Xylose assimilation enhances the production of isobutanol in engineered Saccharomyces cerevisiae

Bioconversion of xylose—the second most abundant sugar in nature—into high-value fuels and chemicals by engineered Saccharomyces cerevisiae has been a long-term goal of the metabolic engineering community. Although most efforts have heavily focused on the production of ethanol by engineered S. cerevisiae, yields and productivities of ethanol produced from xylose have remained inferior as compared with ethanol produced from glucose. However, this entrenched focus on ethanol has concealed the fact that many aspects of xylose metabolism favor the production of nonethanol products. Through reduced overall metabolic flux, a more respiratory nature of consumption, and evading glucose signaling pathways, the bioconversion of xylose can be more amenable to redirecting flux away from ethanol towards the desired target product. In this report, we show that coupling xylose consumption via the oxidoreductive pathway with a mitochondrially-targeted isobutanol biosynthesis pathway leads to enhanced product yields and titers as compared to cultures utilizing glucose or galactose as a carbon source. Through the optimization of culture conditions, we achieve 2.6 g/L of isobutanol in the fed-batch flask and bioreactor fermentations. These results suggest that there may be synergistic benefits of coupling xylose assimilation with the production of nonethanol value-added products.     

In silico analysis of bioethanol production from glucose/xylose mixtures during fed-batch fermentation of co-culture and mono-culture systems

This study presents a detailed in silico analysis of bioethanol production from glucose/xylose mixtures of various compositions by fed-batch co-culture and mono-culture fermentation of specialized microbes. The mono-culture consists of recombinant Saccharomyces cerevisise that can metabolize both hexose and pentose sugars while the co-culture system consists of substrate-selective microbes. Dynamic flux balance models based on available genome-scale reconstructions of the microorganisms have been used to analyze bioethanol production in fed-batch culture with constant feed rates and the maximization of ethanol productivity is addressed by computing optimal aerobic-anaerobic switching times. The simulation results clearly point to the superior performance of fed-batch fermentation of microbial co-culture against fed-batch fermentation of mono-culture for bioethanol production from glucose/xylose mixtures. A set of potential genetic engineering strategies for enhancement of S. cerevisiae and Escherichia coli strains performance have been identified. Such in silico predictions using genome-scale models provide valuable guidance for conducting in vivo metabolic engineering experiments.     

Specific ethanol production rate in ethanologenic Escherichia coli strain KO11 Is limited by pyruvate decarboxylase

Modification of ethanol productivity and yield, using mineral medium supplemented with glucose or xylose as carbon sources, was studied in ethanologenic Escherichia coli KO11 by increasing the activity of five key carbon metabolism enzymes. KO11 efficiently converted glucose or xylose to ethanol with a yield close to 100% of the theoretical maximum when growing in rich medium. However, when KO11 ferments glucose or xylose in mineral medium, the ethanol yields decreased to only 70 and 60%, respectively. An increase in GALP(Ec) (permease of galactose-glucose-xylose) or PGK(Ec) (phosphoglycerate kinase) activities did not change xylose or glucose and ethanol flux. However, when PDC(Zm) (pyruvate decarboxylase from Zymomonas mobilis) activity was increased 7-fold, the yields of ethanol from glucose or xylose were increased to 85 and 75%, respectively, and organic acid formation rates were reduced. Furthermore, as a response to a reduction in acetate and ATP yield, and a limited PDC(Zm) activity, an increase in PFK(Ec) (phosphofructokinase) or PYK(Bs) (pyruvate kinase from Bacillus stearothermophilus) activity drastically reduced glucose or xylose consumption and ethanol formation flux. This experimental metabolic control analysis showed that ethanol flux in KO11 is negatively controlled by phosphofructokinase and pyruvate kinase, and positively influenced by the PDC(Zm) activity level.     

Genome-scale modeling and in silico analysis of ethanologenic bacteria Zymomonas mobilis

Bioethanol has been recognized as a potential alternative energy source. Among various ethanol-producing microbes, Zymomonas mobilis has acquired special attention due to its higher ethanol yield and tolerance. However, cellular metabolism in Z. mobilis remains unclear, hindering its practical application for bioethanol production. To elucidate such physiological characteristics, we reconstructed and validated a genome-scale metabolic network (iZM363) of Z. mobilis ATCC31821 (ZM4) based on its annotated genome and biochemical information. The phenotypic behaviors and metabolic states predicted by our genome-scale model were highly consistent with the experimental observations of Z. mobilis ZM4 strain growing on glucose as well as NMR-measured intracellular fluxes of an engineered strain utilizing glucose, fructose, and xylose. Subsequent comparative analysis with Escherichia coli and Saccharomyces cerevisiae as well as gene essentiality and flux coupling analyses have also confirmed the functional role of pdc and adh genes in the ethanologenic activity of Z. mobilis, thus leading to better understanding of this natural ethanol producer. In future, the current model could be employed to identify potential cell engineering targets, thereby enhancing the productivity of ethanol in Z. mobilis.     

Repeated-batch fermentations of xylose and glucose-xylose mixtures using a respiration-deficient Saccharomyces cerevisiae engineered for xylose metabolism

Xylose-fermenting Saccharomyces strains are needed for commercialization of ethanol production from lignocellulosic biomass. Engineered Saccharomyces cerevisiae strains expressing XYL1, XYL2 and XYL3 from Pichia stipitis, however, utilize xylose in an oxidative manner, which results in significantly lower ethanol yields from xylose as compared to glucose. As such, we hypothesized that reconfiguration of xylose metabolism from oxidative into fermentative manner might lead to efficient ethanol production from xylose. To this end, we generated a respiration-deficient (RD) mutant in order to enforce engineered S. cerevisiae to utilize xylose only through fermentative metabolic routes. Three different repeated-batch fermentations were performed to characterize characteristics of the respiration-deficient mutant. When fermenting glucose as a sole carbon source, the RD mutant exhibited near theoretical ethanol yields (0.46 g g(-1)) during repeated-batch fermentations by recycling the cells. As the repeated-batch fermentation progressed, the volumetric ethanol productivity increased (from 7.5 to 8.3 g L(-1)h(-1)) because of the increased biomass from previous cultures. On the contrary, the mutant showed decreasing volumetric ethanol productivities during the repeated-batch fermentations using xylose as sole carbon source (from 0.4 to 0.3 g L(-1)h(-1)). The mutant did not grow on xylose and lost fermenting ability gradually, indicating that the RD mutant cannot maintain a good fermenting ability on xylose as a sole carbon source. However, the RD mutant was capable of fermenting a mixture of glucose and xylose with stable yields (0.35 g g(-1)) and productivities (0.52 g L(-1)h(-1)) during the repeated-batch fermentation. In addition, ethanol yields from xylose during the mixed sugar fermentation (0.30 g g(-1)) were higher than ethanol yields from xylose as a sole carbon source (0.21 g g(-1)). These results suggest that a strategy for increasing ethanol yield through respiration-deficiency can be applied for the fermentation of lignocellulosic hydrolyzates containing glucose and xylose.     

Simultaneous saccharification and co-fermentation of glucose and xylose in steam-pretreated corn stover at high fiber content with Saccharomyces cerevisiae TMB3400

The two main sugars in the agricultural by-product corn stover are glucose and xylose. Co-fermentation of glucose and xylose at high content of water-insoluble solids (WIS) without detoxification is a prerequisite to obtain high ethanol concentration and to reduce production costs. A recombinant strain of Saccharomyces cerevisiae, TMB3400, was used in simultaneous saccharification and fermentation (SSF) of whole pretreated slurry of corn stover at high WIS. TMB3400 co-fermented glucose and xylose with relatively high ethanol yields giving high final ethanol concentration. The ethanol productivity increased with increasing concentration of pretreatment hydrolysate in the yeast production medium and when SSF was performed in a fed-batch mode.     

酿酒酵母转化木糖生产化学品的研究进展

木糖的有效利用是木质纤维素生产生物燃料或化学品经济性转化的基础。30年来,通过理性代谢改造和适应性进化等工程策略,显著提高了传统乙醇发酵微生物——酿酒酵母Saccharomyces cerevisiae的木糖代谢能力。因此,近年来在酿酒酵母中利用木糖生产化学品的研究逐步展开。研究发现,酿酒酵母分别以木糖和葡萄糖为碳源时,其转录组和代谢组存在明显差异。与葡萄糖相比,木糖代谢过程中细胞整体呈现出Crabtree-negative代谢特征,如有限的糖酵解途径活性减少了丙酮酸到乙醇的代谢通量,以及增强的胞质乙酰辅酶A合成和呼吸能量代谢等,这都有利于以丙酮酸或乙酰辅酶A为前体的下游产物的有效合成。文中对酿酒酵母木糖代谢途径改造与优化、木糖代谢特征以及以木糖为碳源合成化学品的细胞工厂构建等方面进行了详细综述,并对木糖作为重要碳源在大宗化学品生物合成中存在的困难和挑战以及未来研究方向进行了总结与展望。     

Sequential Targeting of Xylose and Glucose Conversion in <Emphasis Type="Bold">F</Emphasis>ed-Batch Simultaneous Saccharification and <Emphasis Type="Bold">C</Emphasis>o-fermentation of Steam-Pretreated Wheat Straw for Improved Xylose Conversion to Ethanol

Efficient conversion of both glucose and xylose in lignocellulosic biomass is necessary to make second-generation bioethanol from agricultural residues competitive with first-generation bioethanol and gasoline. Simultaneous saccharification and co-fermentation (SSCF) is a promising strategy for obtaining high ethanol yields. However, with this method, the xylose-fermenting capacity and viability of yeast tend to decline over time and restrict the xylose utilization. In this study, we examined the ethanol production from steam-pretreated wheat straw using an established SSCF strategy with substrate and enzyme feeding that was previously applied to steam-pretreated corn cobs. Based on our findings, we propose an alternative SSCF strategy to sustain the xylose-fermenting capacity and improve the ethanol yield. The xylose-rich hydrolyzate liquor was separated from the glucose-rich solids, and phases were co-fermented sequentially. By prefermentation of the hydrolyzate liquor followed fed-batch SSCF, xylose, and glucose conversion could be targeted in succession. Because the xylose-fermenting capacity declines over time, while glucose is still converted, it was advantageous to target xylose conversion upfront. With our strategy, an overall ethanol yield of 84% of the theoretical maximum based on both xylose and glucose was reached for a slurry with higher inhibitor concentrations, versus 92% for a slurry with lower inhibitor concentrations. Xylose utilization exceeded 90% after SSCF for both slurries. Sequential targeting of xylose and glucose conversion sustained xylose fermentation and improved xylose utilization and ethanol yield compared with fed-batch SSCF of whole slurry.     

Sugar fermentation by <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> to produce ethanol

As a first step in the research on ethanol production from lignocellulose residues, sugar fermentation by Fusarium oxysporum in oxygen-limited conditions is studied in this work. As a substrate, solutions of arabinose, glucose, xylose and glucose/xylose mixtures are employed. The main kinetic and yield parameters of the process are determined according to a time-dependent model. The microorganism growth is characterized by the maximum specific growth rate and biomass productivity, the substrate consumption is studied through the specific consumption rate and biomass yield, and the product formation via the specific production rate and product yields. In conclusion, F. oxysporum can convert glucose and xylose into ethanol with product yields of 0.38 and 0.25, respectively; when using a glucose/xylose mixture as carbon source, the sugars are utilized sequentially and a maximum value of 0.28 g/g ethanol yield is determined from a 50% glucose/50% xylose mixture. Although fermentation performance by F.␣oxysporum is somewhat lower than that of other fermenting microorganisms, its ability for simultaneous lignocellulose-residue saccharification and fermentation is considered as a potential advantage.     

Systems Metabolic Engineering of Escherichia coli Improves Coconversion of Lignocellulose‐Derived Sugars

Currently, microbial conversion of lignocellulose‐derived glucose and xylose to biofuels is hindered by the fact that most microbes (including Escherichia coli [E. coli], Saccharomyces cerevisiae, and Zymomonas mobilis) preferentially consume glucose first and consume xylose slowly after glucose is depleted in lignocellulosic hydrolysates. In this study, E. coli strains are developed that simultaneously utilize glucose and xylose in lignocellulosic biomass hydrolysate using genome‐scale models and adaptive laboratory evolution. E. coli strains are designed and constructed that coutilize glucose and xylose and adaptively evolve them to improve glucose and xylose utilization. Whole‐genome resequencing of the evolved strains find relevant mutations in metabolic and regulatory genes and the mutations’ involvement in sugar coutilization is investigated. The developed strains show significantly improved coconversion of sugars in lignocellulosic biomass hydrolysates and provide a promising platform for producing next‐generation biofuels.     

Kinetic and nuclear magnetic resonance studies of xylose metabolism by recombinant Zymomonas mobilis ZM4(pZB5)

The specific rates of growth, substrate utilization, and ethanol production as well as yields of biomass and ethanol production on xylose for the recombinant Zymomonas mobilis ZM4(pZB5) were shown to be much less than those on glucose or glucose-xylose mixtures. Typical fermentations with ZM4(pZB5) growing on glucose-xylose mixtures followed two-phase growth kinetics with the initial uptakes of glucose and xylose being followed by slower growth and metabolic uncoupling on xylose after glucose depletion. The reductions in rates and yields from xylose metabolism were considered in the present investigation and may be due to a number of factors, including the following: (i) the increased metabolic burden from maintenance of plasmid-related functions, (ii) the production of by-products identified as xylitol, acetate, lactate, acetoin, and dihydroxyacetone by (13)C-nuclear magnetic resonance (NMR) spectroscopy and high-performance liquid chromatography, (iii) growth inhibition due to xylitol by the putative inhibitory compound xylitol phosphate, and (iv) the less energized state of ZM4(pZB5). In vivo (31)P-NMR studies have established that the levels of NTP and UDP sugars on xylose were less than those on glucose, and this energy limitation is likely to restrict the growth of the recombinant strain on xylose media.     

Dynamic flux balance modeling of microbial co-cultures for efficient batch fermentation of glucose and xylose mixtures

Sequential uptake of pentose and hexose sugars that compose lignocellulosic biomass limits the ability of pure microbial cultures to efficiently produce value-added bioproducts. In this work, we used dynamic flux balance modeling to examine the capability of mixed cultures of substrate-selective microbes to improve the utilization of glucose/xylose mixtures and to convert these mixed substrates into products. Co-culture simulations of Escherichia coli strains ALS1008 and ZSC113, engineered for glucose and xylose only uptake respectively, indicated that improvements in batch substrate consumption observed in previous experimental studies resulted primarily from an increase in ZSC113 xylose uptake relative to wild-type E. coli. The E. coli strain ZSC113 engineered for the elimination of glucose uptake was computationally co-cultured with wild-type Saccharomyces cerevisiae, which can only metabolize glucose, to determine if the co-culture was capable of enhanced ethanol production compared to pure cultures of wild-type E. coli and the S. cerevisiae strain RWB218 engineered for combined glucose and xylose uptake. Under the simplifying assumption that both microbes grow optimally under common environmental conditions, optimization of the strain inoculum and the aerobic to anaerobic switching time produced an almost twofold increase in ethanol productivity over the pure cultures. To examine the effect of reduced strain growth rates at non-optimal pH and temperature values, a break even analysis was performed to determine possible reductions in individual strain substrate uptake rates that resulted in the same predicted ethanol productivity as the best pure culture.     

Characterization of two-substrate fermentation processes for xylitol production using recombinant Saccharomyces cerevisiae containing xylose reductase gene

Fermentation characteristics of recombinant Saccharomyces cerevisiae containing a xylose reductase gene from Pichia stipitis were investigated in an attempt to convert xylose to xylitol, a natural five-carbon sugar alcohol used as a sweetener. Xylitol was produced with a maximum yield of 0.95 g g⁻¹ xylitol xylose consumed in the presence of glucose used as a co-substrate for co-factor regeneration. Addition of glucose caused inhibition of xylose transport and accumulation of ethanol. Such problems were solved by adopting glucose-limited fed-batch fermentations where a high ratio of xylose to glucose was maintained during the bioconversion phase. The optimized two-substrate fed-batch fermentation carried out with S. cerevisiae EH13.15:pY2XR at 30°C resulted in 105.2 g l⁻¹ xylitol concentration with 1.69 g l⁻¹ h⁻¹ productivity.     

Cofermentation of glucose, xylose, and cellobiose by the beetle-associated yeast Spathaspora passalidarum

Fermentation of cellulosic and hemicellulosic sugars from biomass could resolve food-versus-fuel conflicts inherent in the bioconversion of grains. However, the inability to coferment glucose and xylose is a major challenge to the economical use of lignocellulose as a feedstock. Simultaneous cofermentation of glucose, xylose, and cellobiose is problematic for most microbes because glucose represses utilization of the other saccharides. Surprisingly, the ascomycetous, beetle-associated yeast Spathaspora passalidarum, which ferments xylose and cellobiose natively, can also coferment these two sugars in the presence of 30 g/liter glucose. S. passalidarum simultaneously assimilates glucose and xylose aerobically, it simultaneously coferments glucose, cellobiose, and xylose with an ethanol yield of 0.42 g/g, and it has a specific ethanol production rate on xylose more than 3 times that of the corresponding rate on glucose. Moreover, an adapted strain of S. passalidarum produced 39 g/liter ethanol with a yield of 0.37 g/g sugars from a hardwood hydrolysate. Metabolome analysis of S. passalidarum before onset and during the fermentations of glucose and xylose showed that the flux of glycolytic intermediates is significantly higher on xylose than on glucose. The high affinity of its xylose reductase activities for NADH and xylose combined with allosteric activation of glycolysis probably accounts in part for its unusual capacities. These features make S. passalidarum very attractive for studying regulatory mechanisms enabling bioconversion of lignocellulosic materials by yeasts.     

Kinetic and Nuclear Magnetic Resonance Studies of Xylose Metabolism by Recombinant Zymomonas mobilis ZM4(pZB5)

The specific rates of growth, substrate utilization, and ethanol production as well as yields of biomass and ethanol production on xylose for the recombinant Zymomonas mobilis ZM4(pZB5) were shown to be much less than those on glucose or glucose-xylose mixtures. Typical fermentations with ZM4(pZB5) growing on glucose-xylose mixtures followed two-phase growth kinetics with the initial uptakes of glucose and xylose being followed by slower growth and metabolic uncoupling on xylose after glucose depletion. The reductions in rates and yields from xylose metabolism were considered in the present investigation and may be due to a number of factors, including the following: (i) the increased metabolic burden from maintenance of plasmid-related functions, (ii) the production of by-products identified as xylitol, acetate, lactate, acetoin, and dihydroxyacetone by ¹³C-nuclear magnetic resonance (NMR) spectroscopy and high-performance liquid chromatography, (iii) growth inhibition due to xylitol by the putative inhibitory compound xylitol phosphate, and (iv) the less energized state of ZM4(pZB5). In vivo ³¹P-NMR studies have established that the levels of NTP and UDP sugars on xylose were less than those on glucose, and this energy limitation is likely to restrict the growth of the recombinant strain on xylose media.     

Improved ethanol production from xylose with glucose isomerase and Saccharomyces cerevisiae using the respiratory inhibitor azide

Summary Ethanol was produced from xylose, using the enzyme glucose isomerase (xylose isomerase) and Saccharomyces cerevisiae. The influence of aeration, pH, enzyme concentration, cell mass and the concentration of the respiratory inhibitor sodium azide on the production of ethanol and the formation of by-products was investigated. Anaerobic conditions at pH 6.0, 10 g/l enzyme, 75 g/l dry weight cell mass and 4.6 mM sodium azide were found to be optimal. Under these conditions theoretical yields of ethanol were obtained from 42 g/l xylose within 24 hours.In a fed-batch culture, 62 g/l ethanol was produced from 127 g/l xylose with a yield of 0.49 and a productivity of 1.35 g/l·h.     

Systems metabolic engineering of Corynebacterium glutamicum for high-level production of 1,3-propanediol from glucose and xylose

Corynebacterium glutamicum is a versatile chassis which has been widely used to produce various amino acids and organic acids. In this study, we report the development of an efficient C. glutamicum strain to produce 1,3-propanediol (1,3-PDO) from glucose and xylose by systems metabolic engineering approaches, including (1) construction and optimization of two different glycerol synthesis modules; (2) combining glycerol and 1,3-PDO synthesis modules; (3) reducing 3-hydroxypropionate accumulation by clarifying a mechanism involving 1,3-PDO re-consumption; (4) reducing the accumulation of toxic 3-hydroxypropionaldehyde by pathway engineering; (5) engineering NADPH generation pathway and anaplerotic pathway. The final engineered strain can efficiently produce 1,3-PDO from glucose with a titer of 110.4 g/L, a yield of 0.42 g/g glucose, and a productivity of 2.30 g/L/h in fed-batch fermentation. By further introducing an optimized xylose metabolism module, the engineered strain can simultaneously utilize glucose and xylose to produce 1,3-PDO with a titer of 98.2 g/L and a yield of 0.38 g/g sugars. This result demonstrates that C. glutamicum is a potential chassis for the industrial production of 1,3-PDO from abundant lignocellulosic feedstocks.