Genome-scale consequences of cofactor balancing in engineered pentose utilization pathways in Saccharomyces cerevisiae

Biofuels derived from lignocellulosic biomass offer promising alternative renewable energy sources for transportation fuels. Significant effort has been made to engineer Saccharomyces cerevisiae to efficiently ferment pentose sugars such as D-xylose and L-arabinose into biofuels such as ethanol through heterologous expression of the fungal D-xylose and L-arabinose pathways. However, one of the major bottlenecks in these fungal pathways is that the cofactors are not balanced, which contributes to inefficient utilization of pentose sugars. We utilized a genome-scale model of S. cerevisiae to predict the maximal achievable growth rate for cofactor balanced and imbalanced D-xylose and L-arabinose utilization pathways. Dynamic flux balance analysis (DFBA) was used to simulate batch fermentation of glucose, D-xylose, and L-arabinose. The dynamic models and experimental results are in good agreement for the wild type and for the engineered D-xylose utilization pathway. Cofactor balancing the engineered D-xylose and L-arabinose utilization pathways simulated an increase in ethanol batch production of 24.7% while simultaneously reducing the predicted substrate utilization time by 70%. Furthermore, the effects of cofactor balancing the engineered pentose utilization pathways were evaluated throughout the genome-scale metabolic network. This work not only provides new insights to the global network effects of cofactor balancing but also provides useful guidelines for engineering a recombinant yeast strain with cofactor balanced engineered pathways that efficiently co-utilizes pentose and hexose sugars for biofuels production. Experimental switching of cofactor usage in enzymes has been demonstrated, but is a time-consuming effort. Therefore, systems biology models that can predict the likely outcome of such strain engineering efforts are highly useful for motivating which efforts are likely to be worth the significant time investment.     

Stress-related challenges in pentose fermentation to ethanol by the yeast Saccharomyces cerevisiae

Conversion of agricultural residues, energy crops and forest residues into bioethanol requires hydrolysis of the biomass and fermentation of the released sugars. During the hydrolysis of the hemicellulose fraction, substantial amounts of pentose sugars, in particular xylose, are released. Fermentation of these pentose sugars to ethanol by engineered Saccharomyces cerevisiae under industrial process conditions is the subject of this review. First, fermentation challenges originating from the main steps of ethanol production from lignocellulosic feedstocks are discussed, followed by genetic modifications that have been implemented in S. cerevisiae to obtain xylose and arabinose fermenting capacity per se. Finally, the fermentation of a real lignocellulosic medium is discussed in terms of inhibitory effects of furaldehydes, phenolics and weak acids and the presence of contaminating microbiota.     

Engineering redox cofactor utilization for detoxification of glycolaldehyde, a key inhibitor of bioethanol production, in yeast Saccharomyces cerevisiae

Hot-compressed water treatment of lignocellulose liberates numerous inhibitors that prevent ethanol fermentation of yeast Saccharomyces cerevisiae. Glycolaldehyde is one of the strongest fermentation inhibitors and we developed a tolerant strain by overexpressing ADH1 encoding an NADH-dependent reductase; however, its recovery was partial. In this study, to overcome this technical barrier, redox cofactor preference of glycolaldehyde detoxification was investigated. Glycolaldehyde-reducing activity of the ADH1-overexpressing strain was NADH-dependent but not NADPH-dependent. Moreover, genes encoding components of the pentose phosphate pathway, which generates intracellular NADPH, was upregulated in response to high concentrations of glycolaldehyde. Mutants defective in pentose phosphate pathways were sensitive to glycolaldehyde. Genome-wide survey identified GRE2 encoding a NADPH-dependent reductase as the gene that confers tolerance to glycolaldehyde. Overexpression of GRE2 in addition to ADH1 further improved the tolerance to glycolaldehyde. NADPH-dependent glycolaldehyde conversion to ethylene glycol and NADP⁺ content of the strain overexpressing both ADH1 and GRE2 were increased at 5 mM glycolaldehyde. Expression of GRE2 was increased in response to glycolaldehyde. Carbon metabolism of the strain was rerouted from glycerol to ethanol. Thus, it was concluded that the overexpression of GRE2 together with ADH1 restores glycolaldehyde tolerance by augmenting the NADPH-dependent reduction pathway in addition to NADH-dependent reduction pathway. The redox cofactor control for detoxification of glycolaldehyde proposed in this study could influence strategies for improving the tolerance of other fermentation inhibitors.     

Metabolic engineering for improved microbial pentose fermentation

Global concern over the depletion of fossil fuel reserves, and the detrimental impact that combustion of these materials has on the environment, is focusing attention on initiatives to create sustainable approaches for the production and use of biofuels from various biomass substrates. The development of a low-cost, safe and eco-friendly process for the utilization of renewable resources to generate value-added products with biotechnological potential as well as robust microorganisms capable of efficient fermentation of all types of sugars are essential to underpin the economic production of biofuels from biomass feedstocks. Saccharomyces cerevisiae, the most established fermentation yeast used in large scale bioconversion strategies, does not however metabolise the pentose sugars, xylose and arabinose and bioengineering is required for introduction of efficient pentose metabolic pathways and pentose sugar transport proteins for bioconversion of these substrates. Our approach provided a basis for future experiments that may ultimately lead to the development of industrial S. cerevisiae strains engineered to express pentose metabolising proteins from thermophilic fungi living on decaying plant material and here we expand our original article and discuss the strategies implemented to improve pentose fermentation.     

Engineering cofactor and transport mechanisms in Saccharomyces cerevisiae for enhanced acetyl-CoA and polyketide biosynthesis

Synthesis of polyketides at high titer and yield is important for producing pharmaceuticals and biorenewable chemical precursors. In this work, we engineered cofactor and transport pathways in Saccharomyces cerevisiae to increase acetyl-CoA, an important polyketide building block. The highly regulated yeast pyruvate dehydrogenase bypass pathway was supplemented by overexpressing a modified Escherichia coli pyruvate dehydrogenase complex (PDHm) that accepts NADP⁺ for acetyl-CoA production. After 24 h of cultivation, a 3.7-fold increase in NADPH/NADP⁺ ratio was observed relative to the base strain, and a 2.2-fold increase relative to introduction of the native E. coli PDH. Both E. coli pathways increased acetyl-CoA levels approximately 2-fold relative to the yeast base strain. Combining PDHm with a ZWF1 deletion to block the major yeast NADPH biosynthesis pathway resulted in a 12-fold NADPH boost and a 2.2-fold increase in acetyl-CoA. At 48 h, only this coupled approach showed increased acetyl-CoA levels, 3.0-fold higher than that of the base strain. The impact on polyketide synthesis was evaluated in a S. cerevisiae strain expressing the Gerbera hybrida 2-pyrone synthase (2-PS) for the production of the polyketide triacetic acid lactone (TAL). Titers of TAL relative to the base strain improved only 30% with the native E. coli PDH, but 3.0-fold with PDHm and 4.4-fold with PDHm in the Δzwf1 strain. Carbon was further routed toward TAL production by reducing mitochondrial transport of pyruvate and acetyl-CoA; deletions in genes POR2, MPC2, PDA1, or YAT2 each increased titer 2–3-fold over the base strain (up to 0.8 g/L), and in combination to 1.4 g/L. Combining the two approaches (NADPH-generating acetyl-CoA pathway plus reduced metabolite flux into the mitochondria) resulted in a final TAL titer of 1.6 g/L, a 6.4-fold increase over the non-engineered yeast strain, and 35% of theoretical yield (0.16 g/g glucose), the highest reported to date. These biological driving forces present new avenues for improving high-yield production of acetyl-CoA derived compounds.     

Engineering of Saccharomyces cerevisiae for efficient anaerobic alcoholic fermentation of L-arabinose

For cost-effective and efficient ethanol production from lignocellulosic fractions of plant biomass, the conversion of not only major constituents, such as glucose and xylose, but also less predominant sugars, such as l-arabinose, is required. Wild-type strains of Saccharomyces cerevisiae, the organism used in industrial ethanol production, cannot ferment xylose and arabinose. Although metabolic and evolutionary engineering has enabled the efficient alcoholic fermentation of xylose under anaerobic conditions, the conversion of l-arabinose into ethanol by engineered S. cerevisiae strains has previously been demonstrated only under oxygen-limited conditions. This study reports the first case of fast and efficient anaerobic alcoholic fermentation of l-arabinose by an engineered S. cerevisiae strain. This fermentation was achieved by combining the expression of the structural genes for the l-arabinose utilization pathway of Lactobacillus plantarum, the overexpression of the S. cerevisiae genes encoding the enzymes of the nonoxidative pentose phosphate pathway, and extensive evolutionary engineering. The resulting S. cerevisiae strain exhibited high rates of arabinose consumption (0.70 g h(-1) g [dry weight](-1)) and ethanol production (0.29 g h(-1) g [dry weight](-1)) and a high ethanol yield (0.43 g g(-1)) during anaerobic growth on l-arabinose as the sole carbon source. In addition, efficient ethanol production from sugar mixtures containing glucose and arabinose, which is crucial for application in industrial ethanol production, was achieved.     

Arabinose and xylose fermentation by recombinant Saccharomyces cerevisiae expressing a fungal pentose utilization pathway

Background  

Sustainable and economically viable manufacturing of bioethanol from lignocellulose raw material is dependent on the availability of a robust ethanol producing microorganism, able to ferment all sugars present in the feedstock, including the pentose sugars L-arabinose and D-xylose. Saccharomyces cerevisiae is a robust ethanol producer, but needs to be engineered to achieve pentose sugar fermentation.     

Engineering Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: reflections and perspectives

Conversion of the abundant lignocellulosic biomass into ethanol is an environmentally sustainable solution to the energy crisis. Fermentation of lignocellulosic hydrolysates by Saccharomyces cerevisiae is not cost-effective yet as substantial amounts of xylose in the hydrolysates cannot be utilized by native S. cerevisiae strains. Extensive studies including both metabolic and evolutionary engineering have been carried out to develop an efficient xylose-fermenting S. cerevisiae strain, yet the ethanol yield and productivity from xylose fermentation of the best one are still far below expectation. This review compares the engineering approaches and resulted anaerobic xylose fermentation performance of recently reported xylose-utilizing S. cerevisiae strains, with the aim to understand the intrinsic reason for their low xylose fermentation capabilities. These comparative analyses revealed that some of the current engineering targets and the so-called "hot issues" might be overrated. Our opinions on the underrated parts and future efforts in this field are also presented. Overall, this review serves as a comprehensive reference to understanding xylose fermentation by S. cerevisiae.     

Physiological and genetic engineering of cytosolic redox metabolism in Saccharomyces cerevisiae for improved glycerol production

Previous metabolic engineering strategies for improving glycerol production by Saccharomyces cerevisiae were constrained to a maximum theoretical glycerol yield of 1 mol.(molglucose)(-1) due to the introduction of rigid carbon, ATP or redox stoichiometries. In the present study, we sought to circumvent these constraints by (i) maintaining flexibility at fructose-1,6-bisphosphatase and triosephosphate isomerase, while (ii) eliminating reactions that compete with glycerol formation for cytosolic NADH and (iii) enabling oxidative catabolism within the mitochondrial matrix. In aerobic, glucose-grown batch cultures a S. cerevisiae strain, in which the pyruvate decarboxylases the external NADH dehydrogenases and the respiratory chain-linked glycerol-3-phosphate dehydrogenase were deleted for this purpose, produced glycerol at a yield of 0.90 mol.(molglucose)(-1). In aerobic glucose-limited chemostat cultures, the glycerol yield was ca. 25% lower, suggesting the involvement of an alternative glucose-sensitive mechanism for oxidation of cytosolic NADH. Nevertheless, in vivo generation of additional cytosolic NADH by co-feeding of formate to aerobic, glucose-limited chemostat cultures increased the glycerol yield on glucose to 1.08 mol mol(-1). To our knowledge, this is the highest glycerol yield reported for S. cerevisiae.     

Maltotriose fermentation by Saccharomyces cerevisiae

Maltotriose, the second most abundant sugar of brewer's wort, is not fermented but is respired by several industrial yeast strains. We have isolated a strain capable of growing on a medium containing maltotriose and the respiratory inhibitor, antimycin A. This strain produced equivalent amounts of ethanol from 20 g l⁻¹ glucose, maltose, or maltotriose. We performed a detailed analysis of the rates of active transport and intracellular hydrolysis of maltotriose by this strain, and by a strain that does not ferment this sugar. The kinetics of sugar hydrolysis by both strains was similar, and our results also indicated that yeast cells do not synthesize a maltotriose-specific α-glucosidase. However, when considering active sugar transport, a different pattern was observed. The maltotriose-fermenting strain showed the same rate of active maltose or maltotriose transport, while the strain that could not ferment maltotriose showed a lower rate of maltotriose transport when compared with the rates of active maltose transport. Thus, our results revealed that transport across the plasma membrane, and not intracellular hydrolysis, is the rate-limiting step for the fermentation of maltotriose by these Saccharomyces cerevisiae cells. Journal of Industrial Microbiology & Biotechnology (2001) 27, 34–38. Received 13 January 2001/ Accepted in revised form 29 May 2001     

Metabolic engineering of a phosphoketolase pathway for pentose catabolism in Saccharomyces cerevisiae

Low ethanol yields on xylose hamper economically viable ethanol production from hemicellulose-rich plant material with Saccharomyces cerevisiae. A major obstacle is the limited capacity of yeast for anaerobic reoxidation of NADH. Net reoxidation of NADH could potentially be achieved by channeling carbon fluxes through a recombinant phosphoketolase pathway. By heterologous expression of phosphotransacetylase and acetaldehyde dehydrogenase in combination with the native phosphoketolase, we installed a functional phosphoketolase pathway in the xylose-fermenting Saccharomyces cerevisiae strain TMB3001c. Consequently the ethanol yield was increased by 25% because less of the by-product xylitol was formed. The flux through the recombinant phosphoketolase pathway was about 30% of the optimum flux that would be required to completely eliminate xylitol and glycerol accumulation. Further overexpression of phosphoketolase, however, increased acetate accumulation and reduced the fermentation rate. By combining the phosphoketolase pathway with the ald6 mutation, which reduced acetate formation, a strain with an ethanol yield 20% higher and a xylose fermentation rate 40% higher than those of its parent was engineered.     

Xylose fermentation by Saccharomyces cerevisiae

We have performed a comparative study of xylose utilization in Saccharomyces cerevisiae transformants expressing two key enzymes in xylose metabolism, xylose reductase (XR) and xylitol dehydrogenase (XDH), and in a prototypic xylose-utilizing yeast, Pichia stipitis. In the absence of respiration (see text), baker's yeast cells convert half of the xylose to xylitol and ethanol, whereas P. stipilis cells display rather a homofermentative conversion of xylose to ethanol. Xylitol production by baker's yeast is interpreted as a result of the dual cofactor dependence of the XR and the generation of NADPH by the pentose phosphate pathway. Further limitations of xylose utilization in S. cerevisiae cells are very likely caused by an insufficient capacity of the non-oxidative pentose phosphate pathway, as indicated by accumulation of sedoheptulose-7-phosphate and the absence of fructose-1,6-bisphosphate and pyruvate accumulation. By contrast, uptake at high substrate concentrations probably does not limit xylose conversion in S. cerevisiae XYL1/XYL2 transformants. Correspondence to: M. Ciriacy     

Implementation of a transhydrogenase-like shunt to counter redox imbalance during xylose fermentation in Saccharomyces cerevisiae

Three enzymes responsible for the transhydrogenase-like shunt, including malic enzyme (encoded by MAE1), malate dehydrogenase (MDH2), and pyruvate carboxylase (PYC2), were overexpressed to regulate the redox state in xylose-fermenting recombinant Saccharomyces cerevisiae. The YPH499XU/MAE1 strain was constructed by overexpressing native Mae1p in the YPH499XU strain expressing xylose reductase and xylitol dehydrogenase from Scheffersomyces stipitis, and native xylulokinase. Analysis of the xylose fermentation profile under semi-anaerobic conditions revealed that the ethanol yield in the YPH499XU/MAE1 strain (0.38?±?0.01 g g^?1 xylose consumed) was improved from that of the control strain (0.31?±?0.01 g g^?1 xylose consumed). Reduced xylitol production was also observed in YPH499XU/MAE1, suggesting that the redox balance was altered by Mae1p overexpression. Analysis of intracellular metabolites showed that the redox imbalance during xylose fermentation was partly relieved in the transformant. The specific ethanol production rate in the YPH499XU/MAE1–MDH2 strain was 1.25-fold higher than that of YPH499XU/MAE1 due to the additional overexpression of Mdh2p, whereas the ethanol yield was identical to that of YPH499XU/MAE1. The specific xylose consumption rate was drastically increased in the YPH499XU/MAE1–MDH2–PYC2 strain. However, poor ethanol yield as well as increased production of xylitol was observed. These results demonstrate that the transhydrogenase function implemented in S. cerevisiae can regulate the redox state of yeast cells.     

构建酿酒酵母工程菌合成香紫苏醇

香紫苏醇是一种来源于植物的双环二萜醇,常用于香味成分且具有重要生物学活性。为实现香紫苏醇的微生物生产,以酿酒酵母为宿主,表达焦磷酸赖百当烯二醇酯合酶和香紫苏醇合酶,构建香紫苏醇的人工生物合成途径。发现过表达前体代谢关键酶、蛋白质融合增强底物通道效应及去除异源蛋白信号肽等,有利于香紫苏醇合成。在摇瓶培养条件下,组合优化得到的工程菌株S6的香紫苏醇产量达到8.96 mg/L。研究结果对其他萜类化合物的异源生物合成具有参考价值。     

Engineering Saccharomyces cerevisiae for isoprenol production

Isoprenol (3-methyl-3-butene-1-ol) is a valuable drop-in biofuel and an important precursor of several commodity chemicals. Synthetic microbial systems using the heterologous mevalonate pathway have recently been developed for the production of isoprenol in Escherichia coli, and a significant yield and titer improvement has been achieved through a decade of research. Saccharomyces cerevisiae has been widely used in the biotechnology industry for isoprenoid production, but there has been no good example of isoprenol production reported in this host. In this study, we engineered the budding yeast S. cerevisiae for improved biosynthesis of isoprenol. The strain engineered with the mevalonate pathway achieved isoprenol production at the titer of 36.02 ± 0.92 mg/L in the flask. The IPP (isopentenyl diphosphate)-bypass pathway, which has shown more efficient isoprenol production by avoiding the accumulation of the toxic intermediate in E. coli, was also constructed in S. cerevisiae and improved the isoprenol titer by 2-fold. We further engineered the strains by deleting a promiscuous endogenous kinase that could divert the pathway flux away from the isoprenol production and improved the titer to 130.52 ± 8.01 mg/L. Finally, we identified a pathway bottleneck using metabolomics analysis and overexpressed a promiscuous alkaline phosphatase to relieve this bottleneck. The combined efforts resulted in the titer improvement to 383.1 ± 31.62 mg/L in the flask. This is the highest isoprenol titer up to date in S. cerevisiae and this work provides the key strategies to engineer yeast as an industrial platform for isoprenol production.