一种基因工程改造的NADH高亲和力木糖还原酶突变基因可促进酿酒酵母发酵木糖生成乙醇

在导入表达毕赤酵母(Pichia stipitis)木糖还原酶(xylose reductase,XR)和木糖醇脱氢酶(xylitol dehydrogenase,XDH)基因的重组酿酒酵母中,木糖还原酶活性主要依赖辅酶NADPH,木糖醇脱氢酶活性依赖辅酶 NAD+,两者的辅助因子不同导致细胞内电子氧化还原的不平衡,是造成木糖醇积累,影响木糖代谢和乙醇产量的主要原因之一.将经过基因工程改造获得的NADH高亲和力的木糖还原酶突变基因m1,与毕赤酵母木糖醇脱氢酶(PsXDH)基因xyl2共转染酿酒酵母AH109,以转染毕赤酵母木糖还原酶(PsXR)基因xyl1和xyl2重组质粒的酵母细胞为对照菌株,在SC/-Leu/-Trp营养缺陷型培养基中进行筛选,获得的阳性转化子分别命名为AH-M-XDH和AH-XR-XDH.重组酵母在限制氧通气条件下对木糖和葡萄糖进行共发酵摇瓶培养,HPLC检测发酵底物的消耗和代谢产物的产出情况.结果显示,与对照菌株AH-XR-XDH相比,AH-M-XDH的木糖利用率明显提高,乙醇得率增加了16%,木糖醇产生下降了41.4%.结果证实,通过基因工程改造的木糖代谢关键酶,可用于酿酒酵母发酵木糖生产乙醇,其能通过改善酿酒酵母细胞内氧化还原失衡的问题,提高木糖利用率和乙醇产率.     

代谢木糖产乙醇的酿酒酵母工程菌研究进展

随着能源价格的持续上涨,使用木质纤维素生产燃料乙醇已具有重要的实践意义.木糖是多数木质纤维素水解产物中含量仅次于葡萄糖的一种单糖,传统乙醇生产菌株酿酒酵母不能利用木糖,这为使用以木质纤维素为原料发酵生产乙醇带来了困难.多年以来人们试图通过基因工程和细胞融合等方法对其进行改造使其能够代谢木糖生产乙醇.本文主要介绍这方面的研究进展.     

Genetic Engineering of Zymobacter palmae for Production of Ethanol from Xylose

Its metabolic characteristics suggest that Zymobacter palmae gen. nov., sp. nov. could serve as a useful new ethanol-fermenting bacterium, but its biotechnological exploitation will require certain genetic modifications. We therefore engineered Z. palmae so as to broaden the range of its fermentable sugar substrates to include the pentose sugar xylose. The Escherichia coli genes encoding the xylose catabolic enzymes xylose isomerase, xylulokinase, transaldolase, and transketolase were introduced into Z. palmae, where their expression was driven by the Zymomonas mobilis glyceraldehyde-3-phosphate dehydrogenase promoter. When cultured with 40 g/liter xylose, the recombinant Z. palmae strain was able to ferment 16.4 g/liter xylose within 5 days, producing 91% of the theoretical yield of ethanol with no accumulation of organic acids as metabolic by-products. Notably, xylose acclimation enhanced both the expression of xylose catabolic enzymes and the rate of xylose uptake into recombinant Z. palmae, which enabled the acclimated organism to completely and simultaneously ferment a mixture of 40 g/liter glucose and 40 g/liter xylose within 8 h, producing 95% of the theoretical yield of ethanol. Thus, efficient fermentation of a mixture of glucose and xylose to ethanol can be accomplished by using Z. palmae expressing E. coli xylose catabolic enzymes.     

代谢木糖产乙醇的酿酒酵母工程菌研究进展

随着能源价格的持续上涨, 使用木质纤维素生产燃料乙醇已具有重要的实践意义。木糖是多数木质纤维素水解产物中含量仅次于葡萄糖的一种单糖, 传统乙醇生产菌株酿酒酵母不能利用木糖, 这为使用以木质纤维素为原料发酵生产乙醇带来了困难。多年以来人们试图通过基因工程和细胞融合等方法对其进行改造使其能够代谢木糖生产乙醇。本文主要介绍这方面的研究进展。     

Improvement of Xylose Uptake and Ethanol Production in Recombinant Saccharomyces cerevisiae through an Inverse Metabolic Engineering Approach

We used an inverse metabolic engineering approach to identify gene targets for improved xylose assimilation in recombinant Saccharomyces cerevisiae. Specifically, we created a genomic fragment library from Pichia stipitis and introduced it into recombinant S. cerevisiae expressing XYL1 and XYL2. Through serial subculturing enrichment of the transformant library, 16 transformants were identified and confirmed to have a higher growth rate on xylose. Sequencing of the 16 plasmids isolated from these transformants revealed that the majority of the inserts (10 of 16) contained the XYL3 gene, thus confirming the previous finding that XYL3 is the consensus target for increasing xylose assimilation. Following a sequential search for gene targets, we repeated the complementation enrichment process in a XYL1 XYL2 XYL3 background and identified 15 fast-growing transformants, all of which harbored the same plasmid. This plasmid contained an open reading frame (ORF) designated PsTAL1 based on a high level of homology with S. cerevisiae TAL1. To further investigate whether the newly identified PsTAL1 ORF is responsible for the enhanced-growth phenotype, we constructed an expression cassette containing the PsTAL1 ORF under the control of a constitutive promoter and transformed it into an S. cerevisiae recombinant expressing XYL1, XYL2, and XYL3. The resulting recombinant strain exhibited a 100% increase in the growth rate and a 70% increase in ethanol production (0.033 versus 0.019 g ethanol/g cells · h) on xylose compared to the parental strain. Interestingly, overexpression of PsTAL1 did not cause growth inhibition when cells were grown on glucose, unlike overexpression of the ScTAL1 gene. These results suggest that PsTAL1 is a better gene target for engineering of the pentose phosphate pathway in recombinant S. cerevisiae.     

酿酒酵母木糖发酵酒精途径工程的研究进展

途径工程(Pathway engineering),被称为第三代基因工程,改变代谢流向,开辟新的代谢途径是途径工程的主要目的。利用途径工程理念,对酿酒酵母（Saccharomyces cerevisiae）代谢途径进行理性设计,以拓展这一传统酒精生产菌的底物范围,使其充分利用可再生纤维质水解物中的各种糖分,是酿酒酵母酒精途径工程的研究热点之一。这里介绍了近年来酿酒酵母以木糖为底物的酒精途径工程的研究进展。     

Evolutionary Engineering of Saccharomyces cerevisiae for Anaerobic Growth on Xylose

Xylose utilization is of commercial interest for efficient conversion of abundant plant material to ethanol. Perhaps the most important ethanol-producing organism, Saccharomyces cerevisiae, however, is incapable of xylose utilization. While S. cerevisiae strains have been metabolically engineered to utilize xylose, none of the recombinant strains or any other naturally occurring yeast has been able to grow anaerobically on xylose. Starting with the recombinant S. cerevisiae strain TMB3001 that overexpresses the xylose utilization pathway from Pichia stipitis, in this study we developed a selection procedure for the evolution of strains that are capable of anaerobic growth on xylose alone. Selection was successful only when organisms were first selected for efficient aerobic growth on xylose alone and then slowly adapted to microaerobic conditions and finally anaerobic conditions, which indicated that multiple mutations were necessary. After a total of 460 generations or 266 days of selection, the culture reproduced stably under anaerobic conditions on xylose and consisted primarily of two subpopulations with distinct phenotypes. Clones in the larger subpopulation grew anaerobically on xylose and utilized both xylose and glucose simultaneously in batch culture, but they exhibited impaired growth on glucose. Surprisingly, clones in the smaller subpopulation were incapable of anaerobic growth on xylose. However, as a consequence of their improved xylose catabolism, these clones produced up to 19% more ethanol than the parental TMB3001 strain produced under process-like conditions from a mixture of glucose and xylose.     

Metabolic Engineering of Ammonium Assimilation in Xylose-Fermenting Saccharomyces cerevisiae Improves Ethanol Production

Cofactor imbalance impedes xylose assimilation in Saccharomyces cerevisiae that has been metabolically engineered for xylose utilization. To improve cofactor use, we modified ammonia assimilation in recombinant S. cerevisiae by deleting GDH1, which encodes an NADPH-dependent glutamate dehydrogenase, and by overexpressing either GDH2, which encodes an NADH-dependent glutamate dehydrogenase, or GLT1 and GLN1, which encode the GS-GOGAT complex. Overexpression of GDH2 increased ethanol yield from 0.43 to 0.51 mol of carbon (Cmol) Cmol⁻¹, mainly by reducing xylitol excretion by 44%. Overexpression of the GS-GOGAT complex did not improve conversion of xylose to ethanol during batch cultivation, but it increased ethanol yield by 16% in carbon-limited continuous cultivation at a low dilution rate.     

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     

辅酶工程在酿酒酵母木糖代谢工程中的研究进展

辅酶工程（cofactor engineering）是代谢工程的一个重要分支,它通过改变辅酶的再生途径,达到改变细胞内代谢产物构成的目的。介绍了酿酒酵母(Saccharomyces cerevisiae)木糖代谢工程中,利用辅酶工程解决氧化还原平衡问题的研究进展,包括引入转氢酶系统,增加代谢中可利用的NADPH,实现NADH的厌氧氧化等策略。同时介绍了改变XR、XDH辅酶偏好的研究进展。     

Novel Evolutionary Engineering Approach for Accelerated Utilization of Glucose,Xylose, and Arabinose Mixtures by Engineered Saccharomyces cerevisiae Strains

Lignocellulosic feedstocks are thought to have great economic and environmental significance for future biotechnological production processes. For cost-effective and efficient industrial processes, complete and fast conversion of all sugars derived from these feedstocks is required. Hence, simultaneous or fast sequential fermentation of sugars would greatly contribute to the efficiency of production processes. One of the main challenges emerging from the use of lignocellulosics for the production of ethanol by the yeast Saccharomyces cerevisiae is efficient fermentation of d-xylose and l-arabinose, as these sugars cannot be used by natural S. cerevisiae strains. In this study, we describe the first engineered S. cerevisiae strain (strain IMS0003) capable of fermenting mixtures of glucose, xylose, and arabinose with a high ethanol yield (0.43 g g⁻¹ of total sugar) without formation of the side products xylitol and arabinitol. The kinetics of anaerobic fermentation of glucose-xylose-arabinose mixtures were greatly improved by using a novel evolutionary engineering strategy. This strategy included a regimen consisting of repeated batch cultivation with repeated cycles of consecutive growth in three media with different compositions (glucose, xylose, and arabinose; xylose and arabinose; and only arabinose) and allowed rapid selection of an evolved strain (IMS0010) exhibiting improved specific rates of consumption of xylose and arabinose. This evolution strategy resulted in a 40% reduction in the time required to completely ferment a mixture containing 30 g liter⁻¹ glucose, 15 g liter⁻¹ xylose, and 15 g liter⁻¹ arabinose.In recent years, the need for biotechnological manufacturing based on lignocellulosic feedstocks has become evident (6, 10). In contrast to the readily fermentable, mainly starch- or sucrose-containing feedstocks used in current biotechnological production processes, lignocellulosic biomass requires intensive pretreatment and hydrolysis, which yield complex mixtures of sugars (3, 7, 14, 27). For cost-effective and efficient industrial processes, complete and fast conversion of all sugars present in lignocellulosic hydrolysates is a prerequisite. The major hurdles encountered in implementing these production processes are the conversion of substrates that cannot be utilized by the organism of choice and, even more importantly, the subsequent improvement of sugar conversion rates and product yields.The use of evolutionary engineering has proven to be very valuable for obtaining phenotypes of (industrial) microorganisms with improved properties, such as an expanded substrate range, increased stress tolerance, and efficient substrate utilization (16, 17). Also, for the yeast Saccharomyces cerevisiae, the preferred organism for large-scale ethanol production for the past few decades, evolutionary engineering has been extensively used to select for industrially relevant phenotypes. For ethanol production from lignocellulose by S. cerevisiae, one of the main challenges is efficient conversion of the pentoses d-xylose and l-arabinose to ethanol. To deal with this challenge, S. cerevisiae strains have been metabolically engineered since the early 1990s for the conversion of xylose into ethanol by the introduction of heterologous xylose utilization pathways (for recent reviews, see references 9 and 20). Arabinose utilization, however, has been addressed only quite recently. The most successful approach for obtaining arabinose consumption in S. cerevisiae has been the introduction of a bacterial arabinose utilization pathway (5, 26). In addition to metabolic engineering, extensive evolutionary engineering (by prolonged cultivation of recombinant S. cerevisiae strains in either anaerobic chemostat or repeated anaerobic batch cultures) was required to obtain S. cerevisiae strains that ferment either xylose (13, 19) or arabinose (5, 26) fast or to improve fermentation performance with mixtures containing glucose and xylose (12). In contrast, (evolutionary) engineering has still not resulted in fast and efficient fermentation of both xylose and arabinose to ethanol by a single recombinant S. cerevisiae strain. At best, simultaneous utilization of xylose and arabinose yielded large amounts of the undesirable side products xylitol and arabinitol (11). Hence, a major remaining challenge is the conversion of both xylose and arabinose with high ethanol production rates and yields.In a previous study, an S. cerevisiae strain was metabolically engineered to obtain both xylose and arabinose utilization. For this, the Piromyces XylA, S. cerevisiae XKS1, and Lactobacillus plantarum araA, araB, and araD genes, as well as the endogenous genes of the pentose phosphate pathway (RPE1, RKI1, TKL1, and TAL1), were overexpressed. Selection by sequential batch cultivation under conditions with arabinose as the sole carbon source resulted in strain IMS0002, which is capable of fermenting arabinose to ethanol under anaerobic conditions (26). Unfortunately, the ability to ferment xylose to ethanol was largely lost during long-term selection for improved l-arabinose fermentation. During anaerobic batch cultivation of strain IMS0002 in a glucose-xylose-arabinose mixture, xylose was not consumed completely and was converted to almost equimolar amounts of xylitol. This loss of xylose metabolism illustrates the limitations of selection in media supplemented with a single carbon and energy source.The goal of the present study was to evaluate and optimize selection strategies for evolutionary optimization of the utilization of substrate mixtures. Fermentation of glucose, xylose, and arabinose mixtures by engineered S. cerevisiae strains was used as the model.     

Metabolic Engineering of Glycerol Production in Saccharomyces cerevisiae

Inactivation of TPI1, the Saccharomyces cerevisiae structural gene encoding triose phosphate isomerase, completely eliminates growth on glucose as the sole carbon source. In tpi1-null mutants, intracellular accumulation of dihydroxyacetone phosphate might be prevented if the cytosolic NADH generated in glycolysis by glyceraldehyde-3-phosphate dehydrogenase were quantitatively used to reduce dihydroxyacetone phosphate to glycerol. We hypothesize that the growth defect of tpi1-null mutants is caused by mitochondrial reoxidation of cytosolic NADH, thus rendering it unavailable for dihydroxyacetone-phosphate reduction. To test this hypothesis, a tpi1Δ nde1Δ nde2Δ gut2Δ quadruple mutant was constructed. NDE1 and NDE2 encode isoenzymes of mitochondrial external NADH dehydrogenase; GUT2 encodes a key enzyme of the glycerol-3-phosphate shuttle. It has recently been demonstrated that these two systems are primarily responsible for mitochondrial oxidation of cytosolic NADH in S. cerevisiae. Consistent with the hypothesis, the quadruple mutant grew on glucose as the sole carbon source. The growth on glucose, which was accompanied by glycerol production, was inhibited at high-glucose concentrations. This inhibition was attributed to glucose repression of respiratory enzymes as, in the quadruple mutant, respiratory pyruvate dissimilation is essential for ATP synthesis and growth. Serial transfer of the quadruple mutant on high-glucose media yielded a spontaneous mutant with much higher specific growth rates in high-glucose media (up to 0.10 h⁻¹ at 100 g of glucose·liter⁻¹). In aerated batch cultures grown on 400 g of glucose·liter⁻¹, this engineered S. cerevisiae strain produced over 200 g of glycerol·liter⁻¹, corresponding to a molar yield of glycerol on glucose close to unity.     

Engineering of Cyclodextrin Glucanotransferase on the Cell Surface of Saccharomyces cerevisiae for Improved Cyclodextrin Production

The cyclodextrin glucanotransferase (CGTase) gene (cgt) from Bacillus circulans 251 was cloned into plasmid pYD1, which allowed regulated expression, secretion, and detection. The expression of CGTase with a-agglutinin at the N-terminal end on the extracellular surface of Saccharomyces cerevisiae was confirmed by immunofluorescence microscopy. This surface-anchored CGTase gave the yeast the ability to directly utilize starch as a sole carbon source and the ability to produce the anticipated products, cyclodextrins, as well as glucose and maltose. The resulting glucose and maltose, which are efficient acceptors in the CGTase coupling reaction, could be consumed by yeast fermentation and thus facilitated cyclodextrin production. On the other hand, ethanol produced by the yeast may form a complex with cyclodextrin and shift the equilibrium in favor of cyclodextrin production. The yeast with immobilized CGTase produced 24.07 mg/ml cyclodextrins when it was incubated in yeast medium supplemented with 4% starch.     

啤酒酵母代谢工程研究进展

啤酒工业上应用的啤酒酵母菌株在生产中都会存在着某些方面的缺陷。通过分析啤酒酵母某些代谢产物的代谢途径,寻找改变其代谢流量的方法,然后用分子生物学手段对其代谢流量加以改变,来调节啤酒酵母某些产物的代谢水平已经成为啤酒酵母育种的新方式。对酵母的底物利用、可操作性、控制有害副产物的产量及改善啤酒风味等方面的研究成果进行了综述。     

Xylose Assimilation for the Efficient Production of Biofuels and Chemicals by Engineered Saccharomyces cerevisiae

Microbial conversion of plant biomass into fuels and chemicals offers a practical solution to global concerns over limited natural resources, environmental pollution, and climate change. Pursuant to these goals, researchers have put tremendous efforts and resources toward engineering the yeast Saccharomyces cerevisiae to efficiently convert xylose, the second most abundant sugar in lignocellulosic biomass, into various fuels and chemicals. Here, recent advances in metabolic engineering of yeast is summarized to address bottlenecks on xylose assimilation and to enable simultaneous co-utilization of xylose and other substrates in lignocellulosic hydrolysates. Distinct characteristics of xylose metabolism that can be harnessed to produce advanced biofuels and chemicals are also highlighted. Although many challenges remain, recent research investments have facilitated the efficient fermentation of xylose and simultaneous co-consumption of xylose and glucose. In particular, understanding xylose-induced metabolic rewiring in engineered yeast has encouraged the use of xylose as a carbon source for producing various non-ethanol bioproducts. To boost the lignocellulosic biomass-based bioeconomy, much attention is expected to promote xylose-utilizing efficiency via reprogramming cellular regulatory networks, to attain robust co-fermentation of xylose and other cellulosic carbon sources under industrial conditions, and to exploit the advantageous traits of yeast xylose metabolism for producing diverse fuels and chemicals.     

Ethanol Production by Saccharomyces cerevisiae Immobilized in Hollow-Fiber Membrane Bioreactors

Saccharomyces cerevisiae ATCC 4126 was grown within the macroporous matrix of asymmetric-walled polysulfone hollow-fiber membranes and on the exterior surfaces of isotropic-walled polypropylene hollow-fiber membranes. Nutrients were supplied and products were removed by single-pass perfusion of the fiber lumens. Growth of yeast cells within the macrovoids of the asymmetric-walled membranes attained densities of greater than 10¹⁰ cells per ml and in some regions accounted for nearly 100% of the available macrovoid volume, forming a tissue-like mass. A radial distribution of cell packing existed across the fiber wall, indicating an inadequate glucose supply to cells located beyond 100 μm from the lumen surface. By comparison, yeast cell growth on the exterior surfaces of the isotropic-walled membranes resulted in an average density of 3.5 × 10⁹ viable cells per ml. Ethanol production by reactors containing isotropic polypropylene fibers reached a maximum value of 26 g/liter-h based on the total reactor volume. Reactor performance depended on the fiber packing density and on the glucose medium flow rate and was limited by low nutrient and product transport rates. The inhibition of ethanol production and the reduction in fermentation efficiency arose primarily from the accumulation of CO₂ gas within the sealed reactor shell space.     

Production of Ethanol from Starch by Respiration-Deficient Recombinant Saccharomyces cerevisiae

A 100%-respiration-deficient nuclear petite amylolytic Saccharomyces cerevisiae NPB-G strain was generated, and its employment for direct fermentation of starch into ethanol was investigated. In a comparison of ethanol fermentation performances with the parental respiration-sufficient WTPB-G strain, the NPB-G strain showed an increase of ca. 48% in both ethanol yield and ethanol productivity.     

Metabolic Engineering of Saccharomyces cerevisiae for Caffeine and Theobromine Production

Caffeine (1, 3, 7-trimethylxanthine) and theobromine (3, 7-dimethylxanthine) are the major purine alkaloids in plants, e.g. tea (Camellia sinensis) and coffee (Coffea arabica). Caffeine is a major component of coffee and is used widely in food and beverage industries. Most of the enzymes involved in the caffeine biosynthetic pathway have been reported previously. Here, we demonstrated the biosynthesis of caffeine (0.38 mg/L) by co-expression of Coffea arabica xanthosine methyltransferase (CaXMT) and Camellia sinensis caffeine synthase (TCS) in Saccharomyces cerevisiae. Furthermore, we endeavored to develop this production platform for making other purine-based alkaloids. To increase the catalytic activity of TCS in an effort to increase theobromine production, we identified four amino acid residues based on structural analyses of 3D-model of TCS. Two TCS1 mutants (Val317Met and Phe217Trp) slightly increased in theobromine accumulation and simultaneously decreased in caffeine production. The application and further optimization of this biosynthetic platform are discussed.