Carbon dioxide valorization into resveratrol via lithoautotrophic fermentation using engineered Cupriavidus necator H16.

BACKGROUND: Industrial biomanufacturing of value-added products using CO2 as a carbon source is considered more sustainable, cost-effective and resource-efficient than using common carbohydrate feedstocks. Cupriavidus necator H16 is a representative H2-oxidizing lithoautotrophic bacterium that can be utilized to valorize CO2 into valuable chemicals and has recently gained much attention as a promising platform host for versatile C1-based biomanufacturing. Since this microbial platform is genetically tractable and has a high-flux carbon storage pathway, it has been engineered to produce a variety of valuable compounds from renewable carbon sources. In this study, the bacterium was engineered to produce resveratrol autotrophically using an artificial phenylpropanoid pathway. RESULTS: The heterologous genes involved in the resveratrol biosynthetic pathway-tyrosine ammonia lyase (TAL), 4-coumaroyl CoA ligase (4CL), and stilbene synthase (STS) -were implemented in C. necator H16. The overexpression of acetyl-CoA carboxylase (ACC), disruption of the PHB synthetic pathway, and an increase in the copy number of STS genes enhanced resveratrol production. In particular, the increased copies of VvSTS derived from Vitis vinifera resulted a 2-fold improvement in resveratrol synthesis from fructose. The final engineered CR-5 strain produced 1.9 mg/L of resveratrol from CO2 and tyrosine via lithoautotrophic fermentation. CONCLUSIONS: To the best of our knowledge, this study is the first to describe the valorization of CO2 into polyphenolic compounds by engineering a phenylpropanoid pathway using the lithoautotrophic bacterium C. necator H16, demonstrating the potential of this strain a platform for sustainable chemical production.

Biotechnological Plastic Degradation and Valorization Using Systems Metabolic Engineering.

Various kinds of plastics have been developed over the past century, vastly improving the quality of life. However, the indiscriminate production and irresponsible management of plastics have led to the accumulation of plastic waste, emerging as a pressing environmental concern. To establish a clean and sustainable plastic economy, plastic recycling becomes imperative to mitigate resource depletion and replace non-eco-friendly processes, such as incineration. Although chemical and mechanical recycling technologies exist, the prevalence of composite plastics in product manufacturing complicates recycling efforts. In recent years, the biodegradation of plastics using enzymes and microorganisms has been reported, opening a new possibility for biotechnological plastic degradation and bio-upcycling. This review provides an overview of microbial strains capable of degrading various plastics, highlighting key enzymes and their role. In addition, recent advances in plastic waste valorization technology based on systems metabolic engineering are explored in detail. Finally, future perspectives on systems metabolic engineering strategies to develop a circular plastic bioeconomy are discussed.

Engineering Cupriavidus necator H16 for enhanced lithoautotrophic poly(3-hydroxybutyrate) production from CO2.

BACKGROUND: A representative hydrogen-oxidizing bacterium Cupriavidus necator H16 has attracted much attention as hosts to recycle carbon dioxide (CO2) into a biodegradable polymer, poly(R)-3-hydroxybutyrate (PHB). Although C. necator H16 has been used as a model PHB producer, the PHB production rate from CO2 is still too low for commercialization. RESULTS: Here, we engineer the carbon fixation metabolism to improve CO2 utilization and increase PHB production. We explore the possibilities to enhance the lithoautotrophic cell growth and PHB production by introducing additional copies of transcriptional regulators involved in Calvin Benson Bassham (CBB) cycle. Both cbbR and regA-overexpressing strains showed the positive phenotypes for 11% increased biomass accumulation and 28% increased PHB production. The transcriptional changes of key genes involved in CO2-fixing metabolism and PHB production were investigated. CONCLUSIONS: The global transcriptional regulator RegA plays an important role in the regulation of carbon fixation and shows the possibility to improve autotrophic cell growth and PHB accumulation by increasing its expression level. This work represents another step forward in better understanding and improving the lithoautotrophic PHB production by C. necator H16.

Improving Lipid Production of Yarrowia lipolytica by the Aldehyde Dehydrogenase-Mediated Furfural Detoxification.

Yarrowia lipolytica, the non-conventional yeast capable of high lipogenesis, is a microbial chassis for producing lipid-based biofuels and chemicals from renewable resources such as lignocellulosic biomass. However, the low tolerance of Y. lipolytica against furfural, a major inhibitory furan aldehyde derived from the pretreatment processes of lignocellulosic biomass, has restricted the efficient conversion of lignocellulosic hydrolysates. In this study, the furfural tolerance of Y. lipolytica has been improved by supporting its endogenous detoxification mechanism. Specifically, the endogenous genes encoding the aldehyde dehydrogenase family proteins were overexpressed in Y. lipolytica to support the conversion of furfural to furoic acid. Among them, YALI0E15400p (FALDH2) has shown the highest conversion rate of furfural to furoic acid and resulted in two-fold increased cell growth and lipid production in the presence of 0.4 g/L of furfural. To our knowledge, this is the first report to identify the native furfural detoxification mechanism and increase furfural resistance through rational engineering in Y. lipolytica. Overall, these results will improve the potential of Y. lipolytica to produce lipids and other value-added chemicals from a carbon-neutral feedstock of lignocellulosic biomass.

Xylose utilization in Saccharomyces cerevisiae during conversion of hydrothermally pretreated lignocellulosic biomass to ethanol.

With growing interest in alternative fuels to minimize carbon and particle emissions, research continues on the production of lignocellulosic ethanol and on the development of suitable yeast strains. However, great diversities and continued technical advances in pretreatment methods for lignocellulosic biomass complicate the evaluation of developed yeast strains, and strain development often lags industrial applicability. In this review, recent studies demonstrating developed yeast strains with lignocellulosic biomass hydrolysates are compared. For the pretreatment methods, we highlight hydrothermal pretreatments (dilute acid treatment and autohydrolysis), which are the most commonly used and effective methods for lignocellulosic biomass pretreatment. Rather than pretreatment conditions, the type of biomass most strongly influences the composition of the hydrolysates. Metabolic engineering strategies for yeast strain development, the choice of xylose-metabolic pathway, adaptive evolution, and strain background are highlighted as important factors affecting ethanol yield and productivity from lignocellulosic biomass hydrolysates. A comparison of the parameters from recent studies demonstrating lignocellulosic ethanol production provides useful information for future strain development.

Characterization of the biochemical properties of recombinant Xyn10C from a marine bacterium, Saccharophagus degradans 2-40.

Endo-1,4-ß-xylanases are mostly classified into glycoside hydrolase (GH) family 10 or 11. In this study, we examined the catalytic functions of a recombinant endo-1,4-ß-xylanase belonging to GH10 (Xyn10C) from a marine bacterium, Saccharophagus degradans 2-40. Optimal activity of this enzyme was evident at 30 °C and pH 7.0, but activity remained even at low temperatures, indicating its adaptation to cold. With respect to other xylanases known to be active in cold temperatures, Xyn10C is unique in that it showed maximal activity in the presence of 2 M of NaCl. The action patterns of recombinant Xyn10C on xylans from hardwood and softwood differed in part, but the enzyme hydrolyzed polysaccharidic substrates primarily to xylobiose and xylotriose through xylo-oligosaccharides, releasing a small amount of xylose. The K m and V max values on birchwood xylan were 10.4 mg mL(-1) and 253 µmol mg(-1) min(-1), respectively. The efficient catalytic function of Xyn10C on short-length xylo-oligosaccharide chains was similar to the typical function of other known GH10 xylanases.

Effect of liquid hot water pretreatment severity on properties of hardwood lignin and enzymatic hydrolysis of cellulose.

Lignin, one of the major components of lignocellulosic biomass, plays an inhibitory role on the enzymatic hydrolysis of cellulose. This work examines the role of lignin in pretreated hardwood, where extents of cellulose hydrolysis decrease, rather than increase with increasing severity of liquid hot water pretreatment. Hardwood pretreated with liquid hot water at severities ranging from log Ro = 8.25 to 12.51 resulted in 80-90% recovery of the initial lignin in the residual solids. The ratio of acid insoluble lignin (AIL) to acid soluble lignin (ASL) increased and the formation of spherical lignin droplets on the cell wall surface was observed as previously reported in the literature. When lignins were isolated from hardwoods pretreated at increasing severities and characterized based on glass transition temperature (Tg ), the Tg of isolated lignins was found to increase from 171 to 180°C as the severity increased from log Ro = 10.44 to 12.51. The increase in Tg suggested that the condensation reactions of lignin molecules occurred during pretreatment and altered the lignin structure. The contribution of the changes in lignin properties to enzymatic hydrolysis were examined by carrying out Avicel hydrolysis in the presence of isolated lignins. Lignins derived from more severely pretreated hardwoods had higher Tg values and showed more pronounced inhibition of enzymatic hydrolysis.

Adsorption of enzyme onto lignins of liquid hot water pretreated hardwoods.

The adsorption of cellulase enzymes onto lignin is shown to be non-productive and therefore reduces enzymatic hydrolysis of liquid hot water pretreated cellulose. Among the enzyme components of Trichoderma reesei cellulase cocktail, ß-glucosidase showed the strongest adsorption onto lignin. Only 2-18% of the initial ß-glucosidase activity remained in the supernatant while 50-60% of cellobiohydrolase and endoglucanase activities were recovered after incubation with lignin. By increasing the pH to 5.5 and adding NaCl to a 200 mM, the free enzymes in the supernatant were increased but hydrolysis was not enhanced since optimal pH for enzymatic hydrolysis is at 4.8. Electrostatic interactions contributed to enzyme adsorption and their effect was most pronounced for T. reesei ß-glucosidase which had high molecular weights (78-94 kDa) and high isoelectric points (pI 5.7-6.4). Since the enzyme components which are required to synergistically hydrolyze cellulose have different profiles (molecular weight, hydrophobicity and pI), they exhibit different adsorption behaviors with lignin, and thereby change the ratio of enzyme activities needed for synergism during cellulose hydrolysis. ß-glucosidase from Aspergillus niger exhibits less adsorption than ß-glucosidase from T. reesei. Supplemental addition of A. niger ß-glucosidase to the enzyme mixture increases hydrolysis of pretreated hardwood by a factor of two. The analysis presented in this paper shows that lignins with higher guaiacyl content adsorb more cellulase enzymes, particularly ß-glucosidase, and that adsorption of ß-glucosidase onto lignin indirectly suppresses enzymatic hydrolysis of cellulose in pretreated hardwoods due to decreased hydrolysis of cellobiose which in turn accumulates and inhibits CBH.

Hydrolysis-determining substrate characteristics in liquid hot water pretreated hardwood.

Fundamental characterization of pretreated hardwood and its interactions with cellulolytic enzymes has confirmed that a pathway exists for dramatically reducing the loading of cellulase required for hydrolysis of pretreated biomass. We demonstrate that addition of protein effecting a seven-fold decrease in the specific activity of cellulases enables a ten-fold reduction in enzyme loading while maintaining a high level of cellulose hydrolysis in pretreated hardwood. While use of protein and other additives that adsorb on lignin have been reported previously, the current work demonstrates the effect in a dramatic manner and brings the rationale for this change into clear focus. The key to this result is recognizing and mitigating the pretreatment conundrum where increasingly severe pretreatment conditions enhance accessibility of the enzymes not only to cellulose, but also to lignin. The lignin adsorbs enzyme protein causing loss of cellulase activity. More enzyme, added to compensate for this lost activity, results in a higher cellulase loading. The addition of a different protein, such as BSA, prevents cellulase adsorption on lignin and enables the enzyme itself to better target its glucan substrate. This effect dramatically reduces the amount of cellulase for a given level of conversion with enzyme loadings of 15 FPU and 1.3 FPU/g solids both achieving 80% conversion. The understanding of this phenomenon reinvigorates motivation for the search for other approaches that prevent cellulase adsorption on lignin in order to achieve high glucose yields at low enzyme loadings for pretreated lignocellulose.

Compounds inhibiting the bioconversion of hydrothermally pretreated lignocellulose.

Hydrothermal pretreatment using liquid hot water, steam explosion, or dilute acids enhances the enzymatic digestibility of cellulose by altering the chemical and/or physical structures of lignocellulosic biomass. However, compounds that inhibit both enzymes and microbial activity, including lignin-derived phenolics, soluble sugars, furan aldehydes, and weak acids, are also generated during pretreatment. Insoluble lignin, which predominantly remains within the pretreated solids, also acts as a significant inhibitor of cellulases during hydrolysis of cellulose. Exposed lignin, which is modified to be more recalcitrant to enzymes during pretreatment, adsorbs cellulase nonproductively and reduces the availability of active cellulase for hydrolysis of cellulose. Similarly, lignin-derived phenolics inhibit or deactivate cellulase and ß-glucosidase via irreversible binding or precipitation. Meanwhile, the performance of fermenting microorganisms is negatively affected by phenolics, sugar degradation products, and weak acids. This review describes the current knowledge regarding the contributions of inhibitors present in whole pretreatment slurries to the enzymatic hydrolysis of cellulose and fermentation. Furthermore, we discuss various biological strategies to mitigate the effects of these inhibitors on enzymatic and microbial activity to improve the lignocellulose-to-biofuel process robustness. While the inhibitory effect of lignin on enzymes can be relieved through the use of lignin blockers and by genetically engineering the structure of lignin or of cellulase itself, soluble inhibitors, including phenolics, furan aldehydes, and weak acids, can be detoxified by microorganisms or laccase.

Biocompatible Cu/NiMo Composite Electrocatalyst for Hydrogen Evolution Reaction in Microbial Electrosynthesis; Unveiling the Self-Detoxification Effect of Cu.

H2-driven microbial electrosynthesis (MES) is an emerging bioelectrochemical technology that enables the production of complex compounds from CO2. Although the performance of microbial fermentation in the MES system is closely related to the H2 production rate, high-performing metallic H2-evolving catalysts (HEC) generate cytotoxic H2O2 and metal cations from undesirable side reactions, severely damaging microorganisms. Herein, a novel design for self-detoxifying metallic HEC, resulting in biologically benign H2 production, is reported. Cu/NiMo composite HEC suppresses H2O2 evolution by altering the O2 reduction kinetics to a four-electron pathway and subsequently decomposes the inevitably generated H2O2 in sequential catalytic and electrochemical pathways. Furthermore, in situ generated Cu-rich layer at the surface prevents NiMo from corroding and releasing cytotoxic Ni cations. Consequently, the Cu/NiMo composite HEC in the MES system registers a 50% increase in the performance of lithoautotrophic bacterium Cupriavidus necator H16, for the conversion of CO2 to a biopolymer, poly(3-hydroxybutyrate). This work successfully demonstrates the concept of self-detoxification in designing biocompatible materials for bioelectrochemical applications as well as MES systems.

Tailored polyhydroxyalkanoate production from renewable non-fatty acid carbon sources using engineered Cupriavidus necator H16.

As thermoplastic, nontoxic, and biocompatible polyesters, polyhydroxyalkanoates (PHAs) are considered promising biodegradable plastic candidates for diverse applications. Short-chain-length/medium-chain-length (SCL/MCL) PHA copolymers are flexible and versatile PHAs that are typically produced from fatty acids, which are expensive and toxic. Therefore, to achieve the sustainable biosynthesis of SCL/MCL-PHAs from renewable non-fatty acid carbon sources (e.g., sugar or CO2), we used the lithoautotrophic bacterium Cupriavidus necator H16 as a microbial platform. Specifically, we synthesized tailored PHA copolymers with varying MCL-3-hydroxyalkanoate (3HA) compositions (10-70 mol%) from fructose by rewiring the MCL-3HA biosynthetic pathways, including (i) the thioesterase-mediated free fatty acid biosynthetic pathway coupled with the beta-oxidation cycle and (ii) the hydroxyacyl transferase-mediated fatty acid de novo biosynthetic pathway. In addition to sugar-based feedstocks, engineered strains are also promising platforms for the lithoautotrophic production of SCL/MCL-PHAs from CO2. The set of engineered C. necator strains developed in this study provides greater opportunities to produce customized polymers with controllable monomer compositions from renewable resources.

Biodegradation of oxidized low density polyethylene by Pelosinus fermentans lipase.

Polyethylene (PE) exhibits high resistance to degradation, contributing to plastic pollution. PE discarded into the environment is photo-oxidized by sunlight and oxygen. In this study, a key enzyme capable of degrading oxidized PE is reported for the first time. Twenty different enzymes from various lipase families were evaluated for hydrolytic activity using substrates mimicking oxidized PE. Among them, Pelosinus fermentans lipase 1 (PFL1) specifically cleaved the ester bonds within the oxidized carbon-carbon backbone. Moreover, PFL1 (6 µM) degraded oxidized PE film, reducing the weight average and number average molecular weights by 44.6 and 11.3 %, respectively, within five days. Finally, structural analysis and molecular docking simulations were performed to elucidate the degradation mechanism of PFL1. The oxidized PE-degrading enzyme reported here will provide the groundwork for advancing PE waste treatment technology and for engineering microbes to repurpose PE waste into valuable chemicals.

Effective hexanol production from carbon monoxide using extractive fermentation with Clostridium carboxidivorans P7.

This study achieved high production of hexanol via gas fermentation using Clostridium carboxidivorans P7 by extracting hexanol from the fermentation broth. The hexanol extraction efficiency and inhibitory effects on C. carboxidivorans P7 of 2-butyl-1-octanol, hexyl hexanoate and oleyl alcohol were examined, and oleyl alcohol was selected as the extraction solvent. Oleyl alcohol was added at the beginning of fermentation and during fermentation or a small volume of oleyl alcohol was repeatedly added during fermentation. The addition of a small volume of oleyl alcohol during fermentation was the most effective for CO consumption and hexanol production (5.06 g/L), yielding the highest known hexanol titer through any type of fermentation including gas fermentation. Hexanol production was further enhanced to 8.45 g/L with the repeated addition of oleyl alcohol and ethanol during gas fermentation. The results of this study will enable sustainable and carbon-neutral hexanol production via gas fermentation.

Enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with modulated 3-hydroxyvalerate fraction by overexpressing acetolactate synthase in Cupriavidus necator H16.

The elastomeric properties of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a biodegradable copolymer, strongly depend on the molar composition of 3-hydroxyvalerate (3HV). This paper reports an improved artificial pathway for enhancing the 3HV component during PHBV biosynthesis from a structurally unrelated carbon source by Cupriavidus necator H16. To increase the intracellular accumulation of propionyl-CoA, a key precursor of the 3HV monomer, we developed a recombinant strain by genetically manipulating the branched-chain amino acid (e.g., valine, isoleucine) pathways. Overexpression of the heterologous feedback-resistant acetolactate synthase (alsS), (R)-citramalate synthase (leuA), homologous 3-ketothiolase (bktB), and the deletion of 2-methylcitrate synthase (prpC) resulted in biosynthesis of 42.5 % (g PHBV/g dry cell weight) PHBV with 64.9 mol% 3HV monomer from fructose as the sole carbon source. This recombinant strain also accumulated the highest PHBV content of 54.5 % dry cell weight (DCW) with 24 mol% 3HV monomer from CO2 ever reported. The lithoautotrophic cell growth and PHBV production by the recombinant C. necator were promoted by oxygen stress. The thermal properties of PHBV showed a decreasing trend of the glass transition and melting temperatures with increasing 3HV fraction. The average molecular weights of PHBV with modulated 3HV fractions were between 20 and 26 × 104 g/mol.

Production of Hexanol as the Main Product Through Syngas Fermentation by Clostridium carboxidivorans P7.

The production of hexanol from syngas by acetogens has gained attention as a replacement for petroleum-derived hexanol, which is widely used in the chemical synthesis and plastic industries. However, acetogenic bacteria generally produce C2 compounds (e.g., acetate and ethanol) as the main products. In this study, the gas fermentation conditions favorable for hexanol production were investigated at different temperatures (30-37°C) and CO gas contents (30-70%) in batch gas fermentation. Hexanol production increased from 0.02 to 0.09 g/L when the cultivation temperature was lowered from 37 to 30°C. As the CO content increased from 30 to 70%, the CO consumption rate and hexanol production (yield, titer, and ratio of C6 compound to total products) increased with the CO content. When 70% CO gas was repeatedly provided by flushing the headspace of the bottles at 30°C, the total alcohol production increased to 4.32 g/L at the expense of acids. Notably, hexanol production (1.90 g/L) was higher than that of ethanol (1.20 g/L) and butanol (1.20 g/L); this is the highest level of hexanol produced in gas fermentation to date and the first report of hexanol as the main product. Hexanol production was further enhanced to 2.34 g/L when 2 g/L ethanol was supplemented at the beginning of 70% CO gas refeeding fermentation. Particularly, hexanol productivity was significantly enhanced to 0.18 g/L/day while the supplemented ethanol was consumed, indicating that the conversion of ethanol to acetyl-CoA and reducing equivalents positively affected hexanol production. These optimized culture conditions (gas fermentation at 30°C and refeeding with 70% CO gas) and ethanol supplementation provide an effective and sustainable approach for bio-hexanol production.

Glucose/Xylose Co-Fermenting Saccharomyces cerevisiae Increases the Production of Acetyl-CoA Derived n-Butanol From Lignocellulosic Biomass.

Efficient xylose catabolism in engineered Saccharomyces cerevisiae enables more economical lignocellulosic biorefinery with improved production yields per unit of biomass. Yet, the product profile of glucose/xylose co-fermenting S. cerevisiae is mainly limited to bioethanol and a few other chemicals. Here, we introduced an n-butanol-biosynthesis pathway into a glucose/xylose co-fermenting S. cerevisiae strain (XUSEA) to evaluate its potential on the production of acetyl-CoA derived products. Higher n-butanol production of glucose/xylose co-fermenting strain was explained by the transcriptomic landscape, which revealed strongly increased acetyl-CoA and NADPH pools when compared to a glucose fermenting wild-type strain. The acetate supplementation expected to support acetyl-CoA pool further increased n-butanol production, which was also validated during the fermentation of lignocellulosic hydrolysates containing acetate. Our findings imply the feasibility of lignocellulosic biorefinery for producing fuels and chemicals derived from a key intermediate of acetyl-CoA through glucose/xylose co-fermentation.

Improved simultaneous co-fermentation of glucose and xylose by Saccharomyces cerevisiae for efficient lignocellulosic biorefinery.

BACKGROUND: Lignocellulosic biorefinery offers economical and sustainable production of fuels and chemicals. Saccharomyces cerevisiae, a promising industrial host for biorefinery, has been intensively developed to expand its product profile. However, the sequential and slow conversion of xylose into target products remains one of the main challenges for realizing efficient industrial lignocellulosic biorefinery. RESULTS: In this study, we developed a powerful mixed-sugar co-fermenting strain of S. cerevisiae, XUSEA, with improved xylose conversion capacity during simultaneous glucose/xylose co-fermentation. To reinforce xylose catabolism, the overexpression target in the pentose phosphate pathway was selected using a DNA assembler method and overexpressed increasing xylose consumption and ethanol production by twofold. The performance of the newly engineered strain with improved xylose catabolism was further boosted by elevating fermentation temperature and thus significantly reduced the co-fermentation time by half. Through combined efforts of reinforcing the pathway of xylose catabolism and elevating the fermentation temperature, XUSEA achieved simultaneous co-fermentation of lignocellulosic hydrolysates, composed of 39.6 g L-1 glucose and 23.1 g L-1 xylose, within 24 h producing 30.1 g L-1 ethanol with a yield of 0.48 g g-1. CONCLUSIONS: Owing to its superior co-fermentation performance and ability for further engineering, XUSEA has potential as a platform in a lignocellulosic biorefinery toward realizing a more economical and sustainable process for large-scale bioethanol production.

Improved 2,3-butanediol yield and productivity from lignocellulose biomass hydrolysate in metabolically engineered Enterobacter aerogenes.

We previously engineered Enterobacter aerogenesfor glucose and xylose co-utilization and 2,3-butanediol production. Here, strain EMY-22 was further engineered to improve the 2,3-butanediol titer, productivity, and yield by reducing the production of byproducts. To reduce succinate production, the budABC operon and galP gene were overexpressed, which increased 2,3-butanediol production. For further reduction of succinate and 2-ketogluconate production, maeA was selected and overexpressed in EMY-22. The optimally engineered strain produced 2,3-butanediol for a longer time and showed reduced byproduct formation from sugarcane bagasse hydrolysate under flask cultivation conditions. The engineered strain displayed 66.6, 13.4, and 16.8% improvements in titer, yield, productivity of 2,3-butanediol, respectively, compared to its parental strain under fed-batch fermentation conditions. The data demonstrate that the metabolic engineering to reduce byproduct formation is a promising strategy to improve 2,3-butanediol production from lignocellulosic biomass.

Metabolic engineering considerations for the heterologous expression of xylose-catabolic pathways in Saccharomyces cerevisiae.

Xylose, the second most abundant sugar in lignocellulosic biomass hydrolysates, can be fermented by Saccharomyces cerevisiae expressing one of two heterologous xylose pathways: a xylose oxidoreductase pathway and a xylose isomerase pathway. Depending on the type of the pathway, its optimization strategies and the fermentation efficiencies vary significantly. In the present study, we constructed two isogenic strains expressing either the oxidoreductase pathway (XYL123) or the isomerase pathway (XI-XYL3), and delved into simple and reproducible ways to improve the resulting strains. First, the strains were subjected to the deletion of PHO13, overexpression of TAL1, and adaptive evolution, but those individual approaches were only effective in the XYL123 strain but not in the XI-XYL3 strain. Among other optimization strategies of the XI-XYL3 strain, we found that increasing the copy number of the xylose isomerase gene (xylA) is the most promising but yet preliminary strategy for the improvement. These results suggest that the oxidoreductase pathway might provide a simpler metabolic engineering strategy than the isomerase pathway for the development of efficient xylose-fermenting strains under the conditions tested in the present study.