Glucose-xylose Mixture Research Articles

Elucidating the salt-tolerant mechanism of Halomonas cupida J9 and unsterile ectoine production from lignocellulosic biomass

BackgroundEctoine as an amino acid derivative is widely applied in many fields, such as the food industry, cosmetic manufacturing, biologics, and therapeutic agent. Large-scale production of ectoine is mainly restricted by the cost of fermentation substrates (e.g., carbon sources) and sterilization.ResultsIn this study, Halomonas cupida J9 was shown to be capable of synthesizing ectoine using xylose as the sole carbon source. A pathway was proposed in H. cupida J9 that synergistically utilizes both WBG xylose metabolism and EMP glucose metabolism for the synthesis of ectoine. Transcriptome analysis indicated that expression of ectoine biosynthesis module was enhanced under salt stress. Ectoine production by H. cupida J9 was enhanced by improving the expression of ectoine biosynthesis module, increasing the intracellular supply of the precursor oxaloacetate, and utilizing urea as the nitrogen source. The constructed J9U-P8EC achieved a record ectoine production of 4.12 g/L after 60 h of xylose fermentation. Finally, unsterile production of ectoine by J9U-P8EC from either a glucose-xylose mixture or corn straw hydrolysate was demonstrated, with an output of 8.55 g/L and 1.30 g/L of ectoine, respectively.ConclusionsThis study created a promising H. cupida J9-based cell factory for low-cost production of ectoine. Our results highlight the potential of J9U-P8EC to utilize lignocellulose-rich agriculture waste for open production of ectoine.

Experimental evolution reveals an effective avenue for d-lactic acid production from glucose-xylose mixtures via enhanced Glk activity and a cAMP-independent CRP mutation.

d-Lactic acid holds significant industrial importance due to its versatility and serves as a crucial component in the synthesis of environmentally friendly and biodegradable thermal-resistant poly-lactic acid. This polymer exhibits promising potential as a substitute for nonbiodegradable, petroleum-based plastics. The production of d-lactic acid from lignocellulosic biomass, a type of biorenewable and nonfood resources, can lower costs and improve product competitiveness. Glucose and xylose are the most abundant sugar monomers in lignocellulosic biomass materials. Despite Escherichia coli possessing native xylose catabolic pathways and transport, their ability to effectively utilize xylose is often hindered in the presence of glucose. Here, the E. coli strain Rec1.0, previously engineered to overcome carbon catabolite repression, was selected as the initial strain for reengineering to produce d-lactic acid. An adaptive evolution approach was employed to achieve highly efficient fermentation of glucose-xylose mixtures. The resulting strain, QJL010, could produce d-lactic acid of 87.5 g/L with a carbon yield of 0.99 mol/mol. Notably, the consumption rates of glucose and xylose reached 0.75 and 0.82 g/gDCW/h, respectively. Further analysis revealed that increased Glk activity, resulting from glk mutations (A142V and R188H), along with their upregulated expression, contributed to an elevated glucose consumption rate. Additionally, a CRP G141D mutation, cAMP-independent, stimulated the expression of the xylR, xylE, and galABC* genes, resulting in an accelerated xylose consumption rate. These findings provide valuable support for the utilization of E. coli platform strains in the production of value-added chemicals from lignocellulosic biomass.

Optimization of poly(3-hydroxybutyrate) production with simulating corn stover hydrolysates by Paraburkholderia sacchari

In this study, poly(3-hydroxybutyrate) production by Paraburkholderia sacchari was investigated using glucose-xylose mixtures as simulated corn stover hydrolysates to optimize the culture media. Box-Behnken Design (BBD) was selected to comprehensively understand the effects of four variables including sugar concentration, molar C/N ratio, phosphorus concentration, and sugar ratio on P(3HB) production. The optimized parameters were identified as initial sugar concentration (23 g/L), molar C/N ratio (44), phosphorus concentration (8.7 g/L), and sugar ratio (1.9∶1). Under the optimal conditions, P. sacchari achieved the maximum P(3HB) accumulation of 55.8 % with a P(3HB) titer of 5.16 g/L (0.23 g/g Sugar). The extracted polymer was confirmed as P(3HB) and exhibited enhanced thermostability. Furthermore, P(3HB) was successfully synthesized from ball-milled corn stover hydrolysate under optimized conditions with the P(3HB) titer reached 7.66 g/L (0.33 g/g Sugar), which was higher than the pure mixed sugar system and other reports on lignocellulose hydrolysates with batch fermentation (<5 g/L). Therefore, P. sacchari exhibits the potential for P(3HB) production using corn stover hydrolysates as an inexpensive substrate.

Engineering Escherichia coli for Isobutanol Production from Xylose or Glucose-Xylose Mixture.

Aiming to overcome the depletion of fossil fuels and serious environmental pollution, biofuels such as isobutanol have garnered increased attention. Among different synthesis methods, the microbial fermentation of isobutanol from raw substrate is a promising strategy due to its low cost and environmentally friendly and optically pure products. As an important component of lignocellulosics and the second most common sugar in nature, xylose has become a promising renewable resource for microbial production. However, bottlenecks in xylose utilization limit its wide application as substrates. In this work, an isobutanol synthetic pathway from xylose was first constructed in E. coli MG1655 through the combination of the Ehrlich and Dahms pathways. The engineering of xylose transport and electron transport chain complexes further improved xylose assimilation and isobutanol production. By optimizing xylose supplement concentration, the recombinant E. coli strain BWL4 could produce 485.35 mg/L isobutanol from 20 g/L of xylose. To our knowledge, this is the first report related to isobutanol production using xylose as a sole carbon source in E. coli. Additionally, a glucose-xylose mixture was utilized as the carbon source. The Entner-Doudorof pathway was used to assimilate glucose, and the Ehrlich pathway was applied for isobutanol production. After carefully engineering the recombinant E. coli, strain BWL9 could produce 528.72 mg/L isobutanol from a mixture of 20 g/L glucose and 10 g/L xylose. The engineering strategies applied in this work provide a useful reference for the microbial production of isobutanol from xylose or glucose-xylose mixture.

Engineering co-utilization of glucose and xylose for chemical overproduction from lignocellulose.

Bio-refining lignocellulose could provide a sustainable supply of fuels and fine chemicals; however, the challenges associated with the co-utilization of xylose and glucose typically compromise the efficiency of lignocellulose conversion. Here we engineered the industrial yeast Ogataea polymorpha (Hansenula polymorpha) for lignocellulose biorefinery by facilitating the co-utilization of glucose and xylose to optimize the production of free fatty acids (FFAs) and 3-hydroxypropionic acid (3-HP) from lignocellulose. We rewired the central metabolism for the enhanced supply of acetyl-coenzyme A and nicotinamide adenine dinucleotide phosphate hydrogen, obtaining 30.0 g l-1 of FFAs from glucose, with productivity of up to 0.27 g l-1 h-1. Strengthening xylose uptake and catabolism promoted the synchronous utilization of glucose and xylose, which enabled the production of 38.2 g l-1 and 7.0 g l-1 FFAs from the glucose-xylose mixture and lignocellulosic hydrolysates, respectively. Finally, this efficient cell factory was metabolically transformed for 3-HP production with the highest titer of 79.6 g l-1 in fed-batch fermentation in mixed glucose and xylose.

Bifunctional Solid Lewis Acid–Base Catalysts for Efficient Conversion of the Glucose–Xylose Mixture to Methyl Lactate

Sugars produced from lignocellulose hydrolyzate are usually a mixture, of which glucose and xylose are the most abundant. Herein, a bifunctional solid catalyst (Mg-Sn-Beta zeolite) with Lewis (L) acidity and L basicity is developed, which can efficiently catalyze the glucose–xylose mixture to methyl lactate (MLA). This avoids the economic cost and cumbersome operation caused by separating mixed sugars. The retro-aldol condensation and aldol condensation are two major steps in converting glucose and/or xylose to MLA. L acid sites and L base sites in Mg-Sn-Beta zeolite are favorable for the above two steps. 1Mg-Sn-Beta-x and yMg-Sn-Beta-100 with different contents of L acid sites and L base sites were characterized via various techniques. In the conversion of glucose–xylose mixture to MLA, a considerable yield (52%) was obtained over 2Mg-Sn-Beta-100 compared with that on Sn-Beta-100 (25%). Besides, 2Mg-Sn-Beta-100 exhibited excellent stability and reusability during five reaction runs.

Formate as a supplementary substrate facilitates sugar metabolism and solvent production by Clostridium beijerinckii NCIMB 8052

Microbial utilization and conversion of organic one-carbon compounds, such as formate and methanol that can be easily produced from CO2, has emerged as an attractive approach for biorefinery. In this study, we discovered Clostridium beijerinckii NCIMB 8052, a typical solventogenic Clostridium strain, to be a native formate-utilizing bacterium. 13C isotope analysis showed that formate could be metabolized via both assimilation and dissimilation pathways in C. beijerinckii NCIMB 8052. Notably, the use of formate as the supplementary substrate by this strain could significantly enhance its glucose consumption and ABE (acetone-butanol-ethanol) production, largely due to the up-regulation of genes responsible for glycolysis and glucose transport under formate stress. Based on these findings, we further improved formate tolerance of C. beijerinckii NCIMB 8052 by adaptive laboratory evolution, generating an evolved strain Cbei-FA01. The Cbei-FA01 strain could produce 23.0 g/L of ABE solvents using glucose and formate as dual substrates, ∼50% higher than that of the wild-type strain under the same condition. Moreover, such a promotion effect of formate on ABE production by Cbei-FA01 was also observed in fermenting a glucose-xylose mixture. This work reveals a previously unreported role of formate in biological ABE production, providing a new approach to utilize this one-carbon source.

Co-Fermentation of Glucose-Xylose Mixtures from Agroindustrial Residues by Ethanologenic Escherichia coli: A Study on the Lack of Carbon Catabolite Repression in Strain MS04.

The production of biofuels, such as bioethanol from lignocellulosic biomass, is an important task within the sustainable energy concept. Understanding the metabolism of ethanologenic microorganisms for the consumption of sugar mixtures contained in lignocellulosic hydrolysates could allow the improvement of the fermentation process. In this study, the ethanologenic strain Escherichia coli MS04 was used to ferment hydrolysates from five different lignocellulosic agroindustrial wastes, which contained different glucose and xylose concentrations. The volumetric rates of glucose and xylose consumption and ethanol production depend on the initial concentration of glucose and xylose, concentrations of inhibitors, and the positive effect of acetate in the fermentation to ethanol. Ethanol yields above 80% and productivities up to 1.85 gEtOH/Lh were obtained. Furthermore, in all evaluations, a simultaneous co-consumption of glucose and xylose was observed. The effect of deleting the xyIR regulator was studied, concluding that it plays an important role in the metabolism of monosaccharides and in xylose consumption. Moreover, the importance of acetate was confirmed for the ethanologenic strain, showing the positive effect of acetate on the co-consumption rates of glucose and xylose in cultivation media and hydrolysates containing sugar mixtures.

Controlling catabolite repression for isobutanol production using glucose and xylose by overexpressing the xylose regulator

Using lignocellulosic biomass is immensely beneficial for the economical production of biochemicals. However, utilizing mixed sugars from lignocellulosic biomass is challenging because of bacterial preference for specific sugar such as glucose. Although previous studies have attempted to overcome this challenge, no studies have been reported on isobutanol production from mixed sugars in the Escherichia coli strain. To overcome catabolite repression of xylose and produce isobutanol using mixed sugars, we applied the combination of three strategies: (1) deletion of the gene for the glucose-specific transporter of the phosphotransferase system (ptsG); (2) overexpression of glucose kinase (glk) and glucose facilitator protein (glf); and (3) overexpression of the xylose regulator (xylR). xylR gene overexpression resulted in 100% of glucose and 82.5% of xylose consumption in the glucose–xylose mixture (1:1). Moreover, isobutanol production increased by 192% in the 1:1 medium, equivalent to the amount of isobutanol produced using only glucose. These results indicate the effectiveness of xylR overexpression in isobutanol production. Our findings demonstrated various strategies to overcome catabolite repression for a specific product, isobutanol. The present study suggests that the selected strategy in E. coli could overcome the major challenge using lignocellulosic biomass to produce isobutanol.

Rhizopus oryzae for Fumaric Acid Production: Optimising the Use of a Synthetic Lignocellulosic Hydrolysate

The hydrolysis of lignocellulosic biomass opens an array of bioconversion possibilities for producing fuels and chemicals. Microbial fermentation is particularly suited to the conversion of sugar-rich hydrolysates into biochemicals. Rhizopus oryzae ATCC 20344 was employed to produce fumaric acid from glucose, xylose, and a synthetic lignocellulosic hydrolysate (glucose–xylose mixture) in batch and continuous fermentations. A novel immobilised biomass reactor was used to investigate the co-fermentation of xylose and glucose. Ideal medium conditions and a substrate feed strategy were then employed to optimise the production of fumaric acid. The batch fermentation of the synthetic hydrolysate at optimal conditions (urea feed rate 0.625mgL−1h−1 and pH 4) produced a fumaric acid yield of 0.439gg−1. A specific substrate feed rate (0.164gL−1h−1) that negated ethanol production and selected for fumaric acid was determined. Using this feed rate in a continuous fermentation, a fumaric acid yield of 0.735gg−1 was achieved; this was a 67.4% improvement. A metabolic analysis helped to determine a continuous synthetic lignocellulosic hydrolysate feed rate that selected for fumaric acid production while achieving the co-fermentation of glucose and xylose, thus avoiding the undesirable carbon catabolite repression. This work demonstrates the viability of fumaric acid production from lignocellulosic hydrolysate; the process developments discovered will pave the way for an industrially viable process.

Effect of Zymomonas mobilis and Pichia stipitis presence/absence strategies in a two-stage process on bioethanol production from glucose-xylose mixture

Effect of Zymomonas mobilis and Pichia stipitis presence/absence strategies in a two-stage process on bioethanol production from glucose-xylose mixture

Optimization of a Two-Species Microbial Consortium for Improved Mcl-PHA Production From Glucose-Xylose Mixtures.

Polyhydroxyalkanoates (PHAs) have attracted much attention as a good substitute for petroleum-based plastics, especially mcl-PHA due to their superior physical and mechanical properties with broader applications. Artificial microbial consortia can solve the problems of low metabolic capacity of single engineered strains and low conversion efficiency of natural consortia while expanding the scope of substrate utilization. Therefore, the use of artificial microbial consortia is considered a promising method for the production of mcl-PHA. In this work, we designed and constructed a microbial consortium composed of engineered Escherichia coli MG1655 and Pseudomonas putida KT2440 based on the “nutrition supply–detoxification” concept, which improved mcl-PHA production from glucose-xylose mixtures. An engineered E. coli that preferentially uses xylose was engineered with an enhanced ability to secrete acetic acid and free fatty acids (FFAs), producing 6.44 g/L acetic acid and 2.51 g/L FFAs with 20 g/L xylose as substrate. The mcl-PHA producing strain of P. putida in the microbial consortium has been engineered to enhance its ability to convert acetic acid and FFAs into mcl-PHA, producing 0.75 g/L mcl-PHA with mixed substrates consisting of glucose, acetic acid, and octanoate, while also reducing the growth inhibition of E. coli by acetic acid. The further developed artificial microbial consortium finally produced 1.32 g/L of mcl-PHA from 20 g/L of a glucose–xylose mixture (1:1) after substrate competition control and process optimization. The substrate utilization and product synthesis functions were successfully divided into the two strains in the constructed artificial microbial consortium, and a mutually beneficial symbiosis of “nutrition supply–detoxification” with a relatively high mcl-PHA titer was achieved, enabling the efficient accumulation of mcl-PHA. The consortium developed in this study is a potential platform for mcl-PHA production from lignocellulosic biomass.

Directed evolution of Zymomonas mobilis sugar facilitator Glf to overcome glucose inhibition

Cellular import of D-xylose, the second most abundant sugar in typical lignocellulosic biomass, has been evidenced to be an energy-depriving process in bacterial biocatalysts. The sugar facilitator of Zymomonas mobilis, Glf, is capable of importing xylose at high rates without extra energy input, but is inhibited by D-glucose (the primary biomass sugar), potentially limiting the utility of this transporter for fermentation of sugar mixtures derived from lignocellulose. In this work we developed an Escherichia coli platform strain deficient in glucose and xylose transport to facilitate directed evolution of Glf to overcome glucose inhibition. Using this platform, we isolated nine Glf variants created by both random and site-saturation mutagenesis with increased xylose utilization rates ranging from 4.8-fold to 13-fold relative to wild-type Glf when fermenting 100 g l–1 glucose–xylose mixtures. Diverse point mutations such as A165M and L445I were discovered leading to released glucose inhibition. Most of these mutations likely alter sugar coordinating pocket for the 6-hydroxymethyl group of D-glucose. These discovered glucose-resistant Glf variants can be potentially used as energy-conservative alternatives to the native sugar transport systems of bacterial biocatalysts for fermentation of lignocellulose-derived sugars.

Improvement of Enantiomeric l-Lactic Acid Production from Mixed Hexose-Pentose Sugars by Coculture of Enterococcus mundtii WX1 and Lactobacillus rhamnosus SCJ9

Among 39 pentose-utilizing lactic acid bacteria (LAB) selected from acid-forming bacteria from the midgut of Eri silkworm, the isolate WX1 was selected with the highest capability to produce optically pure l-lactic acid (l-LA) from glucose, xylose and arabinose with furfural-tolerant properties. The isolate WX1 was identified as Enterococcus mundtii based on 16S rDNA sequence analysis. The conversion yields of l-LA from glucose and xylose by E. mundtii WX1 were 0.97 and 0.68 g/g substrate, respectively. Furthermore, l-LA production by E. mundtii WX1 in various glucose-xylose mixtures indicated glucose repression effect on xylose consumption. The coculture of E. mundtii WX1 and Lactobacillus rhamnosus SCJ9, a homofermentative LAB capable of producing l-LA from glucose clearly showed an improvement of l-LA production from 30 g/L total glucose-xylose (6:4). The results from Plackett–Burman design (PBD) indicated that Tween 80, MnSO4 and yeast extract (YE) were three medium components that significantly influenced (p &lt; 0.05) l-LA production using the coculture strategy in the presence of 2 g/L furfural. Optimal concentrations of these variables revealed by central composite design (CCD) and response surface methodology (RSM) were 20.61 g/L YE, 1.44 g/L Tween 80 and 1.27 g/L MnSO4. Based on the optimized medium with 30 g/L total glucose-xylose (6:4), the maximum experimental l-LA value of 23.59 g/L reflecting 0.76 g/g substrate were achieved from 48 h fermentation at 37 °C. l-LA produced by coculture cultivated under standard MRS medium and new optimized conditions were 1.28 and 1.53 times higher than that obtained from single culture by E. mundtii WX1, respectively. This study provides the foundations for practical applications of coculture in bioconversion of lignocellulose particularly glucose-xylose-rich corn stover to l-LA.

Transcriptome profile of carbon catabolite repression in an efficient l-(+)-lactic acid-producing bacterium Enterococcus mundtii QU25 grown in media with combinations of cellobiose, xylose, and glucose.

Enterococcus mundtii QU25, a non-dairy lactic acid bacterium of the phylum Firmicutes, is capable of simultaneously fermenting cellobiose and xylose, and is described as a promising strain for the industrial production of optically pure l-lactic acid (≥ 99.9%) via homo-fermentation of lignocellulosic hydrolysates. Generally, Firmicutes bacteria show preferential consumption of sugar (usually glucose), termed carbon catabolite repression (CCR), while hampering the catabolism of other sugars. In our previous study, QU25 exhibited apparent CCR in a glucose-xylose mixture phenotypically, and transcriptional repression of the xylose operon encoding initial xylose metabolism genes, likely occurred in a CcpA-dependent manner. QU25 did not exhibit CCR phenotypically in a cellobiose-xylose mixture. The aim of the current study is to elucidate the transcriptional change associated with the simultaneous utilization of cellobiose and xylose. To this end, we performed RNA-seq analysis in the exponential growth phase of E. mundtii QU25 cells grown in glucose, cellobiose, and/or xylose as either sole or co-carbon sources. Our transcriptomic data showed that the xylose operon was weakly repressed in cells grown in a cellobiose-xylose mixture compared with that in cells grown in a glucose-xylose mixture. Furthermore, the gene expression of talC, the sole gene encoding transaldolase, is expected to be repressed by CcpA-mediated CCR. QU25 metabolized xylose without using transaldolase, which is necessary for homolactic fermentation from pentoses using the pentose-phosphate pathway. Hence, the metabolism of xylose in the presence of cellobiose by QU25 may have been due to 1) sufficient amounts of proteins encoded by the xylose operon genes for xylose metabolism despite of the slight repression of the operon, and 2) bypassing of the pentose-phosphate pathway without the TalC activity. Accordingly, we have determined the targets of genetic modification in QU25 to metabolize cellobiose, xylose and glucose simultaneously for application of the lactic fermentation from lignocellulosic hydrolysates.

Catabolic Division of Labor Enhances Production of D-Lactate and Succinate From Glucose-Xylose Mixtures in Engineered Escherichia coli Co-culture Systems.

Although biological upgrading of lignocellulosic sugars represents a promising and sustainable route to bioplastics, diverse and variable feedstock compositions (e.g., glucose from the cellulose fraction and xylose from the hemicellulose fraction) present several complex challenges. Specifically, sugar mixtures are often incompletely metabolized due to carbon catabolite repression while composition variability further complicates the optimization of co-utilization rates. Benefiting from several unique features including division of labor, increased metabolic diversity, and modularity, synthetic microbial communities represent a promising platform with the potential to address persistent bioconversion challenges. In this work, two unique and catabolically orthogonal Escherichia coli co-cultures systems were developed and used to enhance the production of D-lactate and succinate (two bioplastic monomers) from glucose–xylose mixtures (100 g L–1 total sugars, 2:1 by mass). In both cases, glucose specialist strains were engineered by deleting xylR (encoding the xylose-specific transcriptional activator, XylR) to disable xylose catabolism, whereas xylose specialist strains were engineered by deleting several key components involved with glucose transport and phosphorylation systems (i.e., ptsI, ptsG, galP, glk) while also increasing xylose utilization by introducing specific xylR mutations. Optimization of initial population ratios between complementary sugar specialists proved a key design variable for each pair of strains. In both cases, ∼91% utilization of total sugars was achieved in mineral salt media by simple batch fermentation. High product titer (88 g L–1 D-lactate, 84 g L–1 succinate) and maximum productivity (2.5 g L–1 h–1 D-lactate, 1.3 g L–1 h–1 succinate) and product yield (0.97 g g-total sugar–1 for D-lactate, 0.95 g g-total sugar–1 for succinate) were also achieved.

Construction of a "nutrition supply-detoxification" coculture consortium for medium-chain-length polyhydroxyalkanoate production with a glucose-xylose mixture.

In this study, we constructed a coculture consortium comprising engineered Pseudomonas putida KT2440 and Escherichia coli MG1655. Provision of "related" carbon sources and synthesis of medium-chain-length polyhydroxyalkanoates (mcl-PHAs) were separately assigned to these strains via a modular construction strategy. To avoid growth competition, a preference for the use of a carbon source was constructed. Further, the main intermediate metabolite acetate played an important role in constructing the expected "nutrition supply-detoxification" relationship between these strains. The coculture consortium showed a remarkable increase in the mcl-PHA titer (0.541g/L) with a glucose-xylose mixture (1:1). Subsequently, the titer of mcl-PHA produced by the coculture consortium when tested with actual lignocellulosic hydrolysate (0.434g/L) was similar to that achieved with laboratory sugars' mixture (0.469g/L). These results indicate a competitive potential of the engineered E. coli-P. putida coculture consortium for mcl-PHA production with lignocellulosic hydrolysate.

Engineering a Synthetic, Catabolically Orthogonal Coculture System for Enhanced Conversion of Lignocellulose-Derived Sugars to Ethanol.

Fermentation of lignocellulosic sugar mixtures is often suboptimal due to inefficient xylose catabolism and sequential sugar utilization caused by carbon catabolite repression. Unlike in conventional applications employing a single engineered strain, the alternative development of synthetic microbial communities facilitates the execution of complex metabolic tasks by exploiting the unique community features, including modularity, division of labor, and facile tunability. A series of synthetic, catabolically orthogonal coculture systems were systematically engineered, as derived from either wild-type Escherichia coli W or ethanologenic LY180. Net catabolic activities were effectively balanced by simple tuning of the inoculum ratio between specialist strains, which enabled coutilization (98% of 100 g L-1 total sugars) of glucose-xylose mixtures (2:1 by mass) for both culture systems in simple batch fermentations. The engineered ethanologenic cocultures achieved ethanol titer (46 g L-1), productivity (488 mg L-1 h-1), and yield (∼90% of theoretical maximum), which were all significantly increased compared to LY180 monocultures.

Laboratory evolution for forced glucose-xylose co-consumption enables identification of mutations that improve mixed-sugar fermentation by xylose-fermenting Saccharomyces cerevisiae.

Simultaneous fermentation of glucose and xylose can contribute to improved productivity and robustness of yeast-based processes for bioethanol production from lignocellulosic hydrolysates. This study explores a novel laboratory evolution strategy for identifying mutations that contribute to simultaneous utilisation of these sugars in batch cultures of Saccharomyces cerevisiae. To force simultaneous utilisation of xylose and glucose, the genes encoding glucose-6-phosphate isomerase (PGI1) and ribulose-5-phosphate epimerase (RPE1) were deleted in a xylose-isomerase-based xylose-fermenting strain with a modified oxidative pentose-phosphate pathway. Laboratory evolution of this strain in serial batch cultures on glucose–xylose mixtures yielded mutants that rapidly co-consumed the two sugars. Whole-genome sequencing of evolved strains identified mutations in HXK2, RSP5 and GAL83, whose introduction into a non-evolved xylose-fermenting S. cerevisiae strain improved co-consumption of xylose and glucose under aerobic and anaerobic conditions. Combined deletion of HXK2 and introduction of a GAL83G673T allele yielded a strain with a 2.5-fold higher xylose and glucose co-consumption ratio than its xylose-fermenting parental strain. These two modifications decreased the time required for full sugar conversion in anaerobic bioreactor batch cultures, grown on 20 g L−1 glucose and 10 g L−1 xylose, by over 24 h. This study demonstrates that laboratory evolution and genome resequencing of microbial strains engineered for forced co-consumption is a powerful approach for studying and improving simultaneous conversion of mixed substrates.

Conversion of glucose-xylose mixtures to pyruvate using a consortium of metabolically engineered Escherichia coli.

Two strains of Escherichia coli were engineered to accumulate pyruvic acid from two sugars found in lignocellulosic hydrolysates by knockouts in the aceE, ppsA, poxB, and ldhA genes. Additionally, since glucose and xylose are typically consumed sequentially due to carbon catabolite repression in E. coli, one strain (MEC590) was engineered to grow only on glucose while a second strain (MEC589) grew only on xylose. On a single substrate, each strain generated pyruvate at a yield of about 0.60g/g in both continuous culture and batch culture. In a glucose-xylose mixture under continuous culture, a consortium of both strains maintained a pyruvate yield greater than 0.60g/g when three different concentrations of glucose and xylose were sequentially fed into the system. In a fed-batch process, both sugars in a glucose-xylose mixture were consumed simultaneously to accumulate 39g/L pyruvate in less than 24h at a yield of 0.59g/g.