbial valorization of pectin is not well developed and requires an efficient expression of key enzymes. Here, a constructed biological system successfully drove pectin-based lipid metabolism by collaborative utilization of two engineered Kluyveromyces marxianus strains. The YKM1013 strain with an overexpression of the PGU1 gene served to break down pectin, resulting in a 65% improvement in conversion rate. And the YKM1015 strain with an additional central D-galacturonic acid (D-galUA) metabolic pathway effectively utilized the available D-galUA components. The developed strategy enabled an effective bioconversion of pectin-based lipid metabolism, with an increasing of 19-fold in medium-chain fatty acid (MCFA) and 6-fold in long-chain fatty acid (LCFA). Collectively, this study provided a feasible and sustainable bioconversion route for transforming pectin into chemicals that can be employed in the construction of a microbial cell factory platform for pectin valorization. metabolic engineering pectin bioconversion Kluyveromyces marxianus sustainable system lipid metabolism modular co-utilization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Pectin is a ubiquitous polysaccharide found in the cell wall and inner layer of fruits, vegetables, and other higher plants (Xiang et al. 2024 ). The majority of pectin is derived from raw materials, such as fruit peels and pomaces (Williams et al. 2019 ; Erian and Sauer 2022 ). However, nearly 50% of those is regarded as waste and discarded, resulting in significant environmental pollution and unsustainable development (Chavan et al. 2019 ; Sharma et al. 2022 ). Following enzymatic depolymerization, pectin is converted to D-galUA (~ 70%) and available neutral sugars (including xylose, galactose, mannose, fucose, or arabinose) (Mohnen 2008 ). These components can be utilized as a bio-active feedstock to yield valuable products. Thus, the development of a green bioconversion route is critical for the sustainable utilization of pectin. Microorganisms engineered through metabolic engineering can serve as effective cell factories offering potential to advance agricultural by-product waste treatment and generate a variety of valuable products from biomass resources (Protzko et al. 2018 ; Wikandari et al. 2022 ; Gao et al. 2023 ; Li et al. 2023 ; Zhang et al. 2024 ). In recent years, it has been demonstrated that the bioconversion of D-galUA components into bio-active products, such as meso -galactaric acid and ethanol, is a viable route for pectin bioconversion, contributing to sustainable valorization and waste utilization, facilitating a circular economy (Protzko et al. 2018 ; Sharma et al. 2022 ). However, pectin bioconversion and utilization has been hindered by a lack of complete bioconversion routes and an efficient expression of key enzymes. There has been an increased focus on acquiring enzymes for pectin metabolization that exhibit high stability and efficiency, notably the polygalacturonase (PG), pectin methylesterase (PME), and pectin lyase (PL) produced by fungi (Bonnin et al. 2014 ; Liang et al. 2015 ; Ma et al. 2018 ; Li et al. 2024 ). Besides pectin degradation, the critical enzymes associated with native D-galUA metabolism has been reported in Filamentous fungi, involving D-galacturonate reductase (GAR1) and L-galactonate dehydratase (LGD1) from Trichoderma reesei , and 2-keto-3-deoxy-L-galactonate aldolase (GAAC) from Aspergillus niger (Motter et al. 2014 ; Protzko et al. 2018 ). Nevertheless, in most yeasts, the D-galUA components are difficult to utilize due to the lack of essential enzymes that catalyze D-galUA (Grohmann et al. 1994 ; Huisjes et al. 2012 ; Biz et al. 2016 ). To tackle this problem, several attempts have been made by employing Saccharomyces cerevisiae as a production host to utilize D-galUA as a carbon source via the heterologous incorporation of D-galUA metabolic pathway, aiming to relieve pressure of pectin-rich waste on environment (Huisjes et al. 2012 ; Biz et al. 2016 ; Protzko et al. 2018 ; Sharma et al. 2022 ). Furthermore, identification of GatA as a powerful D-galUA transporter derived from A. niger has significantly enhanced the production of meso -galactaric acid via a co-fermentation of D-galUA and D-glucose (Protzko et al. 2018 ). Also, it stated an increase of ethanol production using an engineered S. cerevisiae strain in the co-utilization of D-galUA and glycerol (Perpelea et al. 2022 ). Despite the numerous studies of D-galUA metabolism and utilization, a co-fermentation with other carbon sources was invariably required. Moreover, the lack of a comprehensive system for pectin metabolism, including pectin degradation and D-galUA metabolism, has hindered the direct utilization of pectin as a carbon source for an efficient bioconversion. Kluyveromyces marxianus , an unconventional yeast strain, exhibits thermotolerance and rapid growth, utilizing a wide range of carbon sources, including C5, C6, and C12 compounds (Löbs et al. 2016 ; Dekker et al. 2021 ; Li et al. 2021 ). In addition, K. marxianus is edible and is a “generally recognized as safe” (GRAS) strain, representing a viable and attractive host for commercialization and metabolic engineering applications. Unlike conventional S. cerevisiae , the majority of K. marxianus strains are aerobic and less tolerant to alcohol fermentation, which can be exploited in biosynthesis applications to avoid by-product generation (Wagner and Alper 2016 ; Bilal et al. 2022 ). More importantly, numerous reports have demonstrated that K. marxianus is highly effective in secreting endo-polygalacturonase (endoPG) (Siekstele et al. 1999 ; Sieiro et al. 2009 ), which can be employed in pectin catalysis, bridging the current gap in the complete pectin metabolic process. Previous efforts to evaluate the endoPG capacity have established the critical role of K. marxianus in pectin catabolism (Wu et al. 2000 ; Masoud and Jespersen 2006 ). The production of endoPG in K. marxianus Y885 has elevated the hydrolysis capacity in cell wall degradation and grape pomace fermentation by 14%, producing ethanol and glycerol (Williams et al. 2019 ). Therefore, the reported works have established K. marxianus as an essential component in an optimized strategy to combine pectin degradation and D-galUA metabolism, and deliver a complete system for the bioconversion of pectin-rich biomass. In this study, we developed a complete bioconversion route involving two stages: pectin degradation (step 1) and D-galUA metabolism (step 2) (Fig. 1 A). In step 1, engineering endoPG over-production strain (YKM1013) was demonstrated to successfully promote pectin degradation. In step 2, the heterologous biosynthesis and the transporter associated with D-galUA metabolism were incorporated in K. marxianus (YKM1015) for the efficient consumption of D-galUA components. Moreover, a modular system comprising YKM1013 and YKM1015 strains, was synergistically developed to boost pectin utilization for cell growth, rewiring lipid metabolism and improving FAs production. Together, an effective bioconversion route of pectin has been established promoting FAs biosynthesis, which can contribute significantly to the sustainable valorization of pectin. Materials and methods Strains, culture media and condition All the strains used in this study were given in Table 1 . The yeast cells were cultivated in either synthetic defined (SD) or yeast-extract peptone dextrose (YPD) medium. The SD medium contained 1.7 g/L Yeast Nitrogen Base without Amino Acids (BD Difco™), 1.31 g/L Yeast Amino Acids Supplement (Coolaber), and 20 g/L glucose (Coolaber). The SD-H, SD-U, and SD-H-U medium were also used, where 1.29 g/L Dropout (DO) Supplement-His (Coolaber), 1.29 g/L DO Supplement-Ura (Coolaber) and 1.27 g/L DO Supplement-His/-Ura (Coolaber) respectively replaced the Yeast Amino Acids Supplement component in the SD medium. The YPD medium was composed of 20 g/L peptone (Oxoid), 10 g/L yeast extract (Oxoid), and 20 g/L glucose (Coolaber). The yeast cells were cultivated in 250 mL flasks containing 25 mL medium with an initial optical density (OD) of 0.05. The temperature and shaker speed for yeast cell culture was 30 ℃ and 200 rpm, respectively. Table 1 The summary of strains used in this study Strains Strains description Source E. Coli DH5α F − φ80( lacZ )ΔM15Δ( lacZYA-argF )U169 end A1 rec A1 hsd R17( m k − , m k + )supE44λ -thi -1 gyr A96 rel A1 pho A TSINGKE E. Coli Stbl3 F − mc rB mrr hsd S20(r B − , m B − ) rec A13 sup E44 ara -14 gal K2 lac Y1 pro A2 rps L20(Str R ) xyl -5λ − leu mtl -1 endA1 + Thermo Fisher YKM1012 Kluyveromyces marxianus CBS6556 ∆his3 ∆ura3 Obtained from Dr. Ian Wheeldon laboratory (UC Riverside) YKM1013 YKM1012 abz1 :: (P KmTEF3 )PGU1 This study YKM1014 YKM1012 ∆ PGU1 This study YKM1015 YKM1012 xyl2 :: (P KmGPD )TrGAR1 abz1 :: (P KmGPD )TrLGD1 lys1 :: (P KmGPD )AnGAAC sdl1 :: (P KmGPD )AnGatA This study YKM1016 YKM1012 sdl1 :: (P KmGPD )TrGAR1 abz1 :: (P KmGPD )V-TrLGD1 lys1 :: (P KmGPD )AnGAAC xyl2 :: (P KmGPD )AnGatA This study Plasmids construction All the plasmids used in this study were constructed by homologous recombination and were given in Table S1 . And all the gene sequences and used primers were listed in Table S2 and Table S3, respectively. The DNA gene fragments were synthesized or amplified by polymerase chain reaction (PCR) using KOD One TM PCR Master Mix (TOYOBO). The vectors were universally linearized by restriction enzymes (XmaI and XhoI) (NEB) at 37 ℃ for 1 h. In addition, the vector for PGko-HD construction was linearized by SacII and EagI (NEB), and the vector for PGko-sg construction was linearized by PspXI (NEB). After connecting the linearized vector and targeted gene fragment, the resulting plasmids containing the homologous fragment and CRISPR/Cas9 fragment were individually transformed into Escherichia coli ( E. coli ) DH5α or Stbl3 competent cells. The transformed cells were then cultivated on Luria–Bertani (LB) agar supplemented with 100 µg/mL ampicillin at 37 ℃ for 16 h. A randomly chosen single colony was genotyped using a 2×Taq Enzyme Mix (Vazyme), and the colonies with the correct insertion size were then confirmed by Sanger sequencing for plasmids generation. Yeast transformation and strain construction The transformation method has been described in a previous report (Yuan et al. 2024 ). The YKM1012 strain was used for transformation and strain construction. After transformation, the cell mixture was reinoculated in fresh selective media for overnight culture at 30 ℃, and then plated on the YPD agar plate for single colony confirmation by PCR amplification and sequencing. The plasmids containing the URA3 selection marker were eliminated by treating them with 1 g/L 5-fluoroorotic acid (5’-FOA) in liquid YPD medium at 30 ℃ overnight. The colonies were reinoculated on the YPD, SD-H, and SD-U agar plates to ensure the successful removal of plasmids, and stored at -80 ℃. Quantification of endoPG enzymatic activity The assay for endoPG enzymatic activity was conducted by applying the 3,5-dinitrosalicylic acid (DNS) method with some minor adjustments (Miller 1959 ). For each sample, the endoPG secreted from the strains was used to degrade pectin in an enzymatic reaction and the mixture was incubated at 45 ℃. The thermal stability of endoPG was assessed in the 30–90 ℃ temperature range. The same volume of pectin hydrolysate and DNS were combined for the measurement of D-galUA release by treatment in a boiling water bath for 10 min, with data collection on a microplate reader at 540 nm. Different concentrations of D-galUAs were processed to generate the calibration curve, with an associated R 2 > 0.999. The enzymatic activity of endoPG is represented by the D-galUA concentration (µmol) per milliliter and also evaluated using the ratio of pectin to D-galUA. Metabolite preparation and pre-processing After 72 h of cultivation, different groups with equal number of cells were obtained by centrifugation at low temperature and stored at -80°C. All the extracts were pre-cooled at -20 ℃ before use. The samples were allowed to thaw at room temperature, and then transferred to a 1.5 mL tube, with the addition of 1 mL pre-cooled methanol-water (V:V = 4:1) and 4 µg/mL L-2-chlorophenylalanine as internal standard, mixing for 10 s. Subsequently, 200 µL chloroform was added and the sample mixture was allowed to evaporate, followed by ultrasonic treatment (500 W) for 3 min and ultrasonic extraction for 20 min in an ice water bath, and left overnight at -40 ℃. The resultant solution was centrifuged at 12000 rpm at 4 ℃ for 10 min, and 800 µL supernatant was placed in a glass vial and dried in a refrigerated concentration centrifugal dryer. A 300 µL methanol-water (V:V = 1:4) mixture was added to each sample and mixed for 30 s, before leaving at -40 ℃ for 2 h. A subsequent ultrasonic extraction was performed under ice condition for 3 min, with centrifugation at 12000 rpm for 10 min at a low temperature. Each tube containing 150 µL supernatant was collected using syringes, filtered through 0.22 µm microfilters, transferred to a LC sample vial, and stored at -80 ℃ before analysis by liquid chromatography-mass spectrometry (LC-MS). LC-MS/MS detection The metabolomic analysis was performed by Shanghai Luming Biological Technology Co., Ltd (Shanghai, China). An ACQUITY UPLC I-Class plus (Waters Corp., Milford, USA) with a Q-Exactive mass spectrometer, equipped with an ACQUITY UPLC HSS T3 column (100 × 2.1 mm, 1.8 µm) and heated using an electrospray ionization (ESI) source (Thermo Fisher Scientific, Waltham, MA, USA) was employed for metabolic profiling in both ESI positive and negative ion modes. The binary gradient elution system consisted of (A) water (containing 0.1% v/v formic acid) and (B) acetonitrile (containing 0.1% v/v formic acid). Separation of metabolites was achieved using the following gradient: 0.01 min, 95% A and 5% B; 2 min, 95% A and 5% B; 4 min, 70% A and 30% B; 8 min, 50% A and 50% B; 10 min, 20% A and 80% B; 14 min, 100% B; 15 min, 100% B; 15.1 min, 95% A and 5% B; 16 min, 95% A and 5% B. The flow rate was 0.35 mL/min and the column temperature was 45 ℃. All the samples were kept at 10 ℃ and the injection volume was 5 µL. The mass analysis ranged from m/z 70 to 1050. In the full MS and HCD MS/MS scans, the resolution was set at 70000 and 17500, respectively, and the collision energy was set at 10, 20, and 40 eV. The following parameters were employed for mass spectrometry analysis: spray voltage = 3800 V (+) and 3200 V (−); capillary temperature = 320°C; aux gas heater temperature = 350°C; sheath gas flow rate = 35 arb; auxiliary gas flow rate = 8 arb; S-lens RF level = 50. Data pre-processing and statistical analysis The raw LC-MS data were processed using Progenesis QI V2.3 software (Nonlinear, Dynamics, Newcastle, UK) for baseline filtration, peak identification, integration, retention time correction, peak alignment, and normalization. The compounds were identified based on the precise mass-charge ratio (m/z), secondary fragments and isotope distribution employing several databases, including The Human Metabolome Database (HMDB), Lipidmaps (V2.3), Metlin and in-house databases. The extracted data were processed to remove more than 50% of the peaks with a missing value (ion intensity = 0), and replace the zero value with half the minimum value, screening according to qualitative results for the compound. The compounds that scored less than 36 out of 60 were considered inaccurate and removed. The positive and negative ion data were combined in a data matrix, which was imported into R software for analysis. A principle component analysis (PCA) was performed to determine the overall sample distribution and analysis stability. Furthermore, the orthogonal partial least-squares-discriminant analysis (OPLS-DA) and partial least-squares-discriminant analysis (PLS-DA) were used to distinguish the metabolites in each group. In order to avoid over-fitting, a 7-fold cross-validation and 200 response permutation testing (RPT) to evaluate the quality of the model was adopted. Based on the OPLS-DA model, the applied variable importance of projection (VIP) values were used to rank the overall contribution of each variable to the group discrimination. A two-tailed Student’s T-test was also applied to determine if any differences were statistically significant. Differential metabolites with VIP > 1.0 and p < 0.05 were selected. Lipid extraction and FAs quantification The method used in lipid extraction and determination has been described previously, and this study involved some modifications of the earlier procedure (Gao et al. 2018 ; Wang et al. 2022 ). The cell shake-flask culture (20 mL) incubated for 72 h was centrifuged at 5000 g and suspended in 2 mL 4 M hydrochloric acid with incubation at 80°C for 2 h. Following cooling, a mixture of chloroform and methanol (V/V = 2:1) was added and vortexed for 5 min to facilitate lipid extraction. After centrifugation, the organic sub-layer was collected, and dried by a rotary evaporator (Lichen, China). The extracted compounds were transformed to fatty acid methyl esters by replenishing 2 mL hydrochloric acid-methanol solution (V/V = 1:9) at 60°C for 1 h. The methylated compounds were obtained by adding 2 mL saturated sodium chloride and 1 mL hexane, followed by vortexing for 5 min. The resulting hexane layer was transferred to a glass vial via a 0.22 µm filter for subsequent gas chromatography (GC) analysis, using Agilent 7890 B unit equipped with a fame ionization detector (FID). An Agilent J&W DBWAXetr column (30 m × 0.25 mm × 0.25 µm) was employed with nitrogen as the carrier gas. The GC analysis parameters were as follows: initial temperature = 100°C, maintained for 0.5 min, increased to 190°C at 5°C/min, followed by a ramp to 220°C at 2°C/min and held for 10 min. The injector and detector temperatures were set at 250 and 260°C, respectively. A 1 µL sample volume was injected in split mode at a ratio of 15:1. The FAs were identified and quantified using the commercial standards and normalized with respect to methyl nonadecanate. Results and discussion Improving pectin degradation by overexpression of PGU1 in K. marxianus Previously, K. marxianus has been reported as a favorable strain to secrete endoPG for pectin degradation due to the considerable economic advantages with higher efficiency and stability by the cultivation in a simple medium (Siekstele et al. 1999 ; Jia and Wheals 2000 ; da Silva et al. 2005 ; Sieiro et al. 2009 ). Given the endoPG secretion capacity of strains, an increased amount of endoPG was observed in the stationary phase after YKM1012 cultivation for 14 h (Fig. 1 B). Additionally, a high-temperature (37 ℃) can effectively induce endoPG secretion, probably relating to the thermotolerant character of K. marxianus (Fig. S1 ). However, cultivation at extreme high-temperature (45 ℃) did not generate significant endoPG secretion (Fig. S1 ), which could be interpreted as saving energy by reducing the cost of growth (Thorwall et al. 2020 ). To clarify the pectin degradation and utilization in K. marxianus , the growth curve of YKM1012 with different medium compositions was collected. A lower cell biomass accumulation was observed in lower glucose content cultivation (Fig. 1 C). Although endoPG secretion was detected in SD medium (Fig. 1 B), there was essentially no change after adding exogenous pectin, even in the presence of endoPG accumulation (Fig. 1 C). And it also indicated that YKM1012 cannot hydrolyze pectin or grow on pectin (Fig. 1 C). To enhance endoPG secretion for pectin utilization, an engineering strategy was considered, directed at overexpressing the PGU1 gene facilitated by the strong TEF promoter at the ABZ1 locus of the YKM1012 genome (Fig. 1 D left). And a deletion of the PGU1 gene in engineering K. marxianus was also conducted to explore the endoPG function in pectin degradation (Fig. 1 D right). As expected, an overexpression of the PGU1 strain (YKM1013) resulted in a significant improvement in endoPG secretion, and there was no detectable secretion in the case of the PGU1 knock-out engineered strain (YKM1014) (Fig. 1 E). The results indicated that the engineered YKM1013 strain can serve as a platform host to enhance the level of endoPG, which is an important consideration in pectin utilization. Furthermore, an increasing of glucose concentration (from 0.3–2%) resulted in a 50% increase of endoPG level (Fig. 2 A) (Fig. 2 B, left Y-axis), with an associated twofold increase in hydrolysis capacity for the YKM1013 strain (Fig. 2 B, right Y-axis). In addition, YKM1013 strain showed a secondary growth in the stationary phase by supplementing pectin (Fig. 2 C), suggesting an effective utilization of pectin hydrolysate for cell growth. Instead, taking the D-galUA components at an equal concentration, the YKM1013 did not exhibit the secondary growth in the stationary phase (Fig. 2 C). To sum up, the increased endoPG level of the engineered strain (YKM1013) served to promote the pectin hydrolysis, and then the hydrolysate components except D-galUA promoted the secondary growth in yeast. To fully assess pectin hydrolysis efficiency, the influence of varying pectin and endoPG concentrations were considered. As there was no observed effect for a range of endoPG concentration, the D-galUA concentrations were maintained at 10 µmol/mL and 20 µmol/mL in 1% and 2% pectin degradation, respectively (Fig. 2 D, left Y-axis), which was accompanied by a 20% of conversion rate (Fig. 2 D, right Y-axis). On the contrary, an appreciable improvement was found for 4% pectin hydrolysis when the endoPG component was increased (Fig. 2 D, left Y-axis), likely due to the higher concentration of catalytic substrates. Nevertheless, a 12% conversion rate was recorded when increasing the endoPG concentration (Fig. 2 D, right Y-axis), where a contribution due to the high viscosity associated with 4% pectin should be taken into account (Xu et al. 2015 ). Given the influence of multiple factors in the enzyme catalytic system, the optimal reaction condition for pectin degradation must be carefully established. Constructing the D-galUA metabolic pathway by assembling targeted heterologous enzymes in K. marxianus Although the engineered YKM1013 strain can efficiently degrade pectin for the utilization of a small amount of available sugars, most of D-galUA components fail to be employed (Fig. 2 C). In previous reports, the heterologous genes involved in the D-galUA metabolic pathway were shown to be active in S. cerevisiae , including GAR1 , LGD1 , GAAC , and GatA (Fig. 3 A) (Biz et al. 2016 ; Protzko et al. 2018 ; Perpelea et al. 2022 ). We then attempted to drive the D-galUA metabolic flux in K. marxianus by heterologously expressing TrGAR1 , TrLGD1 , AnGAAC , and AnGatA at the high-efficiency integration sites SDL1 , ABZ1 , LYS1 , and XYL2 , respectively (Fig. 3 B) (Li et al. 2021 ), in constructing the engineered YKM1015 strain (Fig. 3 B). Then, the contribution of D-galUA was identified in the engineered YKM1015 strain by separately feeding 0.5% glucose and D-galUA. Notably, an excellent performance of cell growth for YKM1015 strain in D-galUA cultivation demonstrated a 3.6-fold difference by comparison with YKM1012, which preferred to grow in glucose cultivation (Fig. 3 C). Hence, the resulting strain exhibited a significant potential to successfully drive the metabolic direction of D-galUA pathway, ensuring an effective utilization of D-galUA components. Optimizing pectin degradation efficiency by application of engineered K. marxianus Given the low efficiency of pectin hydrolysis, a rational strategy is required to promote pectin hydrolysis capacity. Firstly, the confirmation of cultivation conditions was essential to to produce endoPG with higher activity. Concerning the increased endoPG secretion at 37 ℃ (Fig. S1 ), we compared the PG hydrolysis capacity of YKM1013 at 30 ℃ and 37 ℃. Surprisingly, the endoPG activity was initially repressed at 37 ℃ except 10 h (Fig. S2 ). Further discovery exhibited that the endoPG capacity was promoted over 24 h and subsequently declined (Fig. S2 ). Together, cultivation at 30 ℃ for 24 h ensured sufficient endoPG with the requisite efficiency. Subsequently, to identify the appropriate temperature of reaction, an assessment of endoPG thermal stability was conducted. A marked response was observed in the 45–50 ℃ temperature range over a 30 min period (Fig. 4 A). The system was thermally stable up to 12 h at 45 ℃, with evidence of a loss of activity at 48 ℃ within 30 min (Fig. 4 B). Published studies have probed into optimal K. marxianus incubation between 35 ℃ and 40 ℃ (da Silva et al. 2005 ; Masoud and Jespersen 2006 ). Here, there was no significant change in D-galUA content from 30 ℃ to 45 ℃ (Fig. S3). More surprisingly, 70% of the endoPG activity was maintained after pre-processing at 45 ℃ for 48 h, even half the activity retained after 120 h (Fig. S4). Consequently, 45 ℃ was chosen as the reaction temperature in developing the pectin hydrolysis system construction. With the objective of increasing pectin conversion rate, different endoPG and pectin volume ratios were examined. Reduction of endoPG resulted in a promotion of the hydrolysis efficiency, reaching an optimum at 1:9, whereas excessive pectin inhibited the activity (Fig. 4 C). Compared comprehensively, the highest conversion rate (28%) was achieved in degrading 2% pectin (Fig. 4 C, right Y-axis). Previous works have pointed out an optimum medium pH between 4 and 6 for endoPG secretion in yeasts (Moyo et al. 2003 ; da Silva et al. 2005 ). In the case of K. marxianus strain 166 and CCT 3172, the highest activity of endoPG secretion was recorded at pH 5.5 (da Silva et al. 2005 ; Masoud and Jespersen 2006 ). Here, after adjusting the pH to 5.5, an enhanced activity (33% conversion rate) was achieved for an in vitro 1% pectin catalytic assay (Fig. 4 D, right Y-axis). In contrast, a repression of endoPG activity was discovered when using a sodium acetate buffer (pH 5.5) (Fig. S5). Inspired by the optimized condition as a common strategy, the combined effect of varying temperature, volume ratio and pH on the hydrolysis efficiency of endoPG derived from YKM1013 was compared with the commercial PG. Remarkably, the endoPG exhibited less variation with respect to reaction time and superior activity relative to the commercial PG for shorter times (Fig. 4 E). At 45°C for 2 h, the D-galUA concentration and conversion rate were improved by 75% and 65%, respectively (Fig. 2 D) (Fig. 4 E). Therefore, a optimized strategy enabled a gratifying improvement in pectin hydrolysis. Developing a bioconversion system of pectin by modular co-utilization of two engineered strains Having optimized the pectin hydrolysis system, we next set out to develop a pectin metabolism strategy by modular co-utilization of two engineered strains. To this end, firstly, the engineered strain 1 (YKM1013) incubated in SD medium at 30°C for 24 h was utilized to acquire the endoPG with suitable activity, replacing the medium component with a phosphate buffer solution (PBS) at a final concentration of 0.6 mg/mL (Fig. 5 A). Then, pectin hydrolysis was conducted by combining endoPG and 2% pectin at 45°C for 2 h, to obtain the carbon source by high-speed centrifugation (Fig. 5 A). Lastly, after providing nitrogen base and amino acids, the engineered strain 2 (YKM1015) was cultured at 30°C for 72 h by initial OD 0.05 for the determination of metabolite profiles (Fig. 5 A). As a result, the harboring YKM1015 exhibited a twofold increase biomass relative to YKM1012 (Fig. 5 B), confirming the contribution from pectin hydrolysate due to the D-galUA components. Thus, the results indicated that this developed strategy can facilitate pectin bioconversion through modular co-utilization of two engineered strains. Establishing strategy enhanced pectin-based lipid metabolism and FAs production To evaluate the production performance of metabolic flux transformation in pectin bioconversion, the metabolic profiles resulting from incubating different strains were then assessed. Taking account of the influence of cell growth and metabolism, a pronounced gap was apparent in the loading plot (Fig. 6 A), indicating a significant difference in the metabolic profiles. In detail, the YKM1015 strain presented an improvement of 217 metabolites and a repression of 122 metabolites when compared with YKM1012 (Fig. 6 B). Focused on the pathway enrichment, a greater accumulation enriched in lipid metabolism was detected in YKM1015 (Fig. 6 C), suggesting that this engineered strain directed the metabolic flux in favor of lipid biosynthesis. In contrast to Yarrowia lipolytica , a well-studied cell factory for producing lipids via metabolic engineering strategy (Lazar et al. 2018 ; Wang et al. 2020 ; Chen et al. 2021 ), little research referring to lipids has been carried out in K. marxianus so far. However, the down-regulation of amino acids metabolism was performed in engineered strain fermentation (Fig. 6 C). In the pathway associated with the observed enrichment, the elevated level of metabolites was mainly distributed in the metabolisms relating to glycerophospholipid, arachidonic acid, α-linolenic acid, and sphingolipid (Fig. 6 D left). The phosphatidylcholines (PCs), common in the membranes of most eukaryotes, are involved in the sphingolipid and glycerophospholipid metabolisms to determine the physical properties and strictly regulated (de Kroon 2007 ). Thereby, alteration of PCs implied the difference of membrane profile when utilizing pectin hydrolysate. Although most of the information referring to unsaturated fatty acid (UFA) metabolism has been obtained from mammalian biology, the relevant compounds can also be produced by yeasts, such as Candida albicans , Cryptococcus neoformans (Deva et al. 2000 ; Erb-Downward and Huffnagle 2007 ), and Y. lipolytica (Liu et al. 2019 ). Little information is, however, available regarding the metabolites involved in α-linolenic acid and arachidonic acid production in K. marxianus . Conversely, the metabolites with decreased levels of enrichment accounted for the majority of amino acids associated with biosynthesis pathways (Fig. 6 D right). In yeast, the availability of amino acids is important in cell growth and regulatory processes in a complicated network (Broach 2012 ; Takagi 2019 ). Thus, the engineered YKM1015 strain was possibly influenced by an inhibited cell growth and regulatory processes. Referring to the previous studies, T. reesei LGD1 fused with yellow fluorescent protein (Venus-LGD1, V-TrLGD1) was shown to improve a 60-fold enzyme activity in vitro (Biz et al. 2016 ; Protzko et al. 2018 ). In the case of engineered K. marxianus strains, interestingly, the engineered YKM1016 strain equipped with V-TrLGD1 performed in an analogous manner to YKM1015 with respect to cell growth (Fig. 5 B, S6A). Correspondingly, the YKM1012 and YKM1016 strains demonstrated markedly different responses (Fig. S6B), and the two engineered YKM1016 and YKM1015 strains showed greater commonality with increased lipid metabolism and an inhibited amino acid metabolism (Fig. 6 C, S6C, and S6D). Hence, this result suggested a similar metabolic flux without taking the LGD level into account when applying K. marxianus as a host platform. Ultimately, the heterologous metabolic pathway for pectin bioconversion was successfully designed in K. marxianus , yielding 22 mg/L mid-chain fatty acids (MCFA) and 14 mg/L long-chain fatty acids (LCFA), pointing at an increasing of 19-fold and 6-fold, respectively (Fig. 6 E). Consequently, the pectin utilization route can be tailored to generate a range of products and serve as an effective platform for pectin-rich biomass bioconversion. When compared with pectin hydrolysate cultivation, the metabolic profile associated with incubation in glucose displayed a completely different flux transformation that was principally related to specific amino acid metabolism (Fig. S7). The different strains generate a distinct metabolic flux centering on the metabolism of most amino acids in the presence of glucose condition, resulting in specific growth patterns (Broach 2012 ). As shown here, detail of enhanced histidine metabolism and lysine degradation, and repressed valine, leucine and isoleucine biosynthesis were summarized (Fig. S7). Taken together, the results illustrated that the metabolic transformation can be inclined to facilitate high value-added products accumulation from pectin-rich biomass employing a multifunctional K. marxianus host platform. Conclusions In summary, a rational strategy is proposed involving a synergistic application of two engineered K. marxianus strains to construct a pectin bioconversion system, resulting in a 65% improvement of conversion rate and enhancing lipid metabolism flux with an increasing of 19-fold in MCFA and 6-fold in LCFA production. This strategy facilitates a feasible and sustainable bioconversion route of pectin-rich materials, which can form the basis for diverse microbial cell factories in biomass resources utilization. Abbreviations 5’-FOA, 5-fluoroorotic acid; A . niger , Aspergillus niger ; D-galUA, D-galacturonic acid; DNS, 3,5-dinitrosalicylic acid; DO, Dropout; E. coli, Escherichia coli ; endoPG, endo-polygalacturonase; ESI, electrospray ionization; FID, fame ionization detector; GAAC, 2-keto-3-deoxy-L-galactonate aldolase; GAR1, D-galacturonate reductase; GatA, D-GalUA transporter; GC, gas chromatography; GRAS, generally recognized as safe; HMDB, The Human Metabolome Database; K. marxianus , Kluyveromyces marxianus ; LB, Luria–Bertani; LCFA, long-chain fatty acids; LC-MS, liquid chromatography-mass spectrometry; LGD1, L-galactonate dehydratase; MCFA, mid-chain fatty acids; OD, optical density; OPLS-DA, orthogonal partial least-squares-discriminant analysis; PBS, phosphate buffer solution; PCA, principle component analysis; PCR, polymerase chain reaction; PCs, phosphatidylcholines; PL, pectin lyase; PLS-DA, partial least-squares-discriminant analysis; PME, pectin methylesterase; RPT; response permutation testing; S. cerevisiae, Saccharomyces cerevisiae ; SD, synthetic defined; UFA, unsaturated fatty acids; VIP, variable importance of projection; V-TrLGD1, LGD1 fused with yellow fluorescent protein; YPD, yeast-extract peptone dextrose. Declarations Associated content Supporting information endoPG secretion in YKM1012 (Fig. S1); endoPG activity in YKM1013 (Fig. S2,S3); thermal stability of endoPG in YKM1013 (Fig. S4); endoPG capacity in engineered strain (YKM1013) in sodium acetate buffer (pH 5.5) (Fig. S5); metabolic profile between YKM1012 and YKM1016 strains (Fig. S6); differential metabolites between YKM1012 and YKM1015 strains by the cultivation in 2% glucose (Fig. S7) Availability of data and materials The data and materials that support the findings of this study are available from the corresponding author upon reasonable request. Fundings This research was supported by Zhejiang Provincial Natural Science Foundation of China under Grant No. ZCLQ24C0101, ZJU-Hangzhou Global Scientific and Technological Innovation Center, Zhejiang University (Grant No. 02020200-K02013027 to Xuye Lang, 02020100-K3F113074 to Bofan Yu). Author’s contributions X.L. designed the study. X.L. and B.Y. analyzed the data and wrote the manuscript. B.Y. and H.Q. conducted the experiments. All authors revised and approved the manuscript. Ethics approval and consent to participate Not applicable. Consent for publication All authors have consented for publication. Conflicts of interest The authors declare that they have no known competing financial interests. Acknowledgements Acknowledge the support of the ZJU-Hangzhou Global Scientific and Technological Innovation Center, Zhejiang University (HIC-ZJU), the iBiofoundary, the Core Facility of the Institute for Intelligent Bio/Chem Manufacturing at HIC-ZJU, Acknowledge Professor Ian Wheeldon at UC Riverside for providing the strains and previous knowledge. References Bilal M, Ji L, Xu Y, Xu S, Lin Y, Iqbal HMN, Cheng H (2022) Bioprospecting Kluyveromyces marxianus as a Robust Host for Industrial Biotechnology. Front Bioeng Biotechnol 10:851768 Biz A, Sugai-Guérios MH, Kuivanen J, Maaheimo H, Krieger N, Mitchell DA, Richard P (2016) The introduction of the fungal D-galacturonate pathway enables the consumption of D-galacturonic acid by Saccharomyces cerevisiae . Microb Cell Fact 15(1):144 Bonnin E, Garnier C, Ralet MC (2014) Pectin-modifying enzymes and pectin-derived materials: applications and impacts. Appl Microbiol Biotechnol 98(2):519–532 Broach JR (2012) Nutritional control of growth and development in yeast. Genetics 192(1):73–105 Chavan P, Singh AK, Kaur G (2019) Recent progress in the utilization of industrial waste and by-products of citrus fruits: A review. J Food Process Eng Chen L, Yan W, Qian X, Chen M, Zhang X, Xin F, Zhang W, Jiang M, Ochsenreither K (2021) Increased Lipid Production in Yarrowia lipolytica from Acetate through Metabolic Engineering and Cosubstrate Fermentation. ACS Synth Biol 10(11):3129–3138 da Silva EG, de Fátima Borges M, Medina C, Piccoli RH, Schwan RF (2005) Pectinolytic enzymes secreted by yeasts from tropical fruits. FEMS Yeast Res 5(9):859–865 Dekker WJC, Ortiz-Merino RA, Kaljouw A, Battjes J, Wiering FW, Mooiman C, Torre P, Pronk JT (2021) Engineering the thermotolerant industrial yeast Kluyveromyces marxianus for anaerobic growth. Metab Eng 67:347–364 de Kroon AI (2007) Metabolism of phosphatidylcholine and its implications for lipid acyl chain composition in Saccharomyces cerevisiae . Biochim Biophys Acta 1771(3):343–352 Deva R, Ciccoli R, Schewe T, Kock JL, Nigam S (2000) Arachidonic acid stimulates cell growth and forms a novel oxygenated metabolite in Candida albicans . Biochim Biophys Acta 1486(2–3):299–311 Erb-Downward JR, Huffnagle GB (2007) Cryptococcus neoformans produces authentic prostaglandin E 2 without a cyclooxygenase. Eukaryot Cell 6(2):346–350 Erian AM, Sauer M (2022) Utilizing yeasts for the conversion of renewable feedstocks to sugar alcohols - a review. Bioresour Technol 346:126296 Gao J, Yu W, Li Y, Jin M, Yao L, Zhou YJ (2023) Engineering co-utilization of glucose and xylose for chemical overproduction from lignocellulose. Nat Chem Biol 19(12):1524–1531 Gao Q, Cao X, Huang YY, Yang JL, Chen J, Wei LJ, Hua Q (2018) Overproduction of Fatty Acid Ethyl Esters by the Oleaginous Yeast Yarrowia lipolytica through Metabolic Engineering and Process Optimization. ACS Synth Biol 7(5):1371–1380 Grohmann K, Baldwin EA, Buslig BS (1994) Production of ethanol from enzymatically hydrolyzed orange peel by the yeast Saccharomyces cerevisiae . Appl Biochem Biotechnol 45–46:315–327 Huisjes EH, Luttik MA, Almering MJ, Bisschops MM, Dang DH, Kleerebezem M, Siezen R, van Maris AJ, Pronk JT (2012) Toward pectin fermentation by Saccharomyces cerevisiae : expression of the first two steps of a bacterial pathway for D-galacturonate metabolism. J Biotechnol 162(2–3):303–310 Jia J, Wheals A (2000) Endopolygalacturonase genes and enzymes from Saccharomyces cerevisiae and Kluyveromyces marxianus . Curr Genet 38(5):264–270 Lazar Z, Liu N, Stephanopoulos G (2018) Holistic Approaches in Lipid Production by Yarrowia lipolytica . Trends Biotechnol 36(11):1157–1170 Li J, Peng C, Mao A, Zhong M, Hu Z (2024) An overview of microbial enzymatic approaches for pectin degradation. Int J Biol Macromol 254:127804 Li M, Lang XY, Moran Cabrera M, De Keyser S, Sun X, Da Silva N, Wheeldon I (2021) CRISPR-mediated multigene integration enables Shikimate pathway refactoring for enhanced 2-phenylethanol biosynthesis in Kluyveromyces marxianus . Biotechnol Biofuels 14(1):3 Li S, Su C, Fang M, Cai D, Deng L, Wang F, Liu J (2023) Overproduction of palmitoleic acid from corn stover hydrolysate by engineered Saccharomyces cerevisiae . Bioresour Technol 382:129211 Liang C, Gui X, Zhou C, Xue Y, Ma Y, Tang SY (2015) Improving the thermoactivity and thermostability of pectatelyase from Bacillus pumilus for ramie degumming. Appl Microbiol Biotechnol 99(6):2673–2682 Liu HH, Wang C, Lu XY, Huang H, Tian Y, Ji XJ (2019) Improved Production of Arachidonic Acid by Combined Pathway Engineering and Synthetic Enzyme Fusion in Yarrowia lipolytica . J Agric Food Chem 67(35):9851–9857 Löbs AK, Lin JL, Cook M, Wheeldon I (2016) High throughput, colorimetric screening of microbial ester biosynthesis reveals high ethyl acetate production from Kluyveromyces marxianus on C5, C6, and C12 carbon sources. Biotechnol J 11(10):1274–1281 Ma X, Wang DL, Chen WJ, Ismail BB, Wang WJ, Lv RL, Ding T, Ye XQ, Liu DH (2018) Effects of ultrasound pretreatment on the enzymolysis of pectin: Kinetic study, structural characteristics and anti-cancer activity of the hydrolysates. Food Hydrocolloids 79:90–99 Masoud W, Jespersen L (2006) Pectin degrading enzymes in yeasts involved in fermentation of Coffea arabica in East Africa. Int J Food Microbiol 110(3):291–296 Miller G (1959) Use of dinitrosalicyclic acid reagent for determination of reducing sugars. Anal Chem 31:426–428 Mohnen D (2008) Pectin structure and biosynthesis. Curr Opin Plant Biol 11(3):266–277 Motter FA, Kuivanen J, Keränen H, Hilditch S, Penttilä M, Richard P (2014) Categorisation of sugar acid dehydratases in Aspergillus niger . Fungal Genet Biol 64:67–72 Moyo S, Gashe BA, Collison EK, Mpuchane S (2003) Optimising growth conditions for the pectinolytic activity of Kluyveromyces wickerhamii by using response surface methodology. Int J Food Microbiol 85(1–2):87–100 Perpelea A, Wijaya AW, Martins LC, Rippert D, Klein M, Angelov A, Peltonen K, Teleki A, Liebl W, Richard P, Thevelein JM, Takors R, Sá-Correia I, Nevoigt E (2022) Towards valorization of pectin-rich agro-industrial residues: Engineering of Saccharomyces cerevisiae for co-fermentation of d-galacturonic acid and glycerol. Metab Eng 69:1–14 Protzko RJ, Latimer LN, Martinho Z, de Reus E, Seibert T, Benz JP, Dueber JE (2018) Engineering Saccharomyces cerevisiae for co-utilization of D-galacturonic acid and D-glucose from citrus peel waste. Nat Commun 9(1): 5059 Sharma P, Vishvakarma R, Gautam K, Vimal A, Kumar Gaur V, Farooqui A, Varjani S, Younis K (2022) Valorization of citrus peel waste for the sustainable production of value-added products. Bioresour Technol 351:127064 Sieiro C, Sestelo AB, Villa TG (2009) Cloning, characterization, and functional analysis of the EPG1-2 gene: a new allele coding for an endopolygalacturonase in Kluyveromyces marxianus . J Agric Food Chem 57(19):8921–8926 Siekstele R, Bartkeviciute D, Sasnauskas K (1999) Cloning, targeted disruption and heterologous expression of the Kluyveromyces marxianus endopolygalacturonase gene (EPG1). Yeast 15(4):311–322 Takagi H (2019) Metabolic regulatory mechanisms and physiological roles of functional amino acids and their applications in yeast. Biosci Biotechnol Biochem 83(8):1449–1462 Thorwall S, Schwartz C, Chartron JW, Wheeldon I (2020) Stress-tolerant non-conventional microbes enable next-generation chemical biosynthesis. Nat Chem Biol 16(2):113–121 Wagner JM, Alper HS (2016) Synthetic biology and molecular genetics in non-conventional yeasts: Current tools and future advances. Fungal Genet Biol 89:126–136 Wang K, Shi TQ, Wang J, Wei P, Ledesma-Amaro R, Ji XJ (2022) Engineering the Lipid and Fatty Acid Metabolism in Yarrowia lipolytica for Sustainable Production of High Oleic Oils. ACS Synth Biol 11(4):1542–1554 Wang J, Ledesma-Amaro R, Wei Y, Ji B, Ji XJ (2020) Metabolic engineering for increased lipid accumulation in Yarrowia lipolytica - A Review. Bioresour Technol 313:123707 Wikandari R, Hasniah N, Taherzadeh MJ (2022) The role of filamentous fungi in advancing the development of a sustainable circular bioeconomy. Bioresour Technol 345:126531 Williams DL, Schückel J, Vivier MA (2019) Grape pomace fermentation and cell wall degradation by Kluyveromyces marxianus Y885. Bio Eng J 150:107282 Wu YT, Pereira M, Venâncio A, Teixeira J (2000) Recovery of endo-polygalacturonase using polyethylene glycol-salt aqueous two-phase extraction with polymer recycling. Bioseparation 9(4):247–254 Xiang T, Yang R, Li L, Lin H, Kai G (2024) Research progress and application of pectin: A review. J Food Sci 89(11):6985–7007 Xu S, Qin X, Liu B, Zhang DQ, Zhang YH (2015) An acidic pectin lyase from Aspergillus niger with favourable efficiency in fruit juice clarification. Lett Appl Microbiol 60(2):181–187 Yuan YC, Yu BF, Zhou XZ, Qiao H, Lian JZ, Lang XY, Yao Y (2024) Engineering Living Material for Controlled Fragrance Release Utilizing Kluyveromyces marxianus CBS6556 and Adaptive Hydrogel. ACS Synth Biol 13(10):3188–3196 Zhang K, Jiang Z, Li X, Wang D, Hong J (2024) Enhancing simultaneous saccharification and co-fermentation of corncob by Kluyveromyces marxianus through overexpression of putative transcription regulator. Bioresour Technol 399:130627 Supplementary Files Graphicalabstract.jpeg Supportinginformation20250308.docx Cite Share Download PDF Status: Published Journal Publication published 12 Aug, 2025 Read the published version in Bioresources and Bioprocessing → Version 1 posted Editorial decision: Major revision 20 Apr, 2025 Reviewers agreed at journal 27 Mar, 2025 Reviewers invited by journal 18 Mar, 2025 Editor assigned by journal 18 Mar, 2025 First submitted to journal 13 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6193521","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":430386492,"identity":"e3bb6fec-fc5e-4096-86f7-b5a7fd7d4250","order_by":0,"name":"Bofan Yu","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Bofan","middleName":"","lastName":"Yu","suffix":""},{"id":430386493,"identity":"141edc88-2291-4fb0-8778-c2c0f36cc5a2","order_by":1,"name":"He Qiao","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"He","middleName":"","lastName":"Qiao","suffix":""},{"id":430386494,"identity":"500cd18e-ff8e-4e55-ab0b-da54aba577e5","order_by":2,"name":"Xuye Lang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIiWNgGAWjYLCCB0DMz8CQAKSYidQCUivZQLIWgwNgJhFaDG7kGD5IqLljt/n8gWcSDBXWiQ3sZw/g1SI5I8fYIOHYs+RtBw6kSTCcSU9s4MlLwKuFXyLHTCKB7XCy2cGGNAnGtsOJDRI8Bni1sEnkmP9I+Hc42biZAajlHxFaQLYwJLYdtjNgA2lpIEKLZM+zYonEvsMJEmcYki0SjqUbt/Hk4NdicDx544cP3w7b8/efSbzxocZatp/9DH4tDAwcYAWJDQw8CeAIYiOgHgjYH4BIeyDjAGHFo2AUjIJRMCIBABZJReZF/GOtAAAAAElFTkSuQmCC","orcid":"","institution":"Zhejiang University","correspondingAuthor":true,"prefix":"","firstName":"Xuye","middleName":"","lastName":"Lang","suffix":""}],"badges":[],"createdAt":"2025-03-10 08:48:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6193521/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6193521/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40643-025-00927-z","type":"published","date":"2025-08-12T15:57:08+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79275749,"identity":"d1264a82-8171-4dc8-8044-b15e7b4c945c","added_by":"auto","created_at":"2025-03-26 12:13:55","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":395643,"visible":true,"origin":"","legend":"\u003cp\u003eEngineering \u003cem\u003eK. marxianus \u003c/em\u003efor the over-production of endoPG. (A) A simplified diagram of pectin metabolic pathway involves pectin degradation (step 1) and D-galUA metabolism (step 2). (B) The amount of endoPG secretion pointed by \u003cem\u003ered arrows\u003c/em\u003e during different growth period in YKM1012 strain. (C) The growth curve of YKM1012 in different medium compositions. (D) The schematic diagrams of construction of engineered strains YKM1013 (\u003cem\u003eleft\u003c/em\u003e) and YKM1014 (\u003cem\u003eright\u003c/em\u003e). (E) The amount of endoPG secretion in different\u003cem\u003e K. marxianus \u003c/em\u003estrains.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6193521/v1/eb8ba5979ab4f6eaf9d22d89.jpeg"},{"id":79275743,"identity":"26c1c14b-5173-4d7a-a110-e88b52394a41","added_by":"auto","created_at":"2025-03-26 12:13:55","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":432060,"visible":true,"origin":"","legend":"\u003cp\u003eEngineering \u003cem\u003eK. marxianus\u003c/em\u003efor efficient pectin hydrolysis and utilization. (A) The amount of endoPG secretion under different glucose concentrations. (B) Determination of endoPG secretion (column, left Y-axis) and PG activity (symbol, right Y-axis) under different glucose concentrations. (C) The growth curve of engineered strain (YKM1013) in different medium compositions. (D) The D-galUA concentration (column, left Y-axis) and conversion rate (symbol, right Y-axis) in pectin hydrolysis among various combinations of endoPG and pectin.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6193521/v1/61f84323e6628f07c045585a.jpeg"},{"id":79276780,"identity":"d62fa389-4ec1-43c9-a941-fbadf2bfee5c","added_by":"auto","created_at":"2025-03-26 12:29:55","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":511265,"visible":true,"origin":"","legend":"\u003cp\u003eEngineering \u003cem\u003eK. marxianus\u003c/em\u003e for effective utilization of D-galUA. (A) A simplified diagram of D-galUA metabolic pathway involving four heterologous genes derived from either \u003cem\u003eAspergillus niger\u003c/em\u003e (\u003cem\u003eAn\u003c/em\u003e) or \u003cem\u003eTrichoderma reesei\u003c/em\u003e (\u003cem\u003eTr\u003c/em\u003e), including \u003cem\u003eAnGatA\u003c/em\u003e: D-GalUA transporter; \u003cem\u003eTrGAR1\u003c/em\u003e: D-galacturonate reductase; \u003cem\u003eTrLGD1\u003c/em\u003e: L-galactonate dehydratase; \u003cem\u003eAnGAAC\u003c/em\u003e: 2-keto-3-deoxy-L-galactonate aldolase. (B) Construction process of engineered strain by successive integration of four heterologous genes based on the reference strain YKM1012. (C) The cell growth of YKM1012 and YKM1015 by the separate cultivation of glucose and D-galUA.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6193521/v1/4068a17020b5864c49d99a24.jpeg"},{"id":79275978,"identity":"32771eb5-4003-4ba4-ae14-3ebea66dad39","added_by":"auto","created_at":"2025-03-26 12:21:55","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":635939,"visible":true,"origin":"","legend":"\u003cp\u003eOptimization conditions of pectin hydrolysis efficiency. (A) Determination of thermal stability of endoPG in YKM1013 strain by pre-treatment for 30 min among diverse temperatures. The sample without pre-treatment was used as the blank control. (B) Comparison of thermal stability of endoPG in YKM1013 strain between 45 ℃ and 48 ℃ by time-course pre-treatment. (C) D-galUA concentration (column, left Y-axis) and pectin conversion rate (symbol, right Y-axis) among varying ratios of endoPG and pectin, and (D) in different pH environments. (E) Comparison of endoPG and commercial PG in varying conditions of pectin hydrolysis. Left Y-axis, D-galUA concentration; Right Y-axis, conversion rate.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6193521/v1/db753d739ed77c26013bc872.jpeg"},{"id":79275745,"identity":"f3530b54-6862-4e88-a6d3-3faeac03ae2c","added_by":"auto","created_at":"2025-03-26 12:13:55","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":362765,"visible":true,"origin":"","legend":"\u003cp\u003eModular co-utilization of two engineered \u003cem\u003eK. marxianus\u003c/em\u003e strains for pectin bioconversion. (A) The established process of pectin bioconversion through modular co-utilization of two engineered \u003cem\u003eK. marxianus\u003c/em\u003e strains. Engineered strain 1: YKM1013; Engineered strain 2: YKM1015. (B) The cell growth of YKM1012 and YKM1015 by the cultivation in pectin hydrolysate.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6193521/v1/57945551d023354a82308703.jpeg"},{"id":79275755,"identity":"b5eacf36-8284-4d0a-a612-6b2cc308ffc8","added_by":"auto","created_at":"2025-03-26 12:13:55","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":338224,"visible":true,"origin":"","legend":"\u003cp\u003eTransformation of lipid metabolism flux in pectin bioconversion for FAs production. (A) OPLS-DA load diagram, (B) Volcano plot, (C) Summary of classified metabolism pathway of significantly up- and down-regulated metabolites between YKM1012 and YKM1015 strains. (D) Detail of metabolism pathway enrichment of up-regulated (\u003cem\u003eleft\u003c/em\u003e) and down-regulated (\u003cem\u003eright\u003c/em\u003e) metabolites (VIP\u0026gt;1, p\u0026lt;0.05) between YKM1012 and YKM1015 strains. (E) The concentration of MCFA and LCFA between YKM1012 and YKM1015 strains.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6193521/v1/560dd6ffca1645a87fd55c4d.jpeg"},{"id":89310679,"identity":"068a53c1-2bf9-423f-9ead-8e287ffd2829","added_by":"auto","created_at":"2025-08-18 16:09:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3645375,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6193521/v1/0e3088b5-1be2-454b-a96b-7bd45ed6befb.pdf"},{"id":79275979,"identity":"a9caac98-dd52-48da-9fd5-f494ff2f9aae","added_by":"auto","created_at":"2025-03-26 12:21:55","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":78961,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6193521/v1/d2fc6f679cd9a08c243df2d0.jpeg"},{"id":79277164,"identity":"36e5ca74-829a-4f28-9935-c6380fe0fc88","added_by":"auto","created_at":"2025-03-26 12:37:55","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":614620,"visible":true,"origin":"","legend":"","description":"","filename":"Supportinginformation20250308.docx","url":"https://assets-eu.researchsquare.com/files/rs-6193521/v1/fe9e5c54be76511494d36e9b.docx"}],"financialInterests":"","formattedTitle":"Engineering a sustainable system for pectin-based lipid metabolism through modular co-utilization of two Kluyveromyces marxianus strains","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePectin is a ubiquitous polysaccharide found in the cell wall and inner layer of fruits, vegetables, and other higher plants (Xiang et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The majority of pectin is derived from raw materials, such as fruit peels and pomaces (Williams et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Erian and Sauer \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, nearly 50% of those is regarded as waste and discarded, resulting in significant environmental pollution and unsustainable development (Chavan et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sharma et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Following enzymatic depolymerization, pectin is converted to D-galUA (~\u0026thinsp;70%) and available neutral sugars (including xylose, galactose, mannose, fucose, or arabinose) (Mohnen \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). These components can be utilized as a bio-active feedstock to yield valuable products. Thus, the development of a green bioconversion route is critical for the sustainable utilization of pectin. Microorganisms engineered through metabolic engineering can serve as effective cell factories offering potential to advance agricultural by-product waste treatment and generate a variety of valuable products from biomass resources (Protzko et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wikandari et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Gao et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In recent years, it has been demonstrated that the bioconversion of D-galUA components into bio-active products, such as \u003cem\u003emeso\u003c/em\u003e-galactaric acid and ethanol, is a viable route for pectin bioconversion, contributing to sustainable valorization and waste utilization, facilitating a circular economy (Protzko et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Sharma et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, pectin bioconversion and utilization has been hindered by a lack of complete bioconversion routes and an efficient expression of key enzymes.\u003c/p\u003e \u003cp\u003eThere has been an increased focus on acquiring enzymes for pectin metabolization that exhibit high stability and efficiency, notably the polygalacturonase (PG), pectin methylesterase (PME), and pectin lyase (PL) produced by fungi (Bonnin et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Liang et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Ma et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Besides pectin degradation, the critical enzymes associated with native D-galUA metabolism has been reported in Filamentous fungi, involving D-galacturonate reductase (GAR1) and L-galactonate dehydratase (LGD1) from \u003cem\u003eTrichoderma reesei\u003c/em\u003e, and 2-keto-3-deoxy-L-galactonate aldolase (GAAC) from \u003cem\u003eAspergillus niger\u003c/em\u003e (Motter et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Protzko et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Nevertheless, in most yeasts, the D-galUA components are difficult to utilize due to the lack of essential enzymes that catalyze D-galUA (Grohmann et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Huisjes et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Biz et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). To tackle this problem, several attempts have been made by employing \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e as a production host to utilize D-galUA as a carbon source via the heterologous incorporation of D-galUA metabolic pathway, aiming to relieve pressure of pectin-rich waste on environment (Huisjes et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Biz et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Protzko et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Sharma et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Furthermore, identification of GatA as a powerful D-galUA transporter derived from \u003cem\u003eA. niger\u003c/em\u003e has significantly enhanced the production of \u003cem\u003emeso\u003c/em\u003e-galactaric acid via a co-fermentation of D-galUA and D-glucose (Protzko et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Also, it stated an increase of ethanol production using an engineered \u003cem\u003eS. cerevisiae\u003c/em\u003e strain in the co-utilization of D-galUA and glycerol (Perpelea et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Despite the numerous studies of D-galUA metabolism and utilization, a co-fermentation with other carbon sources was invariably required. Moreover, the lack of a comprehensive system for pectin metabolism, including pectin degradation and D-galUA metabolism, has hindered the direct utilization of pectin as a carbon source for an efficient bioconversion.\u003c/p\u003e \u003cp\u003e \u003cem\u003eKluyveromyces marxianus\u003c/em\u003e, an unconventional yeast strain, exhibits thermotolerance and rapid growth, utilizing a wide range of carbon sources, including C5, C6, and C12 compounds (L\u0026ouml;bs et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Dekker et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition, \u003cem\u003eK. marxianus\u003c/em\u003e is edible and is a \u0026ldquo;generally recognized as safe\u0026rdquo; (GRAS) strain, representing a viable and attractive host for commercialization and metabolic engineering applications. Unlike conventional \u003cem\u003eS. cerevisiae\u003c/em\u003e, the majority of \u003cem\u003eK. marxianus\u003c/em\u003e strains are aerobic and less tolerant to alcohol fermentation, which can be exploited in biosynthesis applications to avoid by-product generation (Wagner and Alper \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Bilal et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). More importantly, numerous reports have demonstrated that \u003cem\u003eK. marxianus\u003c/em\u003e is highly effective in secreting endo-polygalacturonase (endoPG) (Siekstele et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Sieiro et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), which can be employed in pectin catalysis, bridging the current gap in the complete pectin metabolic process. Previous efforts to evaluate the endoPG capacity have established the critical role of \u003cem\u003eK. marxianus\u003c/em\u003e in pectin catabolism (Wu et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Masoud and Jespersen \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The production of endoPG in \u003cem\u003eK. marxianus\u003c/em\u003e Y885 has elevated the hydrolysis capacity in cell wall degradation and grape pomace fermentation by 14%, producing ethanol and glycerol (Williams et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Therefore, the reported works have established \u003cem\u003eK. marxianus\u003c/em\u003e as an essential component in an optimized strategy to combine pectin degradation and D-galUA metabolism, and deliver a complete system for the bioconversion of pectin-rich biomass.\u003c/p\u003e \u003cp\u003eIn this study, we developed a complete bioconversion route involving two stages: pectin degradation (step 1) and D-galUA metabolism (step 2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In step 1, engineering endoPG over-production strain (YKM1013) was demonstrated to successfully promote pectin degradation. In step 2, the heterologous biosynthesis and the transporter associated with D-galUA metabolism were incorporated in \u003cem\u003eK. marxianus\u003c/em\u003e (YKM1015) for the efficient consumption of D-galUA components. Moreover, a modular system comprising YKM1013 and YKM1015 strains, was synergistically developed to boost pectin utilization for cell growth, rewiring lipid metabolism and improving FAs production. Together, an effective bioconversion route of pectin has been established promoting FAs biosynthesis, which can contribute significantly to the sustainable valorization of pectin.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStrains, culture media and condition\u003c/h2\u003e \u003cp\u003eAll the strains used in this study were given in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The yeast cells were cultivated in either synthetic defined (SD) or yeast-extract peptone dextrose (YPD) medium. The SD medium contained 1.7 g/L Yeast Nitrogen Base without Amino Acids (BD Difco\u0026trade;), 1.31 g/L Yeast Amino Acids Supplement (Coolaber), and 20 g/L glucose (Coolaber). The SD-H, SD-U, and SD-H-U medium were also used, where 1.29 g/L Dropout (DO) Supplement-His (Coolaber), 1.29 g/L DO Supplement-Ura (Coolaber) and 1.27 g/L DO Supplement-His/-Ura (Coolaber) respectively replaced the Yeast Amino Acids Supplement component in the SD medium. The YPD medium was composed of 20 g/L peptone (Oxoid), 10 g/L yeast extract (Oxoid), and 20 g/L glucose (Coolaber). The yeast cells were cultivated in 250 mL flasks containing 25 mL medium with an initial optical density (OD) of 0.05. The temperature and shaker speed for yeast cell culture was 30 ℃ and 200 rpm, respectively.\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\u003eThe summary of strains used in this study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrains\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStrains description\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSource\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eE. Coli\u003c/em\u003e DH5α\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF\u003csup\u003e\u0026minus;\u003c/sup\u003eφ80(\u003cem\u003elacZ\u003c/em\u003e)ΔM15Δ(\u003cem\u003elacZYA-argF\u003c/em\u003e)U169\u003cem\u003eend\u003c/em\u003eA1\u003cem\u003erec\u003c/em\u003eA1\u003cem\u003ehsd\u003c/em\u003eR17(\u003cem\u003em\u003c/em\u003e\u003csub\u003e\u003cem\u003ek\u003c/em\u003e\u003c/sub\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e,\u003cem\u003em\u003c/em\u003e\u003csub\u003e\u003cem\u003ek\u003c/em\u003e\u003c/sub\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e)supE44λ\u003cem\u003e-thi\u003c/em\u003e-1 \u003cem\u003egyr\u003c/em\u003eA96 \u003cem\u003erel\u003c/em\u003eA1 \u003cem\u003epho\u003c/em\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTSINGKE\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eE. Coli\u003c/em\u003e Stbl3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF\u003csup\u003e\u0026minus;\u003c/sup\u003e \u003cem\u003emc\u003c/em\u003erB \u003cem\u003emrr hsd\u003c/em\u003eS20(r\u003csub\u003eB\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, m\u003csub\u003eB\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) \u003cem\u003erec\u003c/em\u003eA13 \u003cem\u003esup\u003c/em\u003eE44 \u003cem\u003eara\u003c/em\u003e-14 \u003cem\u003egal\u003c/em\u003eK2 \u003cem\u003elac\u003c/em\u003eY1 \u003cem\u003epro\u003c/em\u003eA2\u003cem\u003erps\u003c/em\u003eL20(Str\u003csup\u003eR\u003c/sup\u003e) \u003cem\u003exyl\u003c/em\u003e-5λ\u003csup\u003e\u0026minus;\u003c/sup\u003e \u003cem\u003eleu mtl\u003c/em\u003e-1 endA1\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThermo Fisher\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYKM1012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eKluyveromyces marxianus\u003c/em\u003e CBS6556 ∆his3 ∆ura3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eObtained from Dr. Ian Wheeldon laboratory (UC Riverside)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYKM1013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYKM1012 \u003cem\u003eabz1\u003c/em\u003e:: (P\u003csub\u003e\u003cem\u003eKmTEF3\u003c/em\u003e\u003c/sub\u003e)PGU1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYKM1014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYKM1012 ∆ PGU1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYKM1015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYKM1012 \u003cem\u003exyl2\u003c/em\u003e:: (P\u003csub\u003e\u003cem\u003eKmGPD\u003c/em\u003e\u003c/sub\u003e)TrGAR1 \u003cem\u003eabz1\u003c/em\u003e:: (P\u003csub\u003e\u003cem\u003eKmGPD\u003c/em\u003e\u003c/sub\u003e)TrLGD1 \u003cem\u003elys1\u003c/em\u003e:: (P\u003csub\u003e\u003cem\u003eKmGPD\u003c/em\u003e\u003c/sub\u003e)AnGAAC \u003cem\u003esdl1\u003c/em\u003e:: (P\u003csub\u003e\u003cem\u003eKmGPD\u003c/em\u003e\u003c/sub\u003e)AnGatA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYKM1016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYKM1012 \u003cem\u003esdl1\u003c/em\u003e:: (P\u003csub\u003e\u003cem\u003eKmGPD\u003c/em\u003e\u003c/sub\u003e)TrGAR1 \u003cem\u003eabz1\u003c/em\u003e:: (P\u003csub\u003e\u003cem\u003eKmGPD\u003c/em\u003e\u003c/sub\u003e)V-TrLGD1 \u003cem\u003elys1\u003c/em\u003e:: (P\u003csub\u003e\u003cem\u003eKmGPD\u003c/em\u003e\u003c/sub\u003e)AnGAAC \u003cem\u003exyl2\u003c/em\u003e:: (P\u003csub\u003e\u003cem\u003eKmGPD\u003c/em\u003e\u003c/sub\u003e)AnGatA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\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\n\u003ch3\u003ePlasmids construction\u003c/h3\u003e\n\u003cp\u003eAll the plasmids used in this study were constructed by homologous recombination and were given in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. And all the gene sequences and used primers were listed in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e and Table S3, respectively. The DNA gene fragments were synthesized or amplified by polymerase chain reaction (PCR) using KOD One TM PCR Master Mix (TOYOBO). The vectors were universally linearized by restriction enzymes (XmaI and XhoI) (NEB) at 37 ℃ for 1 h. In addition, the vector for PGko-HD construction was linearized by SacII and EagI (NEB), and the vector for PGko-sg construction was linearized by PspXI (NEB). After connecting the linearized vector and targeted gene fragment, the resulting plasmids containing the homologous fragment and CRISPR/Cas9 fragment were individually transformed into \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e) \u003cem\u003eDH5α\u003c/em\u003e or \u003cem\u003eStbl3\u003c/em\u003e competent cells. The transformed cells were then cultivated on Luria\u0026ndash;Bertani (LB) agar supplemented with 100 \u0026micro;g/mL ampicillin at 37 ℃ for 16 h. A randomly chosen single colony was genotyped using a 2\u0026times;Taq Enzyme Mix (Vazyme), and the colonies with the correct insertion size were then confirmed by Sanger sequencing for plasmids generation.\u003c/p\u003e\n\u003ch3\u003eYeast transformation and strain construction\u003c/h3\u003e\n\u003cp\u003eThe transformation method has been described in a previous report (Yuan et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The YKM1012 strain was used for transformation and strain construction. After transformation, the cell mixture was reinoculated in fresh selective media for overnight culture at 30 ℃, and then plated on the YPD agar plate for single colony confirmation by PCR amplification and sequencing. The plasmids containing the URA3 selection marker were eliminated by treating them with 1 g/L 5-fluoroorotic acid (5\u0026rsquo;-FOA) in liquid YPD medium at 30 ℃ overnight. The colonies were reinoculated on the YPD, SD-H, and SD-U agar plates to ensure the successful removal of plasmids, and stored at -80 ℃.\u003c/p\u003e\n\u003ch3\u003eQuantification of endoPG enzymatic activity\u003c/h3\u003e\n\u003cp\u003eThe assay for endoPG enzymatic activity was conducted by applying the 3,5-dinitrosalicylic acid (DNS) method with some minor adjustments (Miller \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1959\u003c/span\u003e). For each sample, the endoPG secreted from the strains was used to degrade pectin in an enzymatic reaction and the mixture was incubated at 45 ℃. The thermal stability of endoPG was assessed in the 30\u0026ndash;90 ℃ temperature range. The same volume of pectin hydrolysate and DNS were combined for the measurement of D-galUA release by treatment in a boiling water bath for 10 min, with data collection on a microplate reader at 540 nm. Different concentrations of D-galUAs were processed to generate the calibration curve, with an associated R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.999. The enzymatic activity of endoPG is represented by the D-galUA concentration (\u0026micro;mol) per milliliter and also evaluated using the ratio of pectin to D-galUA.\u003c/p\u003e\n\u003ch3\u003eMetabolite preparation and pre-processing\u003c/h3\u003e\n\u003cp\u003eAfter 72 h of cultivation, different groups with equal number of cells were obtained by centrifugation at low temperature and stored at -80\u0026deg;C. All the extracts were pre-cooled at -20 ℃ before use. The samples were allowed to thaw at room temperature, and then transferred to a 1.5 mL tube, with the addition of 1 mL pre-cooled methanol-water (V:V\u0026thinsp;=\u0026thinsp;4:1) and 4 \u0026micro;g/mL L-2-chlorophenylalanine as internal standard, mixing for 10 s. Subsequently, 200 \u0026micro;L chloroform was added and the sample mixture was allowed to evaporate, followed by ultrasonic treatment (500 W) for 3 min and ultrasonic extraction for 20 min in an ice water bath, and left overnight at -40 ℃. The resultant solution was centrifuged at 12000 rpm at 4 ℃ for 10 min, and 800 \u0026micro;L supernatant was placed in a glass vial and dried in a refrigerated concentration centrifugal dryer. A 300 \u0026micro;L methanol-water (V:V\u0026thinsp;=\u0026thinsp;1:4) mixture was added to each sample and mixed for 30 s, before leaving at -40 ℃ for 2 h. A subsequent ultrasonic extraction was performed under ice condition for 3 min, with centrifugation at 12000 rpm for 10 min at a low temperature. Each tube containing 150 \u0026micro;L supernatant was collected using syringes, filtered through 0.22 \u0026micro;m microfilters, transferred to a LC sample vial, and stored at -80 ℃ before analysis by liquid chromatography-mass spectrometry (LC-MS).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLC-MS/MS detection\u003c/h2\u003e \u003cp\u003eThe metabolomic analysis was performed by Shanghai Luming Biological Technology Co., Ltd (Shanghai, China). An ACQUITY UPLC I-Class plus (Waters Corp., Milford, USA) with a Q-Exactive mass spectrometer, equipped with an ACQUITY UPLC HSS T3 column (100 \u0026times; 2.1 mm, 1.8 \u0026micro;m) and heated using an electrospray ionization (ESI) source (Thermo Fisher Scientific, Waltham, MA, USA) was employed for metabolic profiling in both ESI positive and negative ion modes. The binary gradient elution system consisted of (A) water (containing 0.1% v/v formic acid) and (B) acetonitrile (containing 0.1% v/v formic acid). Separation of metabolites was achieved using the following gradient: 0.01 min, 95% A and 5% B; 2 min, 95% A and 5% B; 4 min, 70% A and 30% B; 8 min, 50% A and 50% B; 10 min, 20% A and 80% B; 14 min, 100% B; 15 min, 100% B; 15.1 min, 95% A and 5% B; 16 min, 95% A and 5% B. The flow rate was 0.35 mL/min and the column temperature was 45 ℃. All the samples were kept at 10 ℃ and the injection volume was 5 \u0026micro;L. The mass analysis ranged from m/z 70 to 1050. In the full MS and HCD MS/MS scans, the resolution was set at 70000 and 17500, respectively, and the collision energy was set at 10, 20, and 40 eV. The following parameters were employed for mass spectrometry analysis: spray voltage\u0026thinsp;=\u0026thinsp;3800 V (+) and 3200 V (\u0026minus;); capillary temperature\u0026thinsp;=\u0026thinsp;320\u0026deg;C; aux gas heater temperature\u0026thinsp;=\u0026thinsp;350\u0026deg;C; sheath gas flow rate\u0026thinsp;=\u0026thinsp;35 arb; auxiliary gas flow rate\u0026thinsp;=\u0026thinsp;8 arb; S-lens RF level\u0026thinsp;=\u0026thinsp;50.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eData pre-processing and statistical analysis\u003c/h3\u003e\n\u003cp\u003eThe raw LC-MS data were processed using Progenesis QI V2.3 software (Nonlinear, Dynamics, Newcastle, UK) for baseline filtration, peak identification, integration, retention time correction, peak alignment, and normalization. The compounds were identified based on the precise mass-charge ratio (m/z), secondary fragments and isotope distribution employing several databases, including The Human Metabolome Database (HMDB), Lipidmaps (V2.3), Metlin and in-house databases. The extracted data were processed to remove more than 50% of the peaks with a missing value (ion intensity\u0026thinsp;=\u0026thinsp;0), and replace the zero value with half the minimum value, screening according to qualitative results for the compound. The compounds that scored less than 36 out of 60 were considered inaccurate and removed. The positive and negative ion data were combined in a data matrix, which was imported into R software for analysis. A principle component analysis (PCA) was performed to determine the overall sample distribution and analysis stability. Furthermore, the orthogonal partial least-squares-discriminant analysis (OPLS-DA) and partial least-squares-discriminant analysis (PLS-DA) were used to distinguish the metabolites in each group. In order to avoid over-fitting, a 7-fold cross-validation and 200 response permutation testing (RPT) to evaluate the quality of the model was adopted. Based on the OPLS-DA model, the applied variable importance of projection (VIP) values were used to rank the overall contribution of each variable to the group discrimination. A two-tailed Student\u0026rsquo;s T-test was also applied to determine if any differences were statistically significant. Differential metabolites with VIP\u0026thinsp;\u0026gt;\u0026thinsp;1.0 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were selected.\u003c/p\u003e\n\u003ch3\u003eLipid extraction and FAs quantification\u003c/h3\u003e\n\u003cp\u003eThe method used in lipid extraction and determination has been described previously, and this study involved some modifications of the earlier procedure (Gao et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The cell shake-flask culture (20 mL) incubated for 72 h was centrifuged at 5000 g and suspended in 2 mL 4 M hydrochloric acid with incubation at 80\u0026deg;C for 2 h. Following cooling, a mixture of chloroform and methanol (V/V\u0026thinsp;=\u0026thinsp;2:1) was added and vortexed for 5 min to facilitate lipid extraction. After centrifugation, the organic sub-layer was collected, and dried by a rotary evaporator (Lichen, China). The extracted compounds were transformed to fatty acid methyl esters by replenishing 2 mL hydrochloric acid-methanol solution (V/V\u0026thinsp;=\u0026thinsp;1:9) at 60\u0026deg;C for 1 h. The methylated compounds were obtained by adding 2 mL saturated sodium chloride and 1 mL hexane, followed by vortexing for 5 min. The resulting hexane layer was transferred to a glass vial via a 0.22 \u0026micro;m filter for subsequent gas chromatography (GC) analysis, using Agilent 7890 B unit equipped with a fame ionization detector (FID). An Agilent J\u0026amp;W DBWAXetr column (30 m \u0026times; 0.25 mm \u0026times; 0.25 \u0026micro;m) was employed with nitrogen as the carrier gas. The GC analysis parameters were as follows: initial temperature\u0026thinsp;=\u0026thinsp;100\u0026deg;C, maintained for 0.5 min, increased to 190\u0026deg;C at 5\u0026deg;C/min, followed by a ramp to 220\u0026deg;C at 2\u0026deg;C/min and held for 10 min. The injector and detector temperatures were set at 250 and 260\u0026deg;C, respectively. A 1 \u0026micro;L sample volume was injected in split mode at a ratio of 15:1. The FAs were identified and quantified using the commercial standards and normalized with respect to methyl nonadecanate.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e \u003cb\u003eImproving pectin degradation by overexpression of\u003c/b\u003e \u003cb\u003ePGU1\u003c/b\u003e \u003cb\u003ein\u003c/b\u003e \u003cb\u003eK. marxianus\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePreviously, \u003cem\u003eK. marxianus\u003c/em\u003e has been reported as a favorable strain to secrete endoPG for pectin degradation due to the considerable economic advantages with higher efficiency and stability by the cultivation in a simple medium (Siekstele et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Jia and Wheals \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; da Silva et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Sieiro et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Given the endoPG secretion capacity of strains, an increased amount of endoPG was observed in the stationary phase after YKM1012 cultivation for 14 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Additionally, a high-temperature (37 ℃) can effectively induce endoPG secretion, probably relating to the thermotolerant character of \u003cem\u003eK. marxianus\u003c/em\u003e (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). However, cultivation at extreme high-temperature (45 ℃) did not generate significant endoPG secretion (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), which could be interpreted as saving energy by reducing the cost of growth (Thorwall et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). To clarify the pectin degradation and utilization in \u003cem\u003eK. marxianus\u003c/em\u003e, the growth curve of YKM1012 with different medium compositions was collected. A lower cell biomass accumulation was observed in lower glucose content cultivation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Although endoPG secretion was detected in SD medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), there was essentially no change after adding exogenous pectin, even in the presence of endoPG accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). And it also indicated that YKM1012 cannot hydrolyze pectin or grow on pectin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eTo enhance endoPG secretion for pectin utilization, an engineering strategy was considered, directed at overexpressing the \u003cem\u003ePGU1\u003c/em\u003e gene facilitated by the strong TEF promoter at the ABZ1 locus of the YKM1012 genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD left). And a deletion of the \u003cem\u003ePGU1\u003c/em\u003e gene in engineering \u003cem\u003eK. marxianus\u003c/em\u003e was also conducted to explore the endoPG function in pectin degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD right). As expected, an overexpression of the \u003cem\u003ePGU1\u003c/em\u003e strain (YKM1013) resulted in a significant improvement in endoPG secretion, and there was no detectable secretion in the case of the \u003cem\u003ePGU1\u003c/em\u003e knock-out engineered strain (YKM1014) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). The results indicated that the engineered YKM1013 strain can serve as a platform host to enhance the level of endoPG, which is an important consideration in pectin utilization. Furthermore, an increasing of glucose concentration (from 0.3\u0026ndash;2%) resulted in a 50% increase of endoPG level (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, left Y-axis), with an associated twofold increase in hydrolysis capacity for the YKM1013 strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, right Y-axis). In addition, YKM1013 strain showed a secondary growth in the stationary phase by supplementing pectin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), suggesting an effective utilization of pectin hydrolysate for cell growth. Instead, taking the D-galUA components at an equal concentration, the YKM1013 did not exhibit the secondary growth in the stationary phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). To sum up, the increased endoPG level of the engineered strain (YKM1013) served to promote the pectin hydrolysis, and then the hydrolysate components except D-galUA promoted the secondary growth in yeast.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo fully assess pectin hydrolysis efficiency, the influence of varying pectin and endoPG concentrations were considered. As there was no observed effect for a range of endoPG concentration, the D-galUA concentrations were maintained at 10 \u0026micro;mol/mL and 20 \u0026micro;mol/mL in 1% and 2% pectin degradation, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, left Y-axis), which was accompanied by a 20% of conversion rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, right Y-axis). On the contrary, an appreciable improvement was found for 4% pectin hydrolysis when the endoPG component was increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, left Y-axis), likely due to the higher concentration of catalytic substrates. Nevertheless, a 12% conversion rate was recorded when increasing the endoPG concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, right Y-axis), where a contribution due to the high viscosity associated with 4% pectin should be taken into account (Xu et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Given the influence of multiple factors in the enzyme catalytic system, the optimal reaction condition for pectin degradation must be carefully established.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConstructing the D-galUA metabolic pathway by assembling targeted heterologous enzymes in\u003c/b\u003e \u003cb\u003eK. marxianus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAlthough the engineered YKM1013 strain can efficiently degrade pectin for the utilization of a small amount of available sugars, most of D-galUA components fail to be employed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). In previous reports, the heterologous genes involved in the D-galUA metabolic pathway were shown to be active in \u003cem\u003eS. cerevisiae\u003c/em\u003e, including \u003cem\u003eGAR1\u003c/em\u003e, \u003cem\u003eLGD1\u003c/em\u003e, \u003cem\u003eGAAC\u003c/em\u003e, and \u003cem\u003eGatA\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) (Biz et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Protzko et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Perpelea et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). We then attempted to drive the D-galUA metabolic flux in \u003cem\u003eK. marxianus\u003c/em\u003e by heterologously expressing \u003cem\u003eTrGAR1\u003c/em\u003e, \u003cem\u003eTrLGD1\u003c/em\u003e, \u003cem\u003eAnGAAC\u003c/em\u003e, and \u003cem\u003eAnGatA\u003c/em\u003e at the high-efficiency integration sites \u003cem\u003eSDL1\u003c/em\u003e, \u003cem\u003eABZ1\u003c/em\u003e, \u003cem\u003eLYS1\u003c/em\u003e, and \u003cem\u003eXYL2\u003c/em\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) (Li et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), in constructing the engineered YKM1015 strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Then, the contribution of D-galUA was identified in the engineered YKM1015 strain by separately feeding 0.5% glucose and D-galUA. Notably, an excellent performance of cell growth for YKM1015 strain in D-galUA cultivation demonstrated a 3.6-fold difference by comparison with YKM1012, which preferred to grow in glucose cultivation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Hence, the resulting strain exhibited a significant potential to successfully drive the metabolic direction of D-galUA pathway, ensuring an effective utilization of D-galUA components.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eOptimizing pectin degradation efficiency by application of engineered\u003c/b\u003e \u003cb\u003eK. marxianus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGiven the low efficiency of pectin hydrolysis, a rational strategy is required to promote pectin hydrolysis capacity. Firstly, the confirmation of cultivation conditions was essential to to produce endoPG with higher activity. Concerning the increased endoPG secretion at 37 ℃ (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), we compared the PG hydrolysis capacity of YKM1013 at 30 ℃ and 37 ℃. Surprisingly, the endoPG activity was initially repressed at 37 ℃ except 10 h (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Further discovery exhibited that the endoPG capacity was promoted over 24 h and subsequently declined (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Together, cultivation at 30 ℃ for 24 h ensured sufficient endoPG with the requisite efficiency. Subsequently, to identify the appropriate temperature of reaction, an assessment of endoPG thermal stability was conducted. A marked response was observed in the 45\u0026ndash;50 ℃ temperature range over a 30 min period (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The system was thermally stable up to 12 h at 45 ℃, with evidence of a loss of activity at 48 ℃ within 30 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Published studies have probed into optimal \u003cem\u003eK. marxianus\u003c/em\u003e incubation between 35 ℃ and 40 ℃ (da Silva et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Masoud and Jespersen \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Here, there was no significant change in D-galUA content from 30 ℃ to 45 ℃ (Fig. S3). More surprisingly, 70% of the endoPG activity was maintained after pre-processing at 45 ℃ for 48 h, even half the activity retained after 120 h (Fig. S4). Consequently, 45 ℃ was chosen as the reaction temperature in developing the pectin hydrolysis system construction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWith the objective of increasing pectin conversion rate, different endoPG and pectin volume ratios were examined. Reduction of endoPG resulted in a promotion of the hydrolysis efficiency, reaching an optimum at 1:9, whereas excessive pectin inhibited the activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Compared comprehensively, the highest conversion rate (28%) was achieved in degrading 2% pectin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, right Y-axis). Previous works have pointed out an optimum medium pH between 4 and 6 for endoPG secretion in yeasts (Moyo et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; da Silva et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In the case of \u003cem\u003eK. marxianus\u003c/em\u003e strain 166 and CCT 3172, the highest activity of endoPG secretion was recorded at pH 5.5 (da Silva et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Masoud and Jespersen \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Here, after adjusting the pH to 5.5, an enhanced activity (33% conversion rate) was achieved for an \u003cem\u003ein vitro\u003c/em\u003e 1% pectin catalytic assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, right Y-axis). In contrast, a repression of endoPG activity was discovered when using a sodium acetate buffer (pH 5.5) (Fig. S5). Inspired by the optimized condition as a common strategy, the combined effect of varying temperature, volume ratio and pH on the hydrolysis efficiency of endoPG derived from YKM1013 was compared with the commercial PG. Remarkably, the endoPG exhibited less variation with respect to reaction time and superior activity relative to the commercial PG for shorter times (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). At 45\u0026deg;C for 2 h, the D-galUA concentration and conversion rate were improved by 75% and 65%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Therefore, a optimized strategy enabled a gratifying improvement in pectin hydrolysis.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDeveloping a bioconversion system of pectin by modular co-utilization of two engineered strains\u003c/h2\u003e \u003cp\u003eHaving optimized the pectin hydrolysis system, we next set out to develop a pectin metabolism strategy by modular co-utilization of two engineered strains. To this end, firstly, the engineered strain 1 (YKM1013) incubated in SD medium at 30\u0026deg;C for 24 h was utilized to acquire the endoPG with suitable activity, replacing the medium component with a phosphate buffer solution (PBS) at a final concentration of 0.6 mg/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Then, pectin hydrolysis was conducted by combining endoPG and 2% pectin at 45\u0026deg;C for 2 h, to obtain the carbon source by high-speed centrifugation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Lastly, after providing nitrogen base and amino acids, the engineered strain 2 (YKM1015) was cultured at 30\u0026deg;C for 72 h by initial OD 0.05 for the determination of metabolite profiles (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). As a result, the harboring YKM1015 exhibited a twofold increase biomass relative to YKM1012 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), confirming the contribution from pectin hydrolysate due to the D-galUA components. Thus, the results indicated that this developed strategy can facilitate pectin bioconversion through modular co-utilization of two engineered strains.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEstablishing strategy enhanced pectin-based lipid metabolism and FAs production\u003c/h2\u003e \u003cp\u003eTo evaluate the production performance of metabolic flux transformation in pectin bioconversion, the metabolic profiles resulting from incubating different strains were then assessed. Taking account of the influence of cell growth and metabolism, a pronounced gap was apparent in the loading plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), indicating a significant difference in the metabolic profiles. In detail, the YKM1015 strain presented an improvement of 217 metabolites and a repression of 122 metabolites when compared with YKM1012 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Focused on the pathway enrichment, a greater accumulation enriched in lipid metabolism was detected in YKM1015 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), suggesting that this engineered strain directed the metabolic flux in favor of lipid biosynthesis. In contrast to \u003cem\u003eYarrowia lipolytica\u003c/em\u003e, a well-studied cell factory for producing lipids via metabolic engineering strategy (Lazar et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), little research referring to lipids has been carried out in \u003cem\u003eK. marxianus\u003c/em\u003e so far. However, the down-regulation of amino acids metabolism was performed in engineered strain fermentation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the pathway associated with the observed enrichment, the elevated level of metabolites was mainly distributed in the metabolisms relating to glycerophospholipid, arachidonic acid, α-linolenic acid, and sphingolipid (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD left). The phosphatidylcholines (PCs), common in the membranes of most eukaryotes, are involved in the sphingolipid and glycerophospholipid metabolisms to determine the physical properties and strictly regulated (de Kroon \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Thereby, alteration of PCs implied the difference of membrane profile when utilizing pectin hydrolysate. Although most of the information referring to unsaturated fatty acid (UFA) metabolism has been obtained from mammalian biology, the relevant compounds can also be produced by yeasts, such as \u003cem\u003eCandida albicans\u003c/em\u003e, \u003cem\u003eCryptococcus neoformans\u003c/em\u003e (Deva et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Erb-Downward and Huffnagle \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), and \u003cem\u003eY. lipolytica\u003c/em\u003e (Liu et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Little information is, however, available regarding the metabolites involved in α-linolenic acid and arachidonic acid production in \u003cem\u003eK. marxianus\u003c/em\u003e. Conversely, the metabolites with decreased levels of enrichment accounted for the majority of amino acids associated with biosynthesis pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD right). In yeast, the availability of amino acids is important in cell growth and regulatory processes in a complicated network (Broach \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Takagi \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Thus, the engineered YKM1015 strain was possibly influenced by an inhibited cell growth and regulatory processes.\u003c/p\u003e \u003cp\u003eReferring to the previous studies, \u003cem\u003eT. reesei\u003c/em\u003e LGD1 fused with yellow fluorescent protein (Venus-LGD1, V-TrLGD1) was shown to improve a 60-fold enzyme activity \u003cem\u003ein vitro\u003c/em\u003e (Biz et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Protzko et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In the case of engineered \u003cem\u003eK. marxianus\u003c/em\u003e strains, interestingly, the engineered YKM1016 strain equipped with V-TrLGD1 performed in an analogous manner to YKM1015 with respect to cell growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, S6A). Correspondingly, the YKM1012 and YKM1016 strains demonstrated markedly different responses (Fig. S6B), and the two engineered YKM1016 and YKM1015 strains showed greater commonality with increased lipid metabolism and an inhibited amino acid metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, S6C, and S6D). Hence, this result suggested a similar metabolic flux without taking the LGD level into account when applying \u003cem\u003eK. marxianus\u003c/em\u003e as a host platform. Ultimately, the heterologous metabolic pathway for pectin bioconversion was successfully designed in \u003cem\u003eK. marxianus\u003c/em\u003e, yielding 22 mg/L mid-chain fatty acids (MCFA) and 14 mg/L long-chain fatty acids (LCFA), pointing at an increasing of 19-fold and 6-fold, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Consequently, the pectin utilization route can be tailored to generate a range of products and serve as an effective platform for pectin-rich biomass bioconversion.\u003c/p\u003e \u003cp\u003eWhen compared with pectin hydrolysate cultivation, the metabolic profile associated with incubation in glucose displayed a completely different flux transformation that was principally related to specific amino acid metabolism (Fig. S7). The different strains generate a distinct metabolic flux centering on the metabolism of most amino acids in the presence of glucose condition, resulting in specific growth patterns (Broach \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). As shown here, detail of enhanced histidine metabolism and lysine degradation, and repressed valine, leucine and isoleucine biosynthesis were summarized (Fig. S7). Taken together, the results illustrated that the metabolic transformation can be inclined to facilitate high value-added products accumulation from pectin-rich biomass employing a multifunctional \u003cem\u003eK. marxianus\u003c/em\u003e host platform.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, a rational strategy is proposed involving a synergistic application of two engineered \u003cem\u003eK. marxianus\u003c/em\u003e strains to construct a pectin bioconversion system, resulting in a 65% improvement of conversion rate and enhancing lipid metabolism flux with an increasing of 19-fold in MCFA and 6-fold in LCFA production. This strategy facilitates a feasible and sustainable bioconversion route of pectin-rich materials, which can form the basis for diverse microbial cell factories in biomass resources utilization.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e5’-FOA, 5-fluoroorotic acid;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eA\u003c/em\u003e. \u003cem\u003eniger\u003c/em\u003e, \u003cem\u003eAspergillus niger\u003c/em\u003e;\u003c/p\u003e\n\u003cp\u003eD-galUA, D-galacturonic acid;\u003c/p\u003e\n\u003cp\u003eDNS, 3,5-dinitrosalicylic acid;\u003c/p\u003e\n\u003cp\u003eDO, Dropout;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eE. coli, Escherichia coli\u003c/em\u003e;\u003c/p\u003e\n\u003cp\u003eendoPG, endo-polygalacturonase;\u003c/p\u003e\n\u003cp\u003eESI, electrospray ionization;\u003c/p\u003e\n\u003cp\u003eFID, fame ionization detector;\u003c/p\u003e\n\u003cp\u003eGAAC, 2-keto-3-deoxy-L-galactonate aldolase;\u003c/p\u003e\n\u003cp\u003eGAR1, D-galacturonate reductase;\u003c/p\u003e\n\u003cp\u003eGatA, D-GalUA transporter;\u003c/p\u003e\n\u003cp\u003eGC, gas chromatography;\u003c/p\u003e\n\u003cp\u003eGRAS, generally recognized as safe;\u003c/p\u003e\n\u003cp\u003eHMDB, The Human Metabolome Database;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eK. marxianus\u003c/em\u003e, \u003cem\u003eKluyveromyces marxianus\u003c/em\u003e;\u003c/p\u003e\n\u003cp\u003eLB, Luria–Bertani;\u003c/p\u003e\n\u003cp\u003eLCFA, long-chain fatty acids;\u003c/p\u003e\n\u003cp\u003eLC-MS, liquid chromatography-mass spectrometry;\u003c/p\u003e\n\u003cp\u003eLGD1, L-galactonate dehydratase;\u003c/p\u003e\n\u003cp\u003eMCFA, mid-chain fatty acids;\u003c/p\u003e\n\u003cp\u003eOD, optical density;\u003c/p\u003e\n\u003cp\u003eOPLS-DA, orthogonal partial least-squares-discriminant analysis;\u003c/p\u003e\n\u003cp\u003ePBS, phosphate buffer solution;\u003c/p\u003e\n\u003cp\u003ePCA, principle component analysis;\u003c/p\u003e\n\u003cp\u003ePCR, polymerase chain reaction;\u003c/p\u003e\n\u003cp\u003ePCs, phosphatidylcholines;\u003c/p\u003e\n\u003cp\u003ePL, pectin lyase;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePLS-DA, partial least-squares-discriminant analysis;\u003c/p\u003e\n\u003cp\u003ePME, pectin methylesterase;\u003c/p\u003e\n\u003cp\u003eRPT; response permutation testing;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eS. cerevisiae, Saccharomyces cerevisiae\u003c/em\u003e;\u003c/p\u003e\n\u003cp\u003eSD, synthetic defined;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUFA, unsaturated fatty acids;\u003c/p\u003e\n\u003cp\u003eVIP, variable importance of projection;\u003c/p\u003e\n\u003cp\u003eV-TrLGD1, LGD1 fused with yellow fluorescent protein;\u003c/p\u003e\n\u003cp\u003eYPD, yeast-extract peptone dextrose.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAssociated content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupporting information\u003c/p\u003e\n\u003cp\u003eendoPG secretion in YKM1012 (Fig. S1); endoPG activity in YKM1013 (Fig. S2,S3); thermal stability of endoPG in YKM1013 (Fig. S4); endoPG capacity in engineered strain (YKM1013) in sodium acetate buffer (pH 5.5) (Fig. S5); metabolic profile between YKM1012 and YKM1016 strains (Fig. S6); differential metabolites between YKM1012 and YKM1015 strains by the cultivation in 2% glucose (Fig. S7)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data and materials that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFundings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by Zhejiang Provincial Natural Science Foundation of China under Grant No. ZCLQ24C0101, ZJU-Hangzhou Global Scientific and Technological Innovation Center, Zhejiang University (Grant No. 02020200-K02013027 to Xuye Lang, 02020100-K3F113074 to Bofan Yu).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor’s contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eX.L. designed the study. X.L. and B.Y. analyzed the data and wrote the manuscript. B.Y. and H.Q. conducted the experiments. All authors revised and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have consented for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAcknowledge the support of the ZJU-Hangzhou Global Scientific and Technological Innovation Center, Zhejiang University (HIC-ZJU), the iBiofoundary, the Core Facility of the Institute for Intelligent Bio/Chem Manufacturing at HIC-ZJU, Acknowledge Professor Ian Wheeldon at UC Riverside for providing the strains and previous knowledge.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBilal M, Ji L, Xu Y, Xu S, Lin Y, Iqbal HMN, Cheng H (2022) Bioprospecting \u003cem\u003eKluyveromyces marxianus\u003c/em\u003e as a Robust Host for Industrial Biotechnology. Front Bioeng Biotechnol 10:851768\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBiz A, Sugai-Gu\u0026eacute;rios MH, Kuivanen J, Maaheimo H, Krieger N, Mitchell DA, Richard P (2016) The introduction of the fungal D-galacturonate pathway enables the consumption of D-galacturonic acid by \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. Microb Cell Fact 15(1):144\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBonnin E, Garnier C, Ralet MC (2014) Pectin-modifying enzymes and pectin-derived materials: applications and impacts. Appl Microbiol Biotechnol 98(2):519\u0026ndash;532\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBroach JR (2012) Nutritional control of growth and development in yeast. Genetics 192(1):73\u0026ndash;105\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChavan P, Singh AK, Kaur G (2019) Recent progress in the utilization of industrial waste and by-products of citrus fruits: A review. J Food Process Eng\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen L, Yan W, Qian X, Chen M, Zhang X, Xin F, Zhang W, Jiang M, Ochsenreither K (2021) Increased Lipid Production in \u003cem\u003eYarrowia lipolytica\u003c/em\u003e from Acetate through Metabolic Engineering and Cosubstrate Fermentation. ACS Synth Biol 10(11):3129\u0026ndash;3138\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eda Silva EG, de F\u0026aacute;tima Borges M, Medina C, Piccoli RH, Schwan RF (2005) Pectinolytic enzymes secreted by yeasts from tropical fruits. FEMS Yeast Res 5(9):859\u0026ndash;865\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDekker WJC, Ortiz-Merino RA, Kaljouw A, Battjes J, Wiering FW, Mooiman C, Torre P, Pronk JT (2021) Engineering the thermotolerant industrial yeast \u003cem\u003eKluyveromyces marxianus\u003c/em\u003e for anaerobic growth. Metab Eng 67:347\u0026ndash;364\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Kroon AI (2007) Metabolism of phosphatidylcholine and its implications for lipid acyl chain composition in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. Biochim Biophys Acta 1771(3):343\u0026ndash;352\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeva R, Ciccoli R, Schewe T, Kock JL, Nigam S (2000) Arachidonic acid stimulates cell growth and forms a novel oxygenated metabolite in \u003cem\u003eCandida albicans\u003c/em\u003e. Biochim Biophys Acta 1486(2\u0026ndash;3):299\u0026ndash;311\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eErb-Downward JR, Huffnagle GB (2007) \u003cem\u003eCryptococcus neoformans\u003c/em\u003e produces authentic prostaglandin E\u003csub\u003e2\u003c/sub\u003e without a cyclooxygenase. Eukaryot Cell 6(2):346\u0026ndash;350\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eErian AM, Sauer M (2022) Utilizing yeasts for the conversion of renewable feedstocks to sugar alcohols - a review. Bioresour Technol 346:126296\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao J, Yu W, Li Y, Jin M, Yao L, Zhou YJ (2023) Engineering co-utilization of glucose and xylose for chemical overproduction from lignocellulose. Nat Chem Biol 19(12):1524\u0026ndash;1531\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao Q, Cao X, Huang YY, Yang JL, Chen J, Wei LJ, Hua Q (2018) Overproduction of Fatty Acid Ethyl Esters by the Oleaginous Yeast \u003cem\u003eYarrowia lipolytica\u003c/em\u003e through Metabolic Engineering and Process Optimization. ACS Synth Biol 7(5):1371\u0026ndash;1380\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrohmann K, Baldwin EA, Buslig BS (1994) Production of ethanol from enzymatically hydrolyzed orange peel by the yeast \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. Appl Biochem Biotechnol 45\u0026ndash;46:315\u0026ndash;327\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuisjes EH, Luttik MA, Almering MJ, Bisschops MM, Dang DH, Kleerebezem M, Siezen R, van Maris AJ, Pronk JT (2012) Toward pectin fermentation by \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e: expression of the first two steps of a bacterial pathway for D-galacturonate metabolism. J Biotechnol 162(2\u0026ndash;3):303\u0026ndash;310\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJia J, Wheals A (2000) Endopolygalacturonase genes and enzymes from \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e and \u003cem\u003eKluyveromyces marxianus\u003c/em\u003e. Curr Genet 38(5):264\u0026ndash;270\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLazar Z, Liu N, Stephanopoulos G (2018) Holistic Approaches in Lipid Production by \u003cem\u003eYarrowia lipolytica\u003c/em\u003e. Trends Biotechnol 36(11):1157\u0026ndash;1170\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi J, Peng C, Mao A, Zhong M, Hu Z (2024) An overview of microbial enzymatic approaches for pectin degradation. Int J Biol Macromol 254:127804\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi M, Lang XY, Moran Cabrera M, De Keyser S, Sun X, Da Silva N, Wheeldon I (2021) CRISPR-mediated multigene integration enables Shikimate pathway refactoring for enhanced 2-phenylethanol biosynthesis in \u003cem\u003eKluyveromyces marxianus\u003c/em\u003e. Biotechnol Biofuels 14(1):3\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi S, Su C, Fang M, Cai D, Deng L, Wang F, Liu J (2023) Overproduction of palmitoleic acid from corn stover hydrolysate by engineered \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. Bioresour Technol 382:129211\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang C, Gui X, Zhou C, Xue Y, Ma Y, Tang SY (2015) Improving the thermoactivity and thermostability of pectatelyase from \u003cem\u003eBacillus pumilus\u003c/em\u003e for ramie degumming. Appl Microbiol Biotechnol 99(6):2673\u0026ndash;2682\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu HH, Wang C, Lu XY, Huang H, Tian Y, Ji XJ (2019) Improved Production of Arachidonic Acid by Combined Pathway Engineering and Synthetic Enzyme Fusion in \u003cem\u003eYarrowia lipolytica\u003c/em\u003e. J Agric Food Chem 67(35):9851\u0026ndash;9857\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eL\u0026ouml;bs AK, Lin JL, Cook M, Wheeldon I (2016) High throughput, colorimetric screening of microbial ester biosynthesis reveals high ethyl acetate production from \u003cem\u003eKluyveromyces marxianus\u003c/em\u003e on C5, C6, and C12 carbon sources. Biotechnol J 11(10):1274\u0026ndash;1281\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa X, Wang DL, Chen WJ, Ismail BB, Wang WJ, Lv RL, Ding T, Ye XQ, Liu DH (2018) Effects of ultrasound pretreatment on the enzymolysis of pectin: Kinetic study, structural characteristics and anti-cancer activity of the hydrolysates. Food Hydrocolloids 79:90\u0026ndash;99\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMasoud W, Jespersen L (2006) Pectin degrading enzymes in yeasts involved in fermentation of \u003cem\u003eCoffea arabica\u003c/em\u003e in East Africa. Int J Food Microbiol 110(3):291\u0026ndash;296\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiller G (1959) Use of dinitrosalicyclic acid reagent for determination of reducing sugars. Anal Chem 31:426\u0026ndash;428\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohnen D (2008) Pectin structure and biosynthesis. Curr Opin Plant Biol 11(3):266\u0026ndash;277\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMotter FA, Kuivanen J, Ker\u0026auml;nen H, Hilditch S, Penttil\u0026auml; M, Richard P (2014) Categorisation of sugar acid dehydratases in \u003cem\u003eAspergillus niger\u003c/em\u003e. Fungal Genet Biol 64:67\u0026ndash;72\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoyo S, Gashe BA, Collison EK, Mpuchane S (2003) Optimising growth conditions for the pectinolytic activity of \u003cem\u003eKluyveromyces wickerhamii\u003c/em\u003e by using response surface methodology. Int J Food Microbiol 85(1\u0026ndash;2):87\u0026ndash;100\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerpelea A, Wijaya AW, Martins LC, Rippert D, Klein M, Angelov A, Peltonen K, Teleki A, Liebl W, Richard P, Thevelein JM, Takors R, S\u0026aacute;-Correia I, Nevoigt E (2022) Towards valorization of pectin-rich agro-industrial residues: Engineering of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e for co-fermentation of d-galacturonic acid and glycerol. Metab Eng 69:1\u0026ndash;14\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eProtzko RJ, Latimer LN, Martinho Z, de Reus E, Seibert T, Benz JP, Dueber JE (2018) Engineering \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e for co-utilization of D-galacturonic acid and D-glucose from citrus peel waste. Nat Commun 9(1): 5059\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharma P, Vishvakarma R, Gautam K, Vimal A, Kumar Gaur V, Farooqui A, Varjani S, Younis K (2022) Valorization of citrus peel waste for the sustainable production of value-added products. Bioresour Technol 351:127064\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSieiro C, Sestelo AB, Villa TG (2009) Cloning, characterization, and functional analysis of the EPG1-2 gene: a new allele coding for an endopolygalacturonase in \u003cem\u003eKluyveromyces marxianus\u003c/em\u003e. J Agric Food Chem 57(19):8921\u0026ndash;8926\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiekstele R, Bartkeviciute D, Sasnauskas K (1999) Cloning, targeted disruption and heterologous expression of the \u003cem\u003eKluyveromyces marxianus\u003c/em\u003e endopolygalacturonase gene (EPG1). Yeast 15(4):311\u0026ndash;322\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakagi H (2019) Metabolic regulatory mechanisms and physiological roles of functional amino acids and their applications in yeast. Biosci Biotechnol Biochem 83(8):1449\u0026ndash;1462\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThorwall S, Schwartz C, Chartron JW, Wheeldon I (2020) Stress-tolerant non-conventional microbes enable next-generation chemical biosynthesis. Nat Chem Biol 16(2):113\u0026ndash;121\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWagner JM, Alper HS (2016) Synthetic biology and molecular genetics in non-conventional yeasts: Current tools and future advances. Fungal Genet Biol 89:126\u0026ndash;136\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang K, Shi TQ, Wang J, Wei P, Ledesma-Amaro R, Ji XJ (2022) Engineering the Lipid and Fatty Acid Metabolism in \u003cem\u003eYarrowia lipolytica\u003c/em\u003e for Sustainable Production of High Oleic Oils. ACS Synth Biol 11(4):1542\u0026ndash;1554\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J, Ledesma-Amaro R, Wei Y, Ji B, Ji XJ (2020) Metabolic engineering for increased lipid accumulation in \u003cem\u003eYarrowia lipolytica\u003c/em\u003e - A Review. Bioresour Technol 313:123707\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWikandari R, Hasniah N, Taherzadeh MJ (2022) The role of filamentous fungi in advancing the development of a sustainable circular bioeconomy. Bioresour Technol 345:126531\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilliams DL, Sch\u0026uuml;ckel J, Vivier MA (2019) Grape pomace fermentation and cell wall degradation by \u003cem\u003eKluyveromyces marxianus\u003c/em\u003e Y885. Bio Eng J 150:107282\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu YT, Pereira M, Ven\u0026acirc;ncio A, Teixeira J (2000) Recovery of endo-polygalacturonase using polyethylene glycol-salt aqueous two-phase extraction with polymer recycling. Bioseparation 9(4):247\u0026ndash;254\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiang T, Yang R, Li L, Lin H, Kai G (2024) Research progress and application of pectin: A review. J Food Sci 89(11):6985\u0026ndash;7007\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu S, Qin X, Liu B, Zhang DQ, Zhang YH (2015) An acidic pectin lyase from \u003cem\u003eAspergillus niger\u003c/em\u003e with favourable efficiency in fruit juice clarification. Lett Appl Microbiol 60(2):181\u0026ndash;187\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan YC, Yu BF, Zhou XZ, Qiao H, Lian JZ, Lang XY, Yao Y (2024) Engineering Living Material for Controlled Fragrance Release Utilizing \u003cem\u003eKluyveromyces marxianus\u003c/em\u003e CBS6556 and Adaptive Hydrogel. ACS Synth Biol 13(10):3188\u0026ndash;3196\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang K, Jiang Z, Li X, Wang D, Hong J (2024) Enhancing simultaneous saccharification and co-fermentation of corncob by \u003cem\u003eKluyveromyces marxianus\u003c/em\u003e through overexpression of putative transcription regulator. Bioresour Technol 399:130627\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bioresources-and-bioprocessing","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biob","sideBox":"Learn more about [Bioresources and Bioprocessing](http://bioresourcesbioprocessing.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/biob/default.aspx","title":"Bioresources and Bioprocessing","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"metabolic engineering, pectin bioconversion, Kluyveromyces marxianus, sustainable system, lipid metabolism, modular co-utilization","lastPublishedDoi":"10.21203/rs.3.rs-6193521/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6193521/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePectin bioconversion from renewable feedstocks represents an appealing and sustainable production route. However, the microbial valorization of pectin is not well developed and requires an efficient expression of key enzymes. Here, a constructed biological system successfully drove pectin-based lipid metabolism by collaborative utilization of two engineered \u003cem\u003eKluyveromyces marxianus\u003c/em\u003e strains. The YKM1013 strain with an overexpression of the \u003cem\u003ePGU1\u003c/em\u003e gene served to break down pectin, resulting in a 65% improvement in conversion rate. And the YKM1015 strain with an additional central D-galacturonic acid (D-galUA) metabolic pathway effectively utilized the available D-galUA components. The developed strategy enabled an effective bioconversion of pectin-based lipid metabolism, with an increasing of 19-fold in medium-chain fatty acid (MCFA) and 6-fold in long-chain fatty acid (LCFA). Collectively, this study provided a feasible and sustainable bioconversion route for transforming pectin into chemicals that can be employed in the construction of a microbial cell factory platform for pectin valorization.\u003c/p\u003e","manuscriptTitle":"Engineering a sustainable system for pectin-based lipid metabolism through modular co-utilization of two Kluyveromyces marxianus strains","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-26 12:13:50","doi":"10.21203/rs.3.rs-6193521/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2025-04-20T22:19:44+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-03-27T05:21:50+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-18T09:24:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-18T07:45:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bioresources and Bioprocessing","date":"2025-03-14T01:26:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bioresources-and-bioprocessing","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biob","sideBox":"Learn more about [Bioresources and Bioprocessing](http://bioresourcesbioprocessing.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/biob/default.aspx","title":"Bioresources and Bioprocessing","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0e6bcb58-2a1c-4cd4-b206-c53beef5268d","owner":[],"postedDate":"March 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-18T16:06:02+00:00","versionOfRecord":{"articleIdentity":"rs-6193521","link":"https://doi.org/10.1186/s40643-025-00927-z","journal":{"identity":"bioresources-and-bioprocessing","isVorOnly":false,"title":"Bioresources and Bioprocessing"},"publishedOn":"2025-08-12 15:57:08","publishedOnDateReadable":"August 12th, 2025"},"versionCreatedAt":"2025-03-26 12:13:50","video":"","vorDoi":"10.1186/s40643-025-00927-z","vorDoiUrl":"https://doi.org/10.1186/s40643-025-00927-z","workflowStages":[]},"version":"v1","identity":"rs-6193521","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6193521","identity":"rs-6193521","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}