H2-driven xylitol production in Cupriavidus necator H16

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Claassens, Laura Salusjärvi, Antti Nyyssölä This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5062650/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Dec, 2024 Read the published version in Microbial Cell Factories → Version 1 posted 9 You are reading this latest preprint version Abstract Background : Biocatalysis offers a potentially greener alternative to chemical processes. For biocatalytic systems requiring cofactor recycling, hydrogen emerges as an attractive reducing agent. Hydrogen is attractive because all the electrons can be fully transferred to the product, and it can be efficiently produced from water using renewable electricity. In this article, resting cells of Cupriavidus necator H16 harboring a NAD-dependent hydrogenase were employed for cofactor recycling to reduce D-xylose to xylitol, a commonly used sweetener. To enable this bioconversion, D-xylose reductase from Scheffersomyces stipitis was heterologously expressed in C. necator . Results : D-xylose reductase was successfully expressed in C. necator , enabling complete bioconversion of 30 g/L of D-xylose into xylitol within 7 days using resting cells. It was found that over 90% of the energy and protons derived from hydrogen were spent for the bioconversion, demonstrating the efficiency of the system. The highest xylitol productivity reached was 0.7 g L -1 h -1 . Additionally, the same chassis efficiently produced L-arabitol and D-ribitol from L-arabinose and D-ribose, respectively. Conclusions : This study highlights the efficient utilization of renewable hydrogen as a reducing agent to power cofactor recycling. Hydrogen-oxidizing bacteria, such as C. necator , can be promising hosts for performing hydrogen-driven biocatalysis. Biotransformation cofactor recycling cofactor regeneration Ralstonia eutropha Cupriavidus necator hydrogen-oxidizing bacteria Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Biocatalysis is increasingly applied across different industries due to its efficiency and environmental benefits compared to chemical transformations. Biocatalysis using oxidoreductases often requires cofactors, such as nicotinamide nucleotides NADH and NADPH, but their stoichiometric addition to reaction mixtures is not economically feasible ( 1 ). Therefore, cofactor recycling systems are required. Traditionally cofactor recycling of NAD(P)H is performed using sacrificial substrates, such as glucose and formate, which are oxidized during the biocatalysis, while the cofactor is regenerated to its reduced form ( 2 ). This is not carbon-efficient, since D-gluconolactone and carbon dioxide (CO 2 ) are formed as by-products from glucose and formate, respectively. Specifically for the by-product D-gluconolactone, a substantial number of energy-rich electrons are wasted. In addition, glucose is produced by agriculture, which can decrease the overall sustainability of the production process due to, for example, competition with food production and environmental burdens of agriculture. Formate can be made from CO 2 and renewable electricity using electrochemical reduction. However, electrochemical production of formate has not yet been scaled-up and is not as energy-efficient as the electrochemical production of hydrogen (H 2 ) from water and electricity, which is already performed on a large scale ( 3 ). H 2 is an attractive, byproduct-free sacrificial substrate for cofactor recycling. Molecular H 2 can be oxidized by organisms using hydrogenases. Some of the most extensively researched H 2 -uptake hydrogenases are found in the hydrogen-oxidizing bacterium Cupriavidus necator H16 (formerly Ralstonia eutropha ) ( 4 ). This bacterium possesses two types of hydrogenases that provide the cells with reducing power: membrane-bound and soluble hydrogenases ( 5 ). The membrane-bound hydrogenase is located in the cytoplasmic membrane where it directly feeds electrons to the respiratory chain for ATP production by oxidative phosphorylation. The NAD-dependent hydrogenase resides in the cytoplasm and is therefore referred to as soluble hydrogenase (SH) ( 6 , 7 ). The electrons and protons from H 2 can be directly transferred to NAD + by the SH, reducing NAD + to NADH while simultaneously oxidizing hydrogen ( 4 ). C. necator H16 SH has been researched broadly for cofactor recycling using purified enzymes ( 8 – 12 ). Compared to purified enzymes, whole-cell biocatalysts can provide enzyme stabilization, lower production cost and better inhibitor tolerance ( 13 ). In addition, cofactors are readily available inside the cells. Whole cells were used in one of the earliest studies of SH-catalyzed cofactor recycling in the 1980s where native C. necator cells reduced CO 2 to formate with a 30% yield ( 14 ). More recently, whole cells of recombinant C. necator have been employed as a biocatalyst by Oda et al. ( 15 ) and Assil-Companioni et al. ( 16 ) for reduction of hydroxyacetone to ( R )-1,2-propanediol and for asymmetric C = C bond reduction of unsaturated cyclic ketones, respectively. However, given the limited extent of research and number of products, further studies are required to investigate C. necator as a whole-cell biocatalyst. In the current study, we show that the scope of hydrogen-driven reductive bioconversions can be broadened to encompass a new class of products, sugar alcohols, with xylitol as a particular example. Xylitol (C 5 H 12 O 5 ) is a sugar alcohol used widely as a sweetener ( 17 ). It is currently produced chemically from D-xylose (C 5 H 10 O 5 ), which is the second most abundant sugar in lignocellulosic biomass ( 18 ). The chemical production of xylitol requires extensively purified D-xylose to avoid inactivation of the catalyst. In contrast, biotechnological production does not have this requirement and exhibits greater tolerance for inhibitors. Many yeasts, along with some bacteria and filamentous fungi, naturally reduce D-xylose to xylitol ( 18 ). However, numerous studies have focused on heterologous expression of D-xylose reductase (XR), aiming to increase yield and productivity of xylitol ( 19 – 23 ). All these studies use sugars to power the conversion, and to our knowledge H 2 has not been used as the electron and energy source for the D-xylose-to-xylitol conversion. The objective of this study was to develop a whole-cell biocatalyst strain of C. necator for xylitol production using H 2 as the electron donor. To achieve this, XR from Scheffersomyces stipitis (formerly Pichia stipitis ) was expressed in C. necator to allow the cells to convert D-xylose into xylitol. The native SH of C. necator enabled the H 2 -driven cofactor recycling required for bioconversion. Resting (i.e., non-dividing) cells were used in the experiments. These viable cells exhibit reduced metabolic activity, which allows energy to be directed towards bioconversion instead of biomass accumulation ( 24 ). With the experimental set-up used, we achieved nearly full quantitative conversions. Materials and methods Strains and culture media Bacterial strains and plasmids used in this work are listed in Table 1 and the primers in Table S1 (see Additional file 1). C. necator H16 strains were grown in either rich BD BBL™ Trypticase™ Soy Broth (TSB) or minimal AUT medium ( 25 ) supplemented with 2 g/L fructose and glycerol (FG). Compared to the original AUT medium, the amount of NiCl 2 was doubled and SL-6 trace elements were added (1:1000) ( 26 ). Escherichia coli DH10β was used for plasmid construction. Tetracycline was added at a concentration of 10 µg/mL for E. coli and 5 µg/mL for C. necator when required. The sugars and sugar alcohols used in the study were purchased from Sigma-Aldrich. Table 1 Bacterial strains and plasmids used in the study. The strains with △ phaC deletion are unable to produce polyhydroxybutyrate (PHB) for carbon and energy storage. Strain Relevant characteristics Source or reference C. necator H16 △ phaCAB △ phaC1 △ phaA1 △ phaB1 (27) C. necator H16 △ phaC1 △ phaC1 (28) C. necator H16 △ A0006 △ phaC1 △ A0006 This study C. necator H16 △ phaCAB_ gfp △ phaCAB derivative, pPj5:GFP This study C. necator H16 △ phaC_ gfp △ A0006 derivative, pPj5:GFP This study C. necator H16 △ phaCAB_ xr △ phaCAB derivative, pPj5:XRsti This study C. necator H16 △ phaC_ xr △ A0006 derivative, pPj5:XRsti This study Plasmid pLO3 Suicide vector, sacB (29) pLO3- A0006 pLO3 derivative with 1100 bp upstream and downstream regions of A0006 This study pPj5:GFP pSEVA521 containing Pj5 promoter and GFP (30) pPj5:XRsti pPj5:GFP derivative, GFP replaced with D-xylose reductase from Scheffersomyces stipitis (XRsti) This study Strain construction XR from Scheffersomyces ( Pichia ) stipitis was ordered codon-optimized for C. necator from GenScript (Additional file 1: Table S2) and PCR amplified with Q5 High-Fidelity 2X Master Mix (NEB). The backbone plasmid pSEVA521 + Pj5:GFP and the amplified insert were digested with Spe I-HF and Hin dIII-HF (NEB) and ligated using T4 DNA ligase (NEB) to gain pPj5:XRsti. The verified plasmid was transformed into electrocompetent C. necator cells. For preparing competent cells, C. necator was grown in 100 mL of TSB supplemented with 20 mM of fructose to an optical density at 600 nm (OD 600 ) of 0.6 and washed twice with 1 mM MgSO 4 . The pellet was resuspended into 2 mL of 1 mM MgSO4 and 1 mL of 60% glycerol. Aliquots (50 µL) were stored in -80°C. Cells were mixed with plasmids (250 ng) in a 0.2 cm electroporation cuvette (Bio-Rad), incubated 10 min on ice and electroporated with Electro Cell Manipulator ECM®630 (BTX) with the following settings: 2.5 kV, 200Ω and 25µF. Super Optimal Broth with 20 mM fructose (950 µL) was added immediately after electroporation and cells were incubated at 30°C and 180 rpm for 2–3 hours before plating on BBL™ Trypticase™ Soy Agar (TSA). C. necator H16 △ A0006 was constructed from C. necator H16 △ phaC1 by deleting A0006 with pLO3-based suicide vector as previously described ( 29 ) (Additional file 1: Table S3). The △ A0006 restriction enzyme knockout increases electroporation efficiency ( 31 , 32 ) and is not expected to have any metabolic effects. XR activity assay For the XR activity assay, cells were grown overnight in TSB and then harvested by centrifugation (10 min, 2800 g). The pellet was washed once with 50 mM potassium phosphate buffer (pH 7.5), resuspended into 1 mL of the same buffer supplemented with cOmplete protease inhibitor (Roche) and moved into a 2 mL screw-cap tube with 400 µL of 0.5 mm diameter glass beads. The cells were disrupted with FASTPREP-24 5G (MP Biomedicals) for 2x30sec at 6 m/s speed. After disruption, the tube was centrifuged for 10 min at 16000 g, and the supernatant (soluble extract) was collected. Total protein concentration was analyzed from the soluble extract by Quick Start™ Bradford Protein Assay (Bio-Rad) using bovine serum albumin as the standard. XR activity assays were conducted in 96-well plates. The reaction mixture (330 µL) contained 0.15 mM of cofactor (NADH or NADPH), 50 mM potassium phosphate buffer (pH 6.0), 200 mM D-xylose and an appropriate amount of soluble extract. Absorbance was measured at 340 mM using Epoch 2 Microplate Spectrophotometer (BioTek). One unit of xylose reductase activity was defined as µmol of NAD(P)H oxidized per minute. Specific activities were expressed as units per milligram of total protein. The results are given as averages of triplicate assays. Bioconversions with resting cells Precultures were grown overnight in TSB media. FG media with tetracycline was inoculated to an initial OD 600 of 0.1 and grown for three days, reaching a final OD 600 of approximately 4. The culture was centrifuged for 10 min at 2800 g and washed twice with 100 mM sodium phosphate buffer (pH 7.0) to remove carbon and nitrogen sources. The cells were resuspended in the same buffer with 30 g/L of substrate to an OD 600 of 13–17, if not mentioned otherwise. This range of OD 600 corresponds approximately to a cell dry weight of 4.4–5.4 g/L. The prepared cell suspension (5 mL) was transferred into an anaerobic serum bottle with a rubber stopper. D-xylose was the primary substrate, but one bioconversion was also performed using L-arabinose and another using D-ribose. Three replicate bottles per bioconversion condition were prepared. For the first bioconversion experiments, 100 mL serum bottles were filled with H 2 using vacuum-gas cycles to reach specific H 2 and oxygen (O 2 ) concentrations. As negative controls, bioconversions were performed under ambient air. The bottles were incubated at 30°C and 150 rpm. Samples (200 µL) were taken by opening the rubber stopper and the bottles were refilled with gasses after sampling. For bioconversion optimization, 50 mL serum bottles were filled with H 2 at the start of the bioconversion by flushing with 100% H 2 at 0.5 mL min − 1 for 2 minutes. Samples (200 µL) were taken using a needle and syringe through the rubber stopper of sealed bottles. The approximate H 2 gas consumption was measured after bioconversions by filling a 50 mL syringe with air and recording the volume of air aspirated into the bottle through the syringe needle. Samples were centrifuged for 10 min at 16000 g and the supernatants were analyzed for sugars and sugar alcohols. Calculation of H 2 consumption The amount of H 2 consumed was calculated from the approximated H 2 gas consumption (Eq. 1 ): $$\:n\:\left({\text{H}}_{2}\:\text{c}\text{o}\text{n}\text{s}\text{u}\text{m}\text{e}\text{d}\right)=\frac{pV\:}{RT}$$ 1 where p is the pressure (0.98692 atm), V is the gas volume consumed (L), R is the gas constant (0.08206 L atm mol − 1 K − 1 ) and T is the temperature (298.15 K). One mole of H 2 is needed to reduce one mole of xylose (Eq. 2 ): $$\:{\text{H}}_{2}+{\text{C}}_{5}{\text{H}}_{10}{\text{O}}_{5}={\text{C}}_{5}{\text{H}}_{12}{\text{O}}_{5}$$ 2 The total amount of xylitol produced was calculated as the sum of the xylitol at the end of the bioconversion and the xylitol in the fractions taken out during sampling. The ratio of xylitol production to H 2 consumption was calculated to determine the proportion of energy and protons transferred from H 2 to xylitol. Analysis of sugars and sugar alcohols Two different high performance liquid chromatography (HPLC) systems were used for D-xylose and xylitol analysis: Prominence-i LC-2030C (Shimadzu) equipped with Hi-Plex H 7.7x300 mm column (Agilent) at 45°C and 10 mM H 2 SO 4 as eluent at a flow rate of 1 ml min − 1 and Vanquish Flex (Thermo Fisher Scientific) equipped with Aminex Fast Acid Analysis and HPX-87H columns (Bio-Rad) at 55°C and 2 mM H 2 SO 4 as the eluent at a flow rate of 0.5 ml min − 1 . An injection volume of 10 µL was used in both HPLCs and the compounds were detected with a refractive index detector. L-arabinose, L-arabitol, D-ribose and ribitol were analyzed with high pressure ion chromatography (HPIC) Dionex ICS-6000 (Thermo Fisher Scientific) with CarboPac PA20 column (Thermo Fisher Scientific) at 30°C and 10 mM KOH as the eluent at a flow rate of 0.5 ml min − 1 . An injection volume of 2.5 µL was used and the compounds were detected with an electrochemical detector. Conversion yields at specific time points were calculated by dividing the amount of xylitol by the total amount of xylose and xylitol. Results Xylose reductase from S. stipitis is functionally produced in C. necator H16 In this study, two C. necator strains, △ phaC and △ phaCAB , with partial or full knockouts of the native pathway for storage polymer polyhydroxybutyrate (PHB) formation, were used to avoid the accumulation of this by-product. Before constructing the XR expressing strains, it was confirmed that the host strains cannot grow on C5 sugars and sugar alcohols used in the study (Additional file 1: Fig. S1 ). Both strains were then transformed with the plasmids pPj5:XR, carrying the codon-optimized XR from S. stipitis , and pPj5:GFP, as a negative control. Strains were cultivated heterotrophically, and their soluble extracts were tested for XR activity. The soluble extracts of both XR strains showed reductase activity with both NADPH and NADH cofactors, whereas no activity was detected in the negative control strains (Table 2 ). Codon-optimized XR from Candida parapsilosis was also expressed in the host strains, but no activity was detected (data not shown). Table 2 Specific D-xylose reductase activities of soluble extracts with NADPH or NADH as cofactors (U/mg). The average from three measurements is shown with the standard deviation. No activity could be detected in the negative controls. Strain NADPH NADH △ phaCAB_ xr 0.71 ± 0.20 0.48 ± 0.01 △ phaC_ xr 0.49 ± 0.01 0.41 ± 0.03 Xylose is fully converted to xylitol by C. necator △ phaCAB The first bioconversion experiment was performed at 30 g/L D-xylose by both C. necator strains: △ phaCAB_ xr and △ phaC_ xr. The suspensions were incubated under H 2 (85% H 2 + 15% air) or 100% ambient air. Under H 2 , the △ phaCAB_ xr strain reached complete bioconversion to xylitol within 16 days, whereas the △ phaC_ xr strain converted 78% of the provided D-xylose at the same time (Fig. 1 ). Therefore, further bioconversions were performed using the △ phaCAB_ xr strain. D-xylose was also converted to xylitol in the absence of H 2 by both strains, but the conversion yields were under 25% after 16 days. This demonstrated successful cofactor recycling in resting cells of C. necator using H 2 . The oxygen concentration does not influence the bioconversion Since oxygen, acting as an electron acceptor, can provide the cells with energy via oxidative phosphorylation, we examined the effect of oxygen concentration on the conversion rate. Three different oxygen concentrations (0, 1, and 4%) were tested. The conversion rates and yields showed little variation between the different oxygen concentrations (Fig. 2 ), with over 86% conversion yields being reached within 10 days in all conditions. After confirming that oxygen was not a limiting factor, it was tested whether the amount of H 2 would be. Bioconversions with multiple H 2 flushes during the experiment were compared to bioconversions with a single H 2 flush at the start. The results showed that multiple H 2 flushes failed to improve the conversion yield (Additional file 1: Fig. S2). Therefore, further bioconversions were conducted with only an initial H 2 flush. Higher initial sugar concentration can speed up the bioconversion rate Four different D-xylose concentrations were tested to evaluate their impact on xylitol production rates. With increasing xylose concentration, the rate of conversion increased (Fig. 3 ). The highest xylitol productivity in the first 48 hours (0.7 g L − 1 h − 1 ) was reached with the highest xylose concentration used (114 g/L D-xylose). However, complete conversion was reached faster at a lower xylose concentration: 13 g/L of D-xylose was fully converted to xylitol in 10 days while 85% of 34 g/L D-xylose was converted within the same time. In samples with the highest xylose concentrations (66 and 114 g/L), the final xylitol concentration reached 46 g/L. We hypothesized that H 2 in the headspace was limiting and that measuring H 2 consumption could allow us to estimate the electron conversion efficiency of hydrogen into xylitol. On average, 30 mL of gas was consumed under both conditions, equivalent to 1.2 mmol of H 2 . Given that 0.2 mmol of xylitol was produced without H 2 (as shown in Fig. 1 ) and 1.3 mmol was produced in total, it can be assumed that 1.1 mmol of xylitol was produced with the help of H 2 under both conditions. Consequently, more than 90% of the energy derived from H 2 was spent for the bioconversion, as one mole of H 2 is required to reduce one mole of xylose. Increased cell concentration enhances the rate of bioconversion So far, all bioconversions in this study were conducted with a cell concentration range of OD 600 13–17. The effect of the amount of the whole-cell biocatalyst was examined at lower and higher cell concentrations: OD 600 7 and 60. With the highest cell concentration, full conversion of 30 g/L D-xylose to xylitol was achieved in 7 days (Fig. 4 ). Increasing the cell concentration had a positive effect also on the xylitol production rate. In the first 24 hours, the production rates were 0.5 g L − 1 h − 1 and 0.1 g L − 1 h − 1 for the highest and lowest cell concentrations used, respectively. Arabinose and ribose are reduced to sugar alcohols by the resting cells S. stipitis XR is also known to convert L-arabinose and D-ribose into their respective sugar alcohols ( 33 ). Therefore, we examined whether C. necator harboring the xylose reductase could also be used as a biocatalyst for these conversions. Both sugars were successfully reduced to their corresponding sugar alcohols, with the production rates and yields of L-arabitol and ribitol being only slightly lower than those for xylitol (Fig. 5 ). Discussion This study aimed to advance the development of C. necator as a H 2 -driven whole-cell biocatalyst. We demonstrated a full reduction of 30 g/L of D-xylose to xylitol in resting cells using H 2 for cofactor regeneration. The effects of different parameters on xylitol production were studied to improve the production rates. Up to 30 g/L of xylitol was produced in C. necator △ phaCAB_xr within 7 days and 100% conversion yield (Fig. 4 ). Additionally, it was shown that the system can also convert L-arabinose and D-ribose into their respective sugar alcohols (Fig. 5 ). C. necator accumulates polyhydroxybutyrate (PHB) as a carbon and energy storage compound. To avoid this accumulation, which could also lead to cofactor oxidation, PHB-negative strains △ phaCAB and △ phaC of C. necator were used as hosts. Comparison of the △ phaCAB_ xr and △ phaC_ xr strains revealed that deletion of the whole PHB pathway ( phaCAB ) enhanced both the bioconversion rate and yield (Fig. 1 ). In the △ phaC knock-out strain, it is possible that some of the reducing equivalents from H 2 were consumed by the NAD(P)H-utilizing acetoacetyl-CoA reductase (PhaB) of the PHB pathway. The advantage of knocking out more than just the phaC gene has also been observed in earlier studies in non-resting cells of C. necator . For instance, complete deletion of the PHB pathway was found to be beneficial for resveratrol production in C. necator , whereas deletion of only the phaC gene did not improve the titer compared to the wild type ( 34 ). Oxygen is essential for ATP production from H 2 in C. necator . Although the bioconversion reaction itself does not require ATP, it was hypothesized that the cells would require some ATP for cell maintenance. However, the results suggest otherwise (Fig. 2 ). This outcome is advantageous for industrial applications, as oxygen-free production mitigates the risk associated with flammability of H 2 -O 2 mixtures. The experiments also demonstrated efficient transfer of nearly all hydrogen-derived electrons into the product. When enough H 2 was present in the headspace, full bioconversion of xylose could be demonstrated. However, liquid solubility of H 2 is low and hence may still limit the rate of conversion. To test this, bioconversions could be performed under elevated pressure, where H 2 solubility is increased, but unfortunately it was not possible to test this with the current experimental set-up. Additionally, mass transfer of H 2 to the liquid phase can be significantly improved by using optimized bioreactors equipped with specialized gas spargers and impellers ( 35 ). The most significant improvements in the bioconversion rate were achieved by increasing the cell concentration and xylose concentration. This is not surprising, as a higher cell concentration provides more catalyst for the conversion to occur, and an increased substrate concentration boosts the reaction rate until enzyme saturation is reached. The most efficient xylitol production systems reported in the literature have reached higher xylitol productivity than the 0.7 g L − 1 h − 1 achieved in this study. At a similar cell density, recombinant E. coli cells, coexpressing a D-xylose reductase and a glucose dehydrogenase, produced xylitol at 6.4 g L − 1 h − 1 . In this optimized process, a 100% yield was achieved at an initial D-xylose concentration of 200 g/L using glucose for cofactor recycling ( 23 ). Factors that could account for this include lower enzyme activities, slower substrate and product transport into and outside of the cell, and particularly the aforementioned poor H 2 solubility. Observing the SDS-PAGE gel of Jin et al., it seems that their XR level in the cell was much higher than in this study (Additional file 1: Fig. S3). Although the strongest promoter currently known for C. necator was used in the present study ( 30 , 36 ), stronger expression of XR could improve the bioconversion rates. The expression systems for C. necator need further development to achieve the expression levels obtained with E. coli . On the other hand, the xylose and xylitol transport systems of E. coli are likely more efficient than those of C. necator because E. coli can natively grow on xylose whereas C. necator cannot. A BLAST search of the C. necator H16 genome using the D-xylose specific transport systems of E. coli (XylE and XylFGH) yielded no matches, suggesting that C. necator lacks xylose-specific transporters. Xylose is likely transported into the cells by a sugar transporter with side activity for xylose. Hence, heterologous introduction of a xylose transporter could be considered for future studies to further improve bioconversion rates. The specific activity of XR with NADPH was higher compared to NADH (Table 2 ). The same result has been observed previously by Verduyn, Van Kleef ( 33 ). Using NADPH-producing SH, instead of the native NADH-producing SH, with NADPH-dependent oxidoreductases could increase the rate of the bioconversion. The NAD + -specific SH from C. necator has been engineered to also accept NADP + , but its NADP + -reducing activity would need to be increased ( 12 ). Another option is to use NADH-preferring oxidoreductases or to engineer them to have this preference, ensuring high activity towards NADH. This article presented the first whole-cell, H 2 -driven biocatalysis study using a PHB-negative C. necator strain as the host. Direct comparison of this work to the few prior studies is challenging due to differences in experimental setups, product types, enzyme kinetics, and strains used. Oda et al. ( 15 ) reported a productivity of 0.9 g L -1 h -1 for ( R )-1,2-propanediol, which is within the same order of magnitude as our findings. Whole-cell cofactor recycling using SH has not only been done in C. necator . Lonsdale et al. ( 37 ) expressed SH from C. necator in Pseudomonas putida to perform H 2 -driven bioconversion of n-octane to 1-octanol. The cofactor recycling proved to be effective, resulting in a threefold increase in 1-octanol production in the presence of H 2 . However, the yield and rate of bioconversions they achieved were significantly lower than the ones in this study; Lonsdale et al. reported a maximum productivity of 0.01 g L -1 h -1 , about 100-fold lower than the rates we and Oda et al. achieved. The limited amount of research in this area offers a wide range of opportunities for improving these organisms to perform H 2 -driven bioconversions towards industrial applications. Bioconversion rates can likely be significantly improved using hosts with improved enzyme activities, elevated pressures, higher cell concentrations and optimized bioreactor designs that enhance hydrogen solubility. Conclusions Cofactor recycling via hydrogenases represents a promising alternative for traditional bioconversion systems because of its atom efficiency, lack of by-products and the prospects of H 2 becoming a renewable platform chemical of the future. This study demonstrated H 2 -driven bioconversion of D-xylose to xylitol in XR expressing C. necator strain. 30 g/L of D-xylose was fully converted into xylitol in 7 days. It was shown that nearly all the energy from H 2 is harnessed by the bioconversion, demonstrating the potential of the C. necator system as an efficient H 2 -driven biocatalyst for sugar alcohol production and potentially other products. Declarations Competing interests The authors declare that they have no competing interests. Funding This work was supported by the Research Council of Finland (KNALLRED—Hydrogen powered reductive biosyntheses and biotransformations by an engineered Knallgas bacterium, grant number 342124). Author Contribution All authors designed research. TJ conducted experiments, data analysis, and wrote the manuscript. All authors revised and approved the manuscript. Acknowledgement The authors thank Enrico Orsi for the C. necator △phaCAB strain, Guillermo Bordanaba Florit for constructing the C. necator H16 △A0006 strain, Victor de Lorenzo’s lab for the SEVA plasmid, and Ton van Gelder for the help with HPLC analyses. We also thank Solar Foods, especially Juha-Pekka Pitkänen, for the fruitful discussions and the bioeconomy research infrastructures of Aalto University for the support. Data Availability The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request. References Wang X, Saba T, Yiu HHP, Howe RF, Anderson JA, Shi J. Cofactor NAD(P)H Regeneration Inspired by Heterogeneous Pathways. Chem. 2017. 10.1016/j.chempr.2017.04.009 . van der Donk WA, Zhao H. Recent developments in pyridine nucleotide regeneration. Curr Opin Biotechnol. 2003. 10.1016/s0958-1669(03)00094-6 . Stöckl M, Claassens NJ, Lindner SN, Klemm E, Holtmann D. 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ChemCatChem. 2020; 10.1002/cctc.202000763 Lin B, Tao Y. Whole-cell biocatalysts by design. Microb Cell Fact. 2017. 10.1186/s12934-017-0724-7 . Klibanov AM, Alberti BN, Zale SE. Enzymatic synthesis of formic acid from H 2 and CO 2 and production of hydrogen from formic acid. Biotechnol Bioeng. 1982. 10.1002/bit.260240104 . Oda T, Oda K, Yamamoto H, Matsuyama A, Ishii M, Igarashi Y, et al. Hydrogen-driven asymmetric reduction of hydroxyacetone to (R)-1,2-propanediol by Ralstonia eutropha transformant expressing alcohol dehydrogenase from Kluyveromyces lactis . Microb Cell Fact. 2013. 10.1186/1475-2859-12-2 . Assil-Companioni L, Schmidt S, Heidinger P, Schwab H, Kourist R. Hydrogen-Driven Cofactor Regeneration for Stereoselective Whole-Cell C = C Bond Reduction in Cupriavidus necator . Chemsuschem. 2019. 10.1002/cssc.201900327 . da Silva S, Chandel A, D-Xylitol. Fermentative production, application and commercialization. 1st ed. Heidelberg: Springer-; 2012. Narisetty V, Cox R, Bommareddy R, Agrawal D, Ahmad E, Pant KK, et al. Valorisation of xylose to renewable fuels and chemicals, an essential step in augmenting the commercial viability of lignocellulosic biorefineries. Sustain Energ Fuels. 2022. 10.1039/d1se00927c . Hallborn J, Walfridsson M, Airaksinen U, Ojamo H, Hahn-Hägerdal B, Penttilä M, et al. Xylitol production by recombinant Saccharomyces cerevisiae . Nat Biotechnol. 1991. 10.1038/nbt1191-1090 . Nyyssölä A, Pihlajaniemi A, Palva A, von Weymarn N, Leisola M. Production of xylitol from D-xylose by recombinant Lactococcus lactis . J Biotechnol. 2005. 10.1016/j.jbiotec.2005.03.014 . Cirino PC, Chin JW, Ingram LO. Engineering Escherichia coli for xylitol production from glucose-xylose mixtures. Biotechnol Bioeng. 2006. 10.1002/bit.21082 . Sasaki M, Jojima T, Inui M, Yukawa H. Xylitol production by recombinant Corynebacterium glutamicum under oxygen deprivation. Appl Biochem Biotechnol. 2010. 10.1007/s00253-009-2372-2 . Jin LQ, Xu W, Yang B, Liu ZQ, Zheng YG. Efficient Biosynthesis of Xylitol from Xylose by Coexpression of Xylose Reductase and Glucose Dehydrogenase in Escherichia coli . Appl Biochem Biotechnol. 2019. 10.1007/s12010-018-2878-0 . Julsing MK, Kuhn D, Schmid A, Buhler B. Resting cells of recombinant E. coli show high epoxidation yields on energy source and high sensitivity to product inhibition. Biotechnol Bioeng. 2012. 10.1002/bit.24404 . Lenz O, Lauterbach L, Frielingsdorf S. O 2 -tolerant [NiFe]-hydrogenases of Ralstonia eutropha H16: Physiology, molecular biology, purification, and biochemical analysis. Methods Enzymol. 2018. 10.1016/bs.mie.2018.10.008 . Atlas RM. Handbook of Microbiological Media. 4th ed. Washington DC: Taylor & Francis; 2010. Collas F, Dronsella BB, Kubis A, Schann K, Binder S, Arto N, et al. Engineering the biological conversion of formate into crotonate in Cupriavidus necator . Metab Eng. 2023. 10.1016/j.ymben.2023.06.015 . Lütte S, Pohlmann A, Zaychikov E, Schwartz E, Becher JR, Heumann H, et al. Autotrophic production of stable-isotope-labeled arginine in Ralstonia eutropha strain H16. Appl Environ Microbiol. 2012. 10.1128/AEM.01972-12 . Lenz O, Friedrich B. A novel multicomponent regulatory system mediates H 2 sensing in Alcaligenes eutrophus . Proc Natl Acad Sci. 1998. 10.1073/pnas.95.21.12474 . Claassens NJ, Bordanaba-Florit G, Cotton CAR, De Maria A, Finger-Bou M, Friedeheim L, et al. Replacing the Calvin cycle with the reductive glycine pathway in Cupriavidus necator . Metab Eng. 2020. 10.1016/j.ymben.2020.08.004 . Xiong B, Li Z, Liu L, Zhao D, Zhang X, Bi C. Genome editing of Ralstonia eutropha using an electroporation-based CRISPR-Cas9 technique. Biotechnol Biofuels. 2018. 10.1186/s13068-018-1170-4 . Vajente M, Clerici R, Ballerstedt H, Blank LM, Schmidt S. Using Cupriavidus necator H16 to provide a roadmap for increasing electroporation efficiency in non-model bacteria. bioRxiv. 2024. 10.1101/2024.05.27.596136 . Verduyn C, Van Kleef R, Frank J, Schreuder H, Van Dijken JP, Scheffers WA. Properties of the NAD(P)H-dependent xylose reductase from the xylose-fermenting yeast Pichia stipitis . Biochem J. 1985. 10.1042/bj2260669 . Jang Y, Lee YJ, Gong G, Lee SM, Um Y, Kim KH et al. Carbon dioxide valorization into resveratrol via lithoautotrophic fermentation using engineered Cupriavidus necator H16. Microb Cell Fact. 2024; 10.1186/s12934-024-02398-x Tanaka K, Ishizaki A, Kanamaru T, Kawano T. Production of poly(D-3-hydroxybutyrate) from CO 2 , H 2 , and O 2 by high cell density autotrophic cultivation of Alcaligenes eutrophus . Biotechnol Bioeng. 1995. 10.1002/bit.260450312 . Gruber S, Hagen J, Schwab H, Koefinger P. Versatile and stable vectors for efficient gene expression in Ralstonia eutropha H16. J Biotechnol. 2014. 10.1016/j.jbiotec.2014.06.030 . Lonsdale TH, Lauterbach L, Honda Malca S, Nestl BM, Hauer B, Lenz O. H 2 -driven biotransformation of n-octane to 1-octanol by a recombinant Pseudomonas putida strain co-synthesizing an O 2 -tolerant hydrogenase and a P450 monooxygenase. Chem Commun. 2015. 10.1039/c5cc06078h . Additional Declarations No competing interests reported. Supplementary Files Additionalfile1.docx Additional file 1: Table S1. Oligonucleotide primers used in the study. Table S2. Synthesized xylose reductase gene used in this study originating from Scheffersomyces stipitis . Table S3. Upstream and downstream regions of A0006 used to create C. necator H16 △ A0006 . Figure S1. The growth of C. necator strains △ phaCAB and △ phaC on different sugars and sugar alcohols (100 mM). Figure S2. Comparison of bioconversion with a single H 2 flush at the start and H 2 flush after every sampling. Figure S3. SDS-PAGE analysis of soluble extracts by C. necator H16 strains. Cite Share Download PDF Status: Published Journal Publication published 23 Dec, 2024 Read the published version in Microbial Cell Factories → Version 1 posted Editorial decision: Revision requested 14 Nov, 2024 Reviews received at journal 12 Nov, 2024 Reviewers agreed at journal 05 Nov, 2024 Reviews received at journal 20 Sep, 2024 Reviewers agreed at journal 14 Sep, 2024 Reviewers invited by journal 14 Sep, 2024 Editor assigned by journal 12 Sep, 2024 Submission checks completed at journal 12 Sep, 2024 First submitted to journal 10 Sep, 2024 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5062650","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":378360373,"identity":"a86d87d1-210b-40c8-9627-e1f7b20fc3aa","order_by":0,"name":"Tytti Jämsä","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYLCCBAY2EMX4AESyNxNQzcPAzNgA1cJswJBgwMBzmBgtUDabBFjLAQJa7CXyjz94wMAnr9t/+Fk1748/DDzshGyRSAY7zHDbjTSz2zwgW5iJ1MK47QaD2c0ZQC32xGqx33b++LfCGaTYkrjtQI4ZwweitJx5bAg0my15242cYokPacY8BLWwtyc++Pij4pgt0GEbPyTYyMnx8B8goEcgAUgYHENYS0A9EEDMrCGscBSMglEwCkYuAABz1DraXgEc0QAAAABJRU5ErkJggg==","orcid":"","institution":"VTT Technical Research Centre of Finland Ltd","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Tytti","middleName":"","lastName":"Jämsä","suffix":""},{"id":378360374,"identity":"0087b03b-64a3-4d06-8894-307b33ea0ad1","order_by":1,"name":"Nico J. Claassens","email":"","orcid":"","institution":"Wageningen University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nico","middleName":"J.","lastName":"Claassens","suffix":""},{"id":378360375,"identity":"dfdae1f5-b390-412c-a7e5-fa6b56e3f721","order_by":2,"name":"Laura Salusjärvi","email":"","orcid":"","institution":"VTT Technical Research Centre of Finland Ltd","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Salusjärvi","suffix":""},{"id":378360377,"identity":"5ce394e2-988d-4070-a6c1-af0a3e3b6af7","order_by":3,"name":"Antti Nyyssölä","email":"","orcid":"","institution":"VTT Technical Research Centre of Finland Ltd","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Antti","middleName":"","lastName":"Nyyssölä","suffix":""}],"badges":[],"createdAt":"2024-09-10 07:34:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5062650/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5062650/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12934-024-02615-7","type":"published","date":"2024-12-23T15:57:21+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":70467520,"identity":"4f1ea4aa-b7a4-4db0-84bc-a5fda489f37f","added_by":"auto","created_at":"2024-12-03 12:44:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":33233,"visible":true,"origin":"","legend":"\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003e-driven production of xylitol by \u003cem\u003eC. necator\u003c/em\u003e △\u003cem\u003ephaCAB_\u003c/em\u003exr and △\u003cem\u003ephaC_\u003c/em\u003exr. The initial concentration of D-xylose was 30 g/L in 100 mM sodium phosphate buffer (pH 7.0) with the cells at OD\u003csub\u003e600\u003c/sub\u003e of 17 (△\u003cem\u003ephaCAB_\u003c/em\u003exr) and 15 (△\u003cem\u003ephaC_\u003c/em\u003exr). The used gas mixture consisted of 85% H\u003csub\u003e2\u003c/sub\u003e, 12% N\u003csub\u003e2\u003c/sub\u003e, and 3% O\u003csub\u003e2\u003c/sub\u003e while ambient air contains 78% N\u003csub\u003e2\u003c/sub\u003e and 21% O\u003csub\u003e2\u003c/sub\u003e. The average from three bioconversions is shown with the standard deviation. The percentages represent the final yields.\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5062650/v1/e1330e77c57d8fcd5bff5ca2.png"},{"id":70466329,"identity":"5ffe44cf-43c1-4c21-a368-5ecaf0f18e76","added_by":"auto","created_at":"2024-12-03 12:36:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":30480,"visible":true,"origin":"","legend":"\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003e-driven production of xylitol by \u003cem\u003eC. necator\u003c/em\u003e △\u003cem\u003ephaCAB \u003c/em\u003eon different oxygen concentrations. The reaction mixtures contained initially 30 g/L of D-xylose in 100 mM sodium phosphate buffer (pH 7.0) with cells at OD\u003csub\u003e600\u003c/sub\u003e of 16. The used gas mixtures consisted of 0% O\u003csub\u003e2\u003c/sub\u003e and 100% H\u003csub\u003e2\u003c/sub\u003e, 1% O\u003csub\u003e2\u003c/sub\u003e, 2% N\u003csub\u003e2\u003c/sub\u003e, and 97% H\u003csub\u003e2\u003c/sub\u003e or 4% O\u003csub\u003e2\u003c/sub\u003e, 13% N\u003csub\u003e2\u003c/sub\u003e, and 83% H\u003csub\u003e2\u003c/sub\u003e. The average from three bioconversions is shown with the standard deviation. The percentages represent the final yields.\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5062650/v1/f1e0f6d6aebcd55ad86ec718.png"},{"id":70466323,"identity":"4fa38589-fba4-45fb-a8b8-ad766a252087","added_by":"auto","created_at":"2024-12-03 12:36:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":34239,"visible":true,"origin":"","legend":"\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003e-driven production of xylitol by \u003cem\u003eC. necator\u003c/em\u003e △\u003cem\u003ephaCAB \u003c/em\u003eat different D-xylose concentrations. The reaction mixtures were composed of 13, 34, 66, and 114 g/L of D-xylose in 100 mM sodium phosphate buffer (pH 7.0) and cells at OD\u003csub\u003e600\u003c/sub\u003e of 13-15. The headspace contained 100% H\u003csub\u003e2\u003c/sub\u003e. The average from three bioconversions is shown with the standard deviation. The percentages represent the final yields.\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5062650/v1/d7b0c927d106a695b6d01b01.png"},{"id":70466325,"identity":"b4ca7ab2-7c6d-4085-b9f4-ec821deca1a6","added_by":"auto","created_at":"2024-12-03 12:36:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":27254,"visible":true,"origin":"","legend":"\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003e-driven production of xylitol by \u003cem\u003eC. necator\u003c/em\u003e △\u003cem\u003ephaCAB \u003c/em\u003eat different cell concentrations. The initial concentration of D-xylose was 30 g/L. The conversions were carried out in a 100 mM sodium phosphate buffer (pH 7.0) under 100% H\u003csub\u003e2\u003c/sub\u003e. The average from three bioconversions is shown with the standard deviation. The percentages represent the final yields.\u003c/p\u003e","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5062650/v1/ba0e6f90855b42eb9a2cf9d6.png"},{"id":70466328,"identity":"bd6f76e3-7483-4dec-8ac4-4113524b503d","added_by":"auto","created_at":"2024-12-03 12:36:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":27034,"visible":true,"origin":"","legend":"\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003e-driven production of L-arabitol, ribitol and xylitol by C. necator △\u003cem\u003ephaCAB\u003c/em\u003e. The initial concentrations of L-arabinose, D-ribose and D-xylose were 28, 30 and 34 g/L, respectively, and the corresponding cell densities (OD\u003csub\u003e600\u003c/sub\u003e) were 15, 15, and 13. The experiments were carried out in a 100 mM sodium phosphate buffer (pH 7.0) under 100% H\u003csub\u003e2\u003c/sub\u003e. The average from three bioconversions is shown with the standard deviation. The percentages represent the final yields.\u003c/p\u003e","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5062650/v1/2613979b44be53aa7908c362.png"},{"id":72640581,"identity":"f6b931d0-8b10-4111-9f13-a4da86a3a56b","added_by":"auto","created_at":"2024-12-30 16:07:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":945932,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5062650/v1/f4b1cbe8-20a4-4352-bea3-5fc0256e8d61.pdf"},{"id":70467521,"identity":"b2912023-1655-4797-884f-a222abc9e8d3","added_by":"auto","created_at":"2024-12-03 12:44:05","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":394905,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 1: Table S1.\u003c/p\u003e\n\u003cp\u003eOligonucleotide primers used in the study.\u0026nbsp;\u003cstrong\u003eTable S2.\u003c/strong\u003e\u0026nbsp;Synthesized xylose reductase gene used in this study originating from\u0026nbsp;\u003cem\u003eScheffersomyces stipitis\u003c/em\u003e.\u0026nbsp;\u003cstrong\u003eTable S3.\u003c/strong\u003e\u0026nbsp;Upstream and downstream regions of\u0026nbsp;\u003cem\u003eA0006\u003c/em\u003e\u0026nbsp;used to create\u0026nbsp;\u003cem\u003eC. necator\u003c/em\u003e\u0026nbsp;H16 △\u003cem\u003eA0006\u003c/em\u003e.\u0026nbsp;\u003cstrong\u003eFigure\u0026nbsp;S1.\u003c/strong\u003e\u0026nbsp;The growth of\u0026nbsp;\u003cem\u003eC. necator\u003c/em\u003e\u0026nbsp;strains △\u003cem\u003ephaCAB\u003c/em\u003e\u0026nbsp;and △\u003cem\u003ephaC\u003c/em\u003e\u0026nbsp;on different sugars and sugar alcohols (100 mM).\u0026nbsp;\u003cstrong\u003eFigure S2.\u003c/strong\u003e\u0026nbsp;Comparison of bioconversion with a single H\u003csub\u003e2\u003c/sub\u003e\u0026nbsp;flush at the start and H\u003csub\u003e2\u003c/sub\u003e\u0026nbsp;flush after every sampling.\u0026nbsp;\u003cstrong\u003eFigure S3.\u003c/strong\u003e\u0026nbsp;SDS-PAGE analysis of soluble extracts by\u0026nbsp;\u003cem\u003eC. necator\u003c/em\u003e\u0026nbsp;H16 strains.\u003c/p\u003e","description":"","filename":"Additionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5062650/v1/4d86cf7f0f49a98fec69c061.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"H2-driven xylitol production in Cupriavidus necator H16","fulltext":[{"header":"Background","content":"\u003cp\u003eBiocatalysis is increasingly applied across different industries due to its efficiency and environmental benefits compared to chemical transformations. Biocatalysis using oxidoreductases often requires cofactors, such as nicotinamide nucleotides NADH and NADPH, but their stoichiometric addition to reaction mixtures is not economically feasible (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Therefore, cofactor recycling systems are required. Traditionally cofactor recycling of NAD(P)H is performed using sacrificial substrates, such as glucose and formate, which are oxidized during the biocatalysis, while the cofactor is regenerated to its reduced form (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). This is not carbon-efficient, since D-gluconolactone and carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e) are formed as by-products from glucose and formate, respectively. Specifically for the by-product D-gluconolactone, a substantial number of energy-rich electrons are wasted. In addition, glucose is produced by agriculture, which can decrease the overall sustainability of the production process due to, for example, competition with food production and environmental burdens of agriculture. Formate can be made from CO\u003csub\u003e2\u003c/sub\u003e and renewable electricity using electrochemical reduction. However, electrochemical production of formate has not yet been scaled-up and is not as energy-efficient as the electrochemical production of hydrogen (H\u003csub\u003e2\u003c/sub\u003e) from water and electricity, which is already performed on a large scale (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003e is an attractive, byproduct-free sacrificial substrate for cofactor recycling. Molecular H\u003csub\u003e2\u003c/sub\u003e can be oxidized by organisms using hydrogenases. Some of the most extensively researched H\u003csub\u003e2\u003c/sub\u003e-uptake hydrogenases are found in the hydrogen-oxidizing bacterium \u003cem\u003eCupriavidus necator\u003c/em\u003e H16 (formerly \u003cem\u003eRalstonia eutropha\u003c/em\u003e) (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). This bacterium possesses two types of hydrogenases that provide the cells with reducing power: membrane-bound and soluble hydrogenases (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). The membrane-bound hydrogenase is located in the cytoplasmic membrane where it directly feeds electrons to the respiratory chain for ATP production by oxidative phosphorylation. The NAD-dependent hydrogenase resides in the cytoplasm and is therefore referred to as soluble hydrogenase (SH) (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). The electrons and protons from H\u003csub\u003e2\u003c/sub\u003e can be directly transferred to NAD\u003csup\u003e+\u003c/sup\u003e by the SH, reducing NAD\u003csup\u003e+\u003c/sup\u003e to NADH while simultaneously oxidizing hydrogen (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eC. necator\u003c/em\u003e H16 SH has been researched broadly for cofactor recycling using purified enzymes (\u003cspan additionalcitationids=\"CR9 CR10 CR11\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Compared to purified enzymes, whole-cell biocatalysts can provide enzyme stabilization, lower production cost and better inhibitor tolerance (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). In addition, cofactors are readily available inside the cells. Whole cells were used in one of the earliest studies of SH-catalyzed cofactor recycling in the 1980s where native \u003cem\u003eC. necator\u003c/em\u003e cells reduced CO\u003csub\u003e2\u003c/sub\u003e to formate with a 30% yield (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). More recently, whole cells of recombinant \u003cem\u003eC. necator\u003c/em\u003e have been employed as a biocatalyst by Oda et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) and Assil-Companioni et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) for reduction of hydroxyacetone to (\u003cem\u003eR\u003c/em\u003e)-1,2-propanediol and for asymmetric C\u0026thinsp;=\u0026thinsp;C bond reduction of unsaturated cyclic ketones, respectively. However, given the limited extent of research and number of products, further studies are required to investigate \u003cem\u003eC. necator\u003c/em\u003e as a whole-cell biocatalyst. In the current study, we show that the scope of hydrogen-driven reductive bioconversions can be broadened to encompass a new class of products, sugar alcohols, with xylitol as a particular example.\u003c/p\u003e \u003cp\u003eXylitol (C\u003csub\u003e5\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e) is a sugar alcohol used widely as a sweetener (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). It is currently produced chemically from D-xylose (C\u003csub\u003e5\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e), which is the second most abundant sugar in lignocellulosic biomass (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). The chemical production of xylitol requires extensively purified D-xylose to avoid inactivation of the catalyst. In contrast, biotechnological production does not have this requirement and exhibits greater tolerance for inhibitors. Many yeasts, along with some bacteria and filamentous fungi, naturally reduce D-xylose to xylitol (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). However, numerous studies have focused on heterologous expression of D-xylose reductase (XR), aiming to increase yield and productivity of xylitol (\u003cspan additionalcitationids=\"CR20 CR21 CR22\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). All these studies use sugars to power the conversion, and to our knowledge H\u003csub\u003e2\u003c/sub\u003e has not been used as the electron and energy source for the D-xylose-to-xylitol conversion.\u003c/p\u003e \u003cp\u003eThe objective of this study was to develop a whole-cell biocatalyst strain of \u003cem\u003eC. necator\u003c/em\u003e for xylitol production using H\u003csub\u003e2\u003c/sub\u003e as the electron donor. To achieve this, XR from \u003cem\u003eScheffersomyces stipitis\u003c/em\u003e (formerly \u003cem\u003ePichia stipitis\u003c/em\u003e) was expressed in \u003cem\u003eC. necator\u003c/em\u003e to allow the cells to convert D-xylose into xylitol. The native SH of \u003cem\u003eC. necator\u003c/em\u003e enabled the H\u003csub\u003e2\u003c/sub\u003e-driven cofactor recycling required for bioconversion. Resting (i.e., non-dividing) cells were used in the experiments. These viable cells exhibit reduced metabolic activity, which allows energy to be directed towards bioconversion instead of biomass accumulation (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). With the experimental set-up used, we achieved nearly full quantitative conversions.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStrains and culture media\u003c/h2\u003e \u003cp\u003eBacterial strains and plasmids used in this work are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and the primers in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e (see Additional file 1). \u003cem\u003eC. necator\u003c/em\u003e H16 strains were grown in either rich BD BBL\u0026trade; Trypticase\u0026trade; Soy Broth (TSB) or minimal AUT medium (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) supplemented with 2 g/L fructose and glycerol (FG). Compared to the original AUT medium, the amount of NiCl\u003csub\u003e2\u003c/sub\u003e was doubled and SL-6 trace elements were added (1:1000) (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). \u003cem\u003eEscherichia coli\u003c/em\u003e DH10β was used for plasmid construction. Tetracycline was added at a concentration of 10 \u0026micro;g/mL for \u003cem\u003eE. coli\u003c/em\u003e and 5 \u0026micro;g/mL for \u003cem\u003eC. necator\u003c/em\u003e when required. The sugars and sugar alcohols used in the study were purchased from Sigma-Aldrich.\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\u003eBacterial strains and plasmids used in the study. The strains with △\u003cem\u003ephaC\u003c/em\u003e deletion are unable to produce polyhydroxybutyrate (PHB) for carbon and energy storage.\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\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRelevant characteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSource or reference\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\u003eC. necator\u003c/em\u003e H16 △\u003cem\u003ephaCAB\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e△\u003cem\u003ephaC1\u003c/em\u003e △\u003cem\u003ephaA1\u003c/em\u003e △\u003cem\u003ephaB1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(27)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eC. necator\u003c/em\u003e H16 △\u003cem\u003ephaC1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e△\u003cem\u003ephaC1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(28)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eC. necator\u003c/em\u003e H16 △\u003cem\u003eA0006\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e△\u003cem\u003ephaC1\u003c/em\u003e △\u003cem\u003eA0006\u003c/em\u003e\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\u003e\u003cem\u003eC. necator\u003c/em\u003e H16 △\u003cem\u003ephaCAB_\u003c/em\u003egfp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e△\u003cem\u003ephaCAB\u003c/em\u003e derivative, pPj5:GFP\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\u003e\u003cem\u003eC. necator\u003c/em\u003e H16 △\u003cem\u003ephaC_\u003c/em\u003egfp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e△\u003cem\u003eA0006\u003c/em\u003e derivative, pPj5:GFP\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\u003e\u003cem\u003eC. necator\u003c/em\u003e H16 △\u003cem\u003ephaCAB_\u003c/em\u003exr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e△\u003cem\u003ephaCAB\u003c/em\u003e derivative, pPj5:XRsti\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\u003e\u003cem\u003eC. necator\u003c/em\u003e H16 △\u003cem\u003ephaC_\u003c/em\u003exr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e△\u003cem\u003eA0006\u003c/em\u003e derivative, pPj5:XRsti\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\u003e\u003cb\u003ePlasmid\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epLO3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSuicide vector, sacB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(29)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epLO3-\u003cem\u003eA0006\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epLO3 derivative with 1100 bp upstream and downstream regions of \u003cem\u003eA0006\u003c/em\u003e\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\u003epPj5:GFP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epSEVA521 containing Pj5 promoter and GFP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(30)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epPj5:XRsti\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epPj5:GFP derivative, GFP replaced with D-xylose reductase from \u003cem\u003eScheffersomyces stipitis\u003c/em\u003e (XRsti)\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 \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eStrain construction\u003c/h2\u003e \u003cp\u003eXR from \u003cem\u003eScheffersomyces\u003c/em\u003e (\u003cem\u003ePichia\u003c/em\u003e) \u003cem\u003estipitis\u003c/em\u003e was ordered codon-optimized for \u003cem\u003eC. necator\u003c/em\u003e from GenScript (Additional file 1: Table S2) and PCR amplified with Q5 High-Fidelity 2X Master Mix (NEB). The backbone plasmid pSEVA521\u0026thinsp;+\u0026thinsp;Pj5:GFP and the amplified insert were digested with \u003cem\u003eSpe\u003c/em\u003eI-HF and \u003cem\u003eHin\u003c/em\u003edIII-HF (NEB) and ligated using T4 DNA ligase (NEB) to gain pPj5:XRsti. The verified plasmid was transformed into electrocompetent \u003cem\u003eC. necator\u003c/em\u003e cells. For preparing competent cells, \u003cem\u003eC. necator\u003c/em\u003e was grown in 100 mL of TSB supplemented with 20 mM of fructose to an optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) of 0.6 and washed twice with 1 mM MgSO\u003csub\u003e4\u003c/sub\u003e. The pellet was resuspended into 2 mL of 1 mM MgSO4 and 1 mL of 60% glycerol. Aliquots (50 \u0026micro;L) were stored in -80\u0026deg;C. Cells were mixed with plasmids (250 ng) in a 0.2 cm electroporation cuvette (Bio-Rad), incubated 10 min on ice and electroporated with Electro Cell Manipulator ECM\u0026reg;630 (BTX) with the following settings: 2.5 kV, 200Ω and 25\u0026micro;F. Super Optimal Broth with 20 mM fructose (950 \u0026micro;L) was added immediately after electroporation and cells were incubated at 30\u0026deg;C and 180 rpm for 2\u0026ndash;3 hours before plating on BBL\u0026trade; Trypticase\u0026trade; Soy Agar (TSA).\u003c/p\u003e \u003cp\u003e \u003cem\u003eC. necator\u003c/em\u003e H16 △\u003cem\u003eA0006\u003c/em\u003e was constructed from \u003cem\u003eC. necator\u003c/em\u003e H16 △\u003cem\u003ephaC1\u003c/em\u003e by deleting \u003cem\u003eA0006\u003c/em\u003e with pLO3-based suicide vector as previously described (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) (Additional file 1: Table S3). The △\u003cem\u003eA0006\u003c/em\u003e restriction enzyme knockout increases electroporation efficiency (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e) and is not expected to have any metabolic effects.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eXR activity assay\u003c/h2\u003e \u003cp\u003eFor the XR activity assay, cells were grown overnight in TSB and then harvested by centrifugation (10 min, 2800 g). The pellet was washed once with 50 mM potassium phosphate buffer (pH 7.5), resuspended into 1 mL of the same buffer supplemented with cOmplete protease inhibitor (Roche) and moved into a 2 mL screw-cap tube with 400 \u0026micro;L of 0.5 mm diameter glass beads. The cells were disrupted with FASTPREP-24 5G (MP Biomedicals) for 2x30sec at 6 m/s speed. After disruption, the tube was centrifuged for 10 min at 16000 g, and the supernatant (soluble extract) was collected. Total protein concentration was analyzed from the soluble extract by Quick Start\u0026trade; Bradford Protein Assay (Bio-Rad) using bovine serum albumin as the standard. XR activity assays were conducted in 96-well plates. The reaction mixture (330 \u0026micro;L) contained 0.15 mM of cofactor (NADH or NADPH), 50 mM potassium phosphate buffer (pH 6.0), 200 mM D-xylose and an appropriate amount of soluble extract. Absorbance was measured at 340 mM using Epoch 2 Microplate Spectrophotometer (BioTek). One unit of xylose reductase activity was defined as \u0026micro;mol of NAD(P)H oxidized per minute. Specific activities were expressed as units per milligram of total protein. The results are given as averages of triplicate assays.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eBioconversions with resting cells\u003c/h2\u003e \u003cp\u003ePrecultures were grown overnight in TSB media. FG media with tetracycline was inoculated to an initial OD\u003csub\u003e600\u003c/sub\u003e of 0.1 and grown for three days, reaching a final OD\u003csub\u003e600\u003c/sub\u003e of approximately 4. The culture was centrifuged for 10 min at 2800 g and washed twice with 100 mM sodium phosphate buffer (pH 7.0) to remove carbon and nitrogen sources. The cells were resuspended in the same buffer with 30 g/L of substrate to an OD\u003csub\u003e600\u003c/sub\u003e of 13\u0026ndash;17, if not mentioned otherwise. This range of OD\u003csub\u003e600\u003c/sub\u003e corresponds approximately to a cell dry weight of 4.4\u0026ndash;5.4 g/L. The prepared cell suspension (5 mL) was transferred into an anaerobic serum bottle with a rubber stopper. D-xylose was the primary substrate, but one bioconversion was also performed using L-arabinose and another using D-ribose. Three replicate bottles per bioconversion condition were prepared.\u003c/p\u003e \u003cp\u003eFor the first bioconversion experiments, 100 mL serum bottles were filled with H\u003csub\u003e2\u003c/sub\u003e using vacuum-gas cycles to reach specific H\u003csub\u003e2\u003c/sub\u003e and oxygen (O\u003csub\u003e2\u003c/sub\u003e) concentrations. As negative controls, bioconversions were performed under ambient air. The bottles were incubated at 30\u0026deg;C and 150 rpm. Samples (200 \u0026micro;L) were taken by opening the rubber stopper and the bottles were refilled with gasses after sampling. For bioconversion optimization, 50 mL serum bottles were filled with H\u003csub\u003e2\u003c/sub\u003e at the start of the bioconversion by flushing with 100% H\u003csub\u003e2\u003c/sub\u003e at 0.5 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for 2 minutes. Samples (200 \u0026micro;L) were taken using a needle and syringe through the rubber stopper of sealed bottles. The approximate H\u003csub\u003e2\u003c/sub\u003e gas consumption was measured after bioconversions by filling a 50 mL syringe with air and recording the volume of air aspirated into the bottle through the syringe needle. Samples were centrifuged for 10 min at 16000 g and the supernatants were analyzed for sugars and sugar alcohols.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCalculation of H\u003csub\u003e2\u003c/sub\u003e consumption\u003c/h2\u003e \u003cp\u003eThe amount of H\u003csub\u003e2\u003c/sub\u003e consumed was calculated from the approximated H\u003csub\u003e2\u003c/sub\u003e gas consumption (Eq.\u0026nbsp;\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e):\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:n\\:\\left({\\text{H}}_{2}\\:\\text{c}\\text{o}\\text{n}\\text{s}\\text{u}\\text{m}\\text{e}\\text{d}\\right)=\\frac{pV\\:}{RT}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003ep\u003c/em\u003e is the pressure (0.98692 atm), \u003cem\u003eV\u003c/em\u003e is the gas volume consumed (L), \u003cem\u003eR\u003c/em\u003e is the gas constant (0.08206 L atm mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e K\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and \u003cem\u003eT\u003c/em\u003e is the temperature (298.15 K).\u003c/p\u003e \u003cp\u003eOne mole of H\u003csub\u003e2\u003c/sub\u003e is needed to reduce one mole of xylose (Eq.\u0026nbsp;\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e):\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:{\\text{H}}_{2}+{\\text{C}}_{5}{\\text{H}}_{10}{\\text{O}}_{5}={\\text{C}}_{5}{\\text{H}}_{12}{\\text{O}}_{5}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThe total amount of xylitol produced was calculated as the sum of the xylitol at the end of the bioconversion and the xylitol in the fractions taken out during sampling. The ratio of xylitol production to H\u003csub\u003e2\u003c/sub\u003e consumption was calculated to determine the proportion of energy and protons transferred from H\u003csub\u003e2\u003c/sub\u003e to xylitol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of sugars and sugar alcohols\u003c/h2\u003e \u003cp\u003eTwo different high performance liquid chromatography (HPLC) systems were used for D-xylose and xylitol analysis: Prominence-i LC-2030C (Shimadzu) equipped with Hi-Plex H 7.7x300 mm column (Agilent) at 45\u0026deg;C and 10 mM H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e as eluent at a flow rate of 1 ml min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and Vanquish Flex (Thermo Fisher Scientific) equipped with Aminex Fast Acid Analysis and HPX-87H columns (Bio-Rad) at 55\u0026deg;C and 2 mM H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e as the eluent at a flow rate of 0.5 ml min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. An injection volume of 10 \u0026micro;L was used in both HPLCs and the compounds were detected with a refractive index detector. L-arabinose, L-arabitol, D-ribose and ribitol were analyzed with high pressure ion chromatography (HPIC) Dionex ICS-6000 (Thermo Fisher Scientific) with CarboPac PA20 column (Thermo Fisher Scientific) at 30\u0026deg;C and 10 mM KOH as the eluent at a flow rate of 0.5 ml min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. An injection volume of 2.5 \u0026micro;L was used and the compounds were detected with an electrochemical detector. Conversion yields at specific time points were calculated by dividing the amount of xylitol by the total amount of xylose and xylitol.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eXylose reductase from\u003c/b\u003e \u003cb\u003eS. stipitis\u003c/b\u003e \u003cb\u003eis functionally produced in\u003c/b\u003e \u003cb\u003eC. necator\u003c/b\u003e \u003cb\u003eH16\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn this study, two \u003cem\u003eC. necator\u003c/em\u003e strains, △\u003cem\u003ephaC\u003c/em\u003e and △\u003cem\u003ephaCAB\u003c/em\u003e, with partial or full knockouts of the native pathway for storage polymer polyhydroxybutyrate (PHB) formation, were used to avoid the accumulation of this by-product. Before constructing the XR expressing strains, it was confirmed that the host strains cannot grow on C5 sugars and sugar alcohols used in the study (Additional file 1: Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Both strains were then transformed with the plasmids pPj5:XR, carrying the codon-optimized XR from \u003cem\u003eS. stipitis\u003c/em\u003e, and pPj5:GFP, as a negative control. Strains were cultivated heterotrophically, and their soluble extracts were tested for XR activity. The soluble extracts of both XR strains showed reductase activity with both NADPH and NADH cofactors, whereas no activity was detected in the negative control strains (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Codon-optimized XR from \u003cem\u003eCandida parapsilosis\u003c/em\u003e was also expressed in the host strains, but no activity was detected (data not shown).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSpecific D-xylose reductase activities of soluble extracts with NADPH or NADH as cofactors (U/mg). The average from three measurements is shown with the standard deviation. No activity could be detected in the negative controls.\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=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNADPH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNADH\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\u003ephaCAB_\u003c/em\u003exr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e△\u003cem\u003ephaC_\u003c/em\u003exr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eXylose is fully converted to xylitol by\u003c/b\u003e \u003cb\u003eC. necator\u003c/b\u003e \u003cb\u003e△\u003c/b\u003e\u003cb\u003ephaCAB\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe first bioconversion experiment was performed at 30 g/L D-xylose by both \u003cem\u003eC. necator\u003c/em\u003e strains: △\u003cem\u003ephaCAB_\u003c/em\u003exr and △\u003cem\u003ephaC_\u003c/em\u003exr. The suspensions were incubated under H\u003csub\u003e2\u003c/sub\u003e (85% H\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;15% air) or 100% ambient air. Under H\u003csub\u003e2\u003c/sub\u003e, the △\u003cem\u003ephaCAB_\u003c/em\u003exr strain reached complete bioconversion to xylitol within 16 days, whereas the △\u003cem\u003ephaC_\u003c/em\u003exr strain converted 78% of the provided D-xylose at the same time (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Therefore, further bioconversions were performed using the △\u003cem\u003ephaCAB_\u003c/em\u003exr strain. D-xylose was also converted to xylitol in the absence of H\u003csub\u003e2\u003c/sub\u003e by both strains, but the conversion yields were under 25% after 16 days. This demonstrated successful cofactor recycling in resting cells of \u003cem\u003eC. necator\u003c/em\u003e using H\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eThe oxygen concentration does not influence the bioconversion\u003c/h2\u003e \u003cp\u003eSince oxygen, acting as an electron acceptor, can provide the cells with energy via oxidative phosphorylation, we examined the effect of oxygen concentration on the conversion rate. Three different oxygen concentrations (0, 1, and 4%) were tested. The conversion rates and yields showed little variation between the different oxygen concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), with over 86% conversion yields being reached within 10 days in all conditions. After confirming that oxygen was not a limiting factor, it was tested whether the amount of H\u003csub\u003e2\u003c/sub\u003e would be. Bioconversions with multiple H\u003csub\u003e2\u003c/sub\u003e flushes during the experiment were compared to bioconversions with a single H\u003csub\u003e2\u003c/sub\u003e flush at the start. The results showed that multiple H\u003csub\u003e2\u003c/sub\u003e flushes failed to improve the conversion yield (Additional file 1: Fig. S2). Therefore, further bioconversions were conducted with only an initial H\u003csub\u003e2\u003c/sub\u003e flush.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHigher initial sugar concentration can speed up the bioconversion rate\u003c/h2\u003e \u003cp\u003eFour different D-xylose concentrations were tested to evaluate their impact on xylitol production rates. With increasing xylose concentration, the rate of conversion increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The highest xylitol productivity in the first 48 hours (0.7 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was reached with the highest xylose concentration used (114 g/L D-xylose). However, complete conversion was reached faster at a lower xylose concentration: 13 g/L of D-xylose was fully converted to xylitol in 10 days while 85% of 34 g/L D-xylose was converted within the same time.\u003c/p\u003e \u003cp\u003eIn samples with the highest xylose concentrations (66 and 114 g/L), the final xylitol concentration reached 46 g/L. We hypothesized that H\u003csub\u003e2\u003c/sub\u003e in the headspace was limiting and that measuring H\u003csub\u003e2\u003c/sub\u003e consumption could allow us to estimate the electron conversion efficiency of hydrogen into xylitol. On average, 30 mL of gas was consumed under both conditions, equivalent to 1.2 mmol of H\u003csub\u003e2\u003c/sub\u003e. Given that 0.2 mmol of xylitol was produced without H\u003csub\u003e2\u003c/sub\u003e (as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and 1.3 mmol was produced in total, it can be assumed that 1.1 mmol of xylitol was produced with the help of H\u003csub\u003e2\u003c/sub\u003e under both conditions. Consequently, more than 90% of the energy derived from H\u003csub\u003e2\u003c/sub\u003e was spent for the bioconversion, as one mole of H\u003csub\u003e2\u003c/sub\u003e is required to reduce one mole of xylose.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eIncreased cell concentration enhances the rate of bioconversion\u003c/h2\u003e \u003cp\u003eSo far, all bioconversions in this study were conducted with a cell concentration range of OD\u003csub\u003e600\u003c/sub\u003e 13\u0026ndash;17. The effect of the amount of the whole-cell biocatalyst was examined at lower and higher cell concentrations: OD\u003csub\u003e600\u003c/sub\u003e 7 and 60. With the highest cell concentration, full conversion of 30 g/L D-xylose to xylitol was achieved in 7 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Increasing the cell concentration had a positive effect also on the xylitol production rate. In the first 24 hours, the production rates were 0.5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 0.1 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for the highest and lowest cell concentrations used, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eArabinose and ribose are reduced to sugar alcohols by the resting cells\u003c/h2\u003e \u003cp\u003e \u003cem\u003eS. stipitis\u003c/em\u003e XR is also known to convert L-arabinose and D-ribose into their respective sugar alcohols (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Therefore, we examined whether \u003cem\u003eC. necator\u003c/em\u003e harboring the xylose reductase could also be used as a biocatalyst for these conversions. Both sugars were successfully reduced to their corresponding sugar alcohols, with the production rates and yields of L-arabitol and ribitol being only slightly lower than those for xylitol (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study aimed to advance the development of \u003cem\u003eC. necator\u003c/em\u003e as a H\u003csub\u003e2\u003c/sub\u003e-driven whole-cell biocatalyst. We demonstrated a full reduction of 30 g/L of D-xylose to xylitol in resting cells using H\u003csub\u003e2\u003c/sub\u003e for cofactor regeneration. The effects of different parameters on xylitol production were studied to improve the production rates. Up to 30 g/L of xylitol was produced in \u003cem\u003eC. necator\u003c/em\u003e △\u003cem\u003ephaCAB_xr\u003c/em\u003e within 7 days and 100% conversion yield (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Additionally, it was shown that the system can also convert L-arabinose and D-ribose into their respective sugar alcohols (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eC. necator\u003c/em\u003e accumulates polyhydroxybutyrate (PHB) as a carbon and energy storage compound. To avoid this accumulation, which could also lead to cofactor oxidation, PHB-negative strains △\u003cem\u003ephaCAB\u003c/em\u003e and △\u003cem\u003ephaC\u003c/em\u003e of \u003cem\u003eC. necator\u003c/em\u003e were used as hosts. Comparison of the △\u003cem\u003ephaCAB_\u003c/em\u003exr \u003cem\u003eand\u003c/em\u003e △\u003cem\u003ephaC_\u003c/em\u003exr strains revealed that deletion of the whole PHB pathway (\u003cem\u003ephaCAB\u003c/em\u003e) enhanced both the bioconversion rate and yield (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In the △\u003cem\u003ephaC\u003c/em\u003e knock-out strain, it is possible that some of the reducing equivalents from H\u003csub\u003e2\u003c/sub\u003e were consumed by the NAD(P)H-utilizing acetoacetyl-CoA reductase (PhaB) of the PHB pathway. The advantage of knocking out more than just the \u003cem\u003ephaC\u003c/em\u003e gene has also been observed in earlier studies in non-resting cells of \u003cem\u003eC. necator\u003c/em\u003e. For instance, complete deletion of the PHB pathway was found to be beneficial for resveratrol production in \u003cem\u003eC. necator\u003c/em\u003e, whereas deletion of only the \u003cem\u003ephaC\u003c/em\u003e gene did not improve the titer compared to the wild type (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOxygen is essential for ATP production from H\u003csub\u003e2\u003c/sub\u003e in \u003cem\u003eC. necator\u003c/em\u003e. Although the bioconversion reaction itself does not require ATP, it was hypothesized that the cells would require some ATP for cell maintenance. However, the results suggest otherwise (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This outcome is advantageous for industrial applications, as oxygen-free production mitigates the risk associated with flammability of H\u003csub\u003e2\u003c/sub\u003e-O\u003csub\u003e2\u003c/sub\u003e mixtures. The experiments also demonstrated efficient transfer of nearly all hydrogen-derived electrons into the product. When enough H\u003csub\u003e2\u003c/sub\u003e was present in the headspace, full bioconversion of xylose could be demonstrated. However, liquid solubility of H\u003csub\u003e2\u003c/sub\u003e is low and hence may still limit the rate of conversion. To test this, bioconversions could be performed under elevated pressure, where H\u003csub\u003e2\u003c/sub\u003e solubility is increased, but unfortunately it was not possible to test this with the current experimental set-up. Additionally, mass transfer of H\u003csub\u003e2\u003c/sub\u003e to the liquid phase can be significantly improved by using optimized bioreactors equipped with specialized gas spargers and impellers (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe most significant improvements in the bioconversion rate were achieved by increasing the cell concentration and xylose concentration. This is not surprising, as a higher cell concentration provides more catalyst for the conversion to occur, and an increased substrate concentration boosts the reaction rate until enzyme saturation is reached. The most efficient xylitol production systems reported in the literature have reached higher xylitol productivity than the 0.7 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e achieved in this study. At a similar cell density, recombinant \u003cem\u003eE. coli\u003c/em\u003e cells, coexpressing a D-xylose reductase and a glucose dehydrogenase, produced xylitol at 6.4 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. In this optimized process, a 100% yield was achieved at an initial D-xylose concentration of 200 g/L using glucose for cofactor recycling (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Factors that could account for this include lower enzyme activities, slower substrate and product transport into and outside of the cell, and particularly the aforementioned poor H\u003csub\u003e2\u003c/sub\u003e solubility. Observing the SDS-PAGE gel of Jin et al., it seems that their XR level in the cell was much higher than in this study (Additional file 1: Fig. S3). Although the strongest promoter currently known for \u003cem\u003eC. necator\u003c/em\u003e was used in the present study (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e), stronger expression of XR could improve the bioconversion rates. The expression systems for \u003cem\u003eC. necator\u003c/em\u003e need further development to achieve the expression levels obtained with \u003cem\u003eE. coli\u003c/em\u003e. On the other hand, the xylose and xylitol transport systems of \u003cem\u003eE. coli\u003c/em\u003e are likely more efficient than those of \u003cem\u003eC. necator\u003c/em\u003e because \u003cem\u003eE. coli\u003c/em\u003e can natively grow on xylose whereas \u003cem\u003eC. necator\u003c/em\u003e cannot. A BLAST search of the \u003cem\u003eC. necator\u003c/em\u003e H16 genome using the D-xylose specific transport systems of \u003cem\u003eE. coli\u003c/em\u003e (XylE and XylFGH) yielded no matches, suggesting that \u003cem\u003eC. necator\u003c/em\u003e lacks xylose-specific transporters. Xylose is likely transported into the cells by a sugar transporter with side activity for xylose. Hence, heterologous introduction of a xylose transporter could be considered for future studies to further improve bioconversion rates.\u003c/p\u003e \u003cp\u003eThe specific activity of XR with NADPH was higher compared to NADH (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The same result has been observed previously by Verduyn, Van Kleef (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Using NADPH-producing SH, instead of the native NADH-producing SH, with NADPH-dependent oxidoreductases could increase the rate of the bioconversion. The NAD\u003csup\u003e+\u003c/sup\u003e-specific SH from \u003cem\u003eC. necator\u003c/em\u003e has been engineered to also accept NADP\u003csup\u003e+\u003c/sup\u003e, but its NADP\u003csup\u003e+\u003c/sup\u003e-reducing activity would need to be increased (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Another option is to use NADH-preferring oxidoreductases or to engineer them to have this preference, ensuring high activity towards NADH.\u003c/p\u003e \u003cp\u003eThis article presented the first whole-cell, H\u003csub\u003e2\u003c/sub\u003e-driven biocatalysis study using a PHB-negative \u003cem\u003eC. necator\u003c/em\u003e strain as the host. Direct comparison of this work to the few prior studies is challenging due to differences in experimental setups, product types, enzyme kinetics, and strains used. Oda et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) reported a productivity of 0.9 g L\u003csup\u003e-1\u003c/sup\u003e h\u003csup\u003e-1\u003c/sup\u003e for (\u003cem\u003eR\u003c/em\u003e)-1,2-propanediol, which is within the same order of magnitude as our findings. Whole-cell cofactor recycling using SH has not only been done in \u003cem\u003eC. necator\u003c/em\u003e. Lonsdale et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) expressed SH from \u003cem\u003eC. necator\u003c/em\u003e in \u003cem\u003ePseudomonas putida\u003c/em\u003e to perform H\u003csub\u003e2\u003c/sub\u003e-driven bioconversion of n-octane to 1-octanol. The cofactor recycling proved to be effective, resulting in a threefold increase in 1-octanol production in the presence of H\u003csub\u003e2\u003c/sub\u003e. However, the yield and rate of bioconversions they achieved were significantly lower than the ones in this study; Lonsdale et al. reported a maximum productivity of 0.01 g L\u003csup\u003e-1\u003c/sup\u003e h\u003csup\u003e-1\u003c/sup\u003e, about 100-fold lower than the rates we and Oda et al. achieved.\u003c/p\u003e \u003cp\u003eThe limited amount of research in this area offers a wide range of opportunities for improving these organisms to perform H\u003csub\u003e2\u003c/sub\u003e-driven bioconversions towards industrial applications. Bioconversion rates can likely be significantly improved using hosts with improved enzyme activities, elevated pressures, higher cell concentrations and optimized bioreactor designs that enhance hydrogen solubility.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eCofactor recycling via hydrogenases represents a promising alternative for traditional bioconversion systems because of its atom efficiency, lack of by-products and the prospects of H\u003csub\u003e2\u003c/sub\u003e becoming a renewable platform chemical of the future. This study demonstrated H\u003csub\u003e2\u003c/sub\u003e-driven bioconversion of D-xylose to xylitol in XR expressing \u003cem\u003eC. necator\u003c/em\u003e strain. 30 g/L of D-xylose was fully converted into xylitol in 7 days. It was shown that nearly all the energy from H\u003csub\u003e2\u003c/sub\u003e is harnessed by the bioconversion, demonstrating the potential of the \u003cem\u003eC. necator\u003c/em\u003e system as an efficient H\u003csub\u003e2\u003c/sub\u003e-driven biocatalyst for sugar alcohol production and potentially other products.\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the Research Council of Finland (KNALLRED\u0026mdash;Hydrogen powered reductive biosyntheses and biotransformations by an engineered Knallgas bacterium, grant number 342124).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors designed research. TJ conducted experiments, data analysis, and wrote the manuscript. All authors revised and approved the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thank Enrico Orsi for the C. necator △phaCAB strain, Guillermo Bordanaba Florit for constructing the C. necator H16 △A0006 strain, Victor de Lorenzo\u0026rsquo;s lab for the SEVA plasmid, and Ton van Gelder for the help with HPLC analyses. We also thank Solar Foods, especially Juha-Pekka Pitk\u0026auml;nen, for the fruitful discussions and the bioeconomy research infrastructures of Aalto University for the support.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWang X, Saba T, Yiu HHP, Howe RF, Anderson JA, Shi J. Cofactor NAD(P)H Regeneration Inspired by Heterogeneous Pathways. 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Chem Commun. 2015. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/c5cc06078h\u003c/span\u003e\u003cspan address=\"10.1039/c5cc06078h\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Biotransformation, cofactor recycling, cofactor regeneration, Ralstonia eutropha, Cupriavidus necator, hydrogen-oxidizing bacteria","lastPublishedDoi":"10.21203/rs.3.rs-5062650/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5062650/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Biocatalysis offers a potentially greener alternative to chemical processes. For biocatalytic systems requiring cofactor recycling, hydrogen emerges as an attractive reducing agent. Hydrogen is attractive because all the electrons can be fully transferred to the product, and it can be efficiently produced from water using renewable electricity. In this article, resting cells of \u003cem\u003eCupriavidus necator\u003c/em\u003e H16 harboring a NAD-dependent hydrogenase were employed for cofactor recycling to reduce D-xylose to xylitol, a commonly used sweetener. To enable this bioconversion, D-xylose reductase from \u003cem\u003eScheffersomyces stipitis \u003c/em\u003ewas heterologously expressed in \u003cem\u003eC. necator\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: D-xylose reductase was successfully expressed in \u003cem\u003eC. necator\u003c/em\u003e, enabling complete bioconversion of 30 g/L of D-xylose into xylitol within 7 days using resting cells. It was found that over 90% of the energy and protons derived from hydrogen were spent for the bioconversion, demonstrating the efficiency of the system. The highest xylitol productivity reached was 0.7 g L\u003csup\u003e-1\u003c/sup\u003e h\u003csup\u003e-1\u003c/sup\u003e. Additionally, the same chassis efficiently produced L-arabitol and D-ribitol from L-arabinose and D-ribose, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: This study highlights the efficient utilization of renewable hydrogen as a reducing agent to power cofactor recycling. Hydrogen-oxidizing bacteria, such as \u003cem\u003eC. necator\u003c/em\u003e, can be promising hosts for performing hydrogen-driven biocatalysis.\u003c/p\u003e","manuscriptTitle":"H2-driven xylitol production in Cupriavidus necator H16","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-03 12:36:00","doi":"10.21203/rs.3.rs-5062650/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-15T02:02:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-12T06:32:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"222991637601415222749333976722771275812","date":"2024-11-05T06:29:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-21T03:21:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"254807015586620045755981238697590338800","date":"2024-09-14T07:37:07+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-14T06:12:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-09-12T15:15:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-09-12T15:11:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbial Cell Factories","date":"2024-09-10T07:33:13+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3e448ff9-0d6d-4ad5-9f1a-af13483ac564","owner":[],"postedDate":"December 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-30T16:00:46+00:00","versionOfRecord":{"articleIdentity":"rs-5062650","link":"https://doi.org/10.1186/s12934-024-02615-7","journal":{"identity":"microbial-cell-factories","isVorOnly":false,"title":"Microbial Cell Factories"},"publishedOn":"2024-12-23 15:57:21","publishedOnDateReadable":"December 23rd, 2024"},"versionCreatedAt":"2024-12-03 12:36:00","video":"","vorDoi":"10.1186/s12934-024-02615-7","vorDoiUrl":"https://doi.org/10.1186/s12934-024-02615-7","workflowStages":[]},"version":"v1","identity":"rs-5062650","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5062650","identity":"rs-5062650","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}