生产聚羟基脂肪酸酯的微生物细胞工厂

聚羟基脂肪酸酯(PHA)是一类由微生物合成的、生物可再生、生物可降解、具有多种材料学性能的高分子聚合物,在很多领域有着广泛的应用前景。以下从辅酶工程、代谢工程、微氧生产等方面综述了微生物法生产PHA的研究进展,并对利用PHA合成基因提高基因工程菌的代谢潜能进行了讨论。     

解决氧化还原酶反应体系中辅酶问题的策略及其应用

氧化还原酶还原醛或酮生成各种手性醇或手性胺类化合物。然而,氧化还原酶的催化过程通常需要价格昂贵的烟酰胺类辅酶提供或接受电子,这严重阻碍了氧化还原酶的工业化进程。因此,如何降低辅酶的成本已成为生物催化领域的研究热点和关键问题。随着工业化应用的实际需求和研究工作的深入,各种解决辅酶问题的策略被相继提出,如构建体外辅酶再生系统,利用发酵工程与代谢工程等手段提高内源性辅酶利用率,研究和开发辅酶替代物等。文中对这些策略的研究概况进行简要介绍,并通过列举相关应用实例分析各自的优、缺点,为进一步拓展氧化还原酶的工业化应用提供借鉴和参考。     

微生物辅因子工程研究进展

微生物细胞中的大部分酶促反应都需要各种辅因子的参与,辅因子平衡对维持细胞内的生化反应稳态非常重要,辅因子供应不足会导致细胞生长和化合物生产的紊乱。近年来,辅因子在生化反应过程中的关键作用备受关注,但由于其价格较昂贵、稳定性差,因此限制了辅因子工程的发展。合成生物学和代谢工程的发展为辅因子的可持续供应提供了可行的解决方案,多种加强辅因子供应的策略有效地推动了目标化合物的生物合成。其中,烟酰胺类辅因子NAD(P)⁺、NAD(P)H是微生物代谢过程中最常见的氧化还原辅因子,它们在所有生物体内作为重要的电子受体或供体推动合成与分解代谢反应,对维持胞内氧化还原动态平衡起着决定性作用。从NAD(P)H的主要来源和NAD(P)⁺/NAD(P)H的平衡对天然产物生物合成中的影响出发,重点从三个不同维度讨论辅因子工程策略,综述代谢途径调节、外源氧化还原酶的引入、蛋白质工程等多种辅因子再生策略的最新研究进展及应用,展望辅因子代谢工程在生物合成中的未来发展方向。     

微藻生物燃料及高价值产物的代谢研究进展

微藻是当今代谢工程领域最具潜力的燃料生物质来源之一.其结构简单、基因可操作性强的特点使其可通过不同的代谢工程手段得到丰富多样的生物燃料和高价值产物.本文系统地总结了近年来国内外利用微藻在脂质、氢气、乙烯、醇类、脂肪醇和脂肪烃、糖类、萜类及其他高价值产物的生产中取得的进展,并通过介绍着眼于改变光合作用关键酶体或复合物以提升生物质产量的相关研究进展,分析了生物质合成代谢途径的改变对上游光合作用的潜在影响.结合系统生物学及生物信息学方法筛选高效的微藻株系,改变碳流方向以提升生物质的合成效率,实现高效地同源重组,提高外源基因在微藻内的表达效率是微藻代谢工程亟待解决的问题.本文在此基础上结合近年来各个交叉学科的发展趋势,提出了若干改良微藻代谢的新模式,以期对微藻代谢研究及后续的工程改造有所启示.     

代谢工程

代谢工程,也称途径工程,是基因工程一个重要分支,一般是多基因的基因工程,与细胞的基因调控、代谢调控和生化工程密切相关。讨论了代谢工程的应用,包括通过改变代谢流和代谢途径提高产量,改善生产过程,构建新的代谢途径和产生新的代谢产物等。     

逆代谢工程——基于有用表型的定向遗传工程策略

代谢工程分为推理性代谢工程和逆代谢工程,逆代谢工程是避开对代谢网络充分认识的一种全新的遗传工程。本简要介绍了逆代谢工程的策略,并以实例详细分析了逆代谢工程的具体应用,最后,分析了逆代谢工程的问题和发展前景。     

代谢工程——生物工程学科的新兴研究领域

代谢工程包括推理性代谢工程及逆代谢工程。针对限制生物活性的因素 ,从不同的途径设计代谢改变策略 ,采用重组DNA技术解除或削弱该影响 ,提高目的产物的产率 ,是生化工程学科提高生物活性的新兴研究领域。讨论了推理性代谢工程及逆代谢工程的设计及应用。     

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。     

历久弥新：进化中的代谢工程

20世纪90年代,Bailey及Stephanopoulos等提出了经典代谢工程的理念,旨在利用DNA重组技术对代谢网络进行改造,以达到细胞性能改善,目标产物增加的目的。自代谢工程诞生以来的30年,生命科学蓬勃发展,基因组学、系统生物学、合成生物学等新学科不断涌现,为代谢工程的发展注入了新的内涵与活力。经典代谢工程研究已进入到前所未有的系统代谢工程阶段。组学技术、基因组代谢模型、元件组装、回路设计、动态控制、基因组编辑等合成生物学工具与策略的应用,大大提升了复杂代谢的设计与合成能力;机器学习的介入以及进化工程与代谢工程的结合,为系统代谢工程的未来开辟了新的方向。文中对过去30年代谢工程的发展趋势作了梳理,介绍了代谢工程在发展中不断创新的理论与方法及其应用。     

嘌呤核苷及其衍生物的代谢工程

嘌呤核苷及其衍生物被广泛应用于食品和医药领域。利用诱变筛选技术可以获得嘌呤核苷类产品的工业生产菌株,但往往耗时,效率低,而且获得的某些高产菌株还存在不稳定的缺陷。菌株代谢调控与生理生化的研究为代谢工程优化嘌呤核苷类产品的合成提供了理论基础,利用代谢工程改造菌株合成嘌呤核苷也引起了研究人员的关注。系统地介绍了微生物嘌呤生物合成途径及其调控机制,综述了嘌呤核苷类产品及其衍生物的代谢工程研究进展,最后讨论了利用代谢工程改造菌株合成这些产品面临的问题及今后的研究方向。     

Impact of overexpressing NADH kinase on glucose and xylose metabolism in recombinant xylose-utilizing <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

During growth of Saccharomyces cerevisiae on glucose, the redox cofactors NADH and NADPH are predominantly involved in catabolism and biosynthesis, respectively. A deviation from the optimal level of these cofactors often results in major changes in the substrate uptake and biomass formation. However, the metabolism of xylose by recombinant S. cerevisiae carrying xylose reductase and xylitol dehydrogenase from the fungal pathway requires both NADH and NADPH and creates cofactor imbalance during growth on xylose. As one possible solution to overcoming this imbalance, the effect of overexpressing the native NADH kinase (encoded by the POS5 gene) in xylose-consuming recombinant S. cerevisiae directed either into the cytosol or to the mitochondria was evaluated. The physiology of the NADH kinase containing strains was also evaluated during growth on glucose. Overexpressing NADH kinase in the cytosol redirected carbon flow from CO₂ to ethanol during aerobic growth on glucose and to ethanol and acetate during anaerobic growth on glucose. However, cytosolic NADH kinase has an opposite effect during anaerobic metabolism of xylose consumption by channeling carbon flow from ethanol to xylitol. In contrast, overexpressing NADH kinase in the mitochondria did not affect the physiology to a large extent. Overall, although NADH kinase did not increase the rate of xylose consumption, we believe that it can provide an important source of NADPH in yeast, which can be useful for metabolic engineering strategies where the redox fluxes are manipulated.     

Engineering redox cofactor regeneration for improved pentose fermentation in Saccharomyces cerevisiae

Pentose fermentation to ethanol with recombinant Saccharomyces cerevisiae is slow and has a low yield. A likely reason for this is that the catabolism of the pentoses D-xylose and L-arabinose through the corresponding fungal pathways creates an imbalance of redox cofactors. The process, although redox neutral, requires NADPH and NAD+, which have to be regenerated in separate processes. NADPH is normally generated through the oxidative part of the pentose phosphate pathway by the action of glucose-6-phosphate dehydrogenase (ZWF1). To facilitate NADPH regeneration, we expressed the recently discovered gene GDP1, which codes for a fungal NADP+-dependent D-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH) (EC 1.2.1.13), in an S. cerevisiae strain with the D-xylose pathway. NADPH regeneration through an NADP-GAPDH is not linked to CO2 production. The resulting strain fermented D-xylose to ethanol with a higher rate and yield than the corresponding strain without GDP1; i.e., the levels of the unwanted side products xylitol and CO2 were lowered. The oxidative part of the pentose phosphate pathway is the main natural path for NADPH regeneration. However, use of this pathway causes wasteful CO2 production and creates a redox imbalance on the path of anaerobic pentose fermentation to ethanol because it does not regenerate NAD+. The deletion of the gene ZWF1 (which codes for glucose-6-phosphate dehydrogenase), in combination with overexpression of GDP1 further stimulated D-xylose fermentation with respect to rate and yield. Through genetic engineering of the redox reactions, the yeast strain was converted from a strain that produced mainly xylitol and CO2 from D-xylose to a strain that produced mainly ethanol under anaerobic conditions.     

Comparative metabolic network analysis of two xylose fermenting recombinant Saccharomyces cerevisiae strains

The recombinant xylose fermenting strain Saccharomyces cerevisiae TMB3001 can grow on xylose, but the xylose utilisation rate is low. One important reason for the inefficient fermentation of xylose to ethanol is believed to be the imbalance of redox co-factors. In the present study, a metabolic flux model was constructed for two recombinant S. cerevisiae strains: TMB3001 and CPB.CR4 which in addition to xylose metabolism have a modulated redox metabolism, i.e. ammonia assimilation was shifted from being NADPH to NADH dependent by deletion of gdh1 and over-expression of GDH2. The intracellular fluxes were estimated for both strains in anaerobic continuous cultivations when the growth limiting feed consisted of glucose (2.5 g L-1) and xylose (13 g L-1). The metabolic network analysis with 13C labelled glucose showed that there was a shift in the specific xylose reductase activity towards use of NADH as co-factor rather than NADPH. This shift is beneficial for solving the redox imbalance and it can therefore partly explain the 25% increase in the ethanol yield observed for CPB.CR4. Furthermore, the analysis indicated that the glyoxylate cycle was activated in CPB.CR4.     

Stoichiometric network constraints on xylose metabolism by recombinant Saccharomyces cerevisiae

Metabolic pathway engineering is constrained by the thermodynamic and stoichiometric feasibility of enzymatic activities of introduced genes. Engineering of xylose metabolism in Saccharomyces cerevisiae has focused on introducing genes for the initial xylose assimilation steps from Pichia stipitis, a xylose-fermenting yeast, into S. cerevisiae, a yeast traditionally used in ethanol production from hexose. However, recombinant S. cerevisiae created in several laboratories have used xylose oxidatively rather than in the fermentative manner that this yeast metabolizes glucose. To understand the differences between glucose and engineered xylose metabolic networks, we performed a flux balance analysis (FBA) and calculated extreme pathways using a stoichiometric model that describes the biochemistry of yeast cell growth. FBA predicted that the ethanol yield from xylose exhibits a maximum under oxygen-limited conditions, and a fermentation experiment confirmed this finding. Fermentation results were largely consistent with in silico phenotypes based on calculated extreme pathways, which displayed several phases of metabolic phenotype with respect to oxygen availability from anaerobic to aerobic conditions. However, in contrast to the model prediction, xylitol production continued even after the optimum aeration level for ethanol production was attained. These results suggest that oxygen (or some other electron accepting system) is required to resolve the redox imbalance caused by cofactor difference between xylose reductase and xylitol dehydrogenase, and that other factors limit glycolytic flux when xylose is the sole carbon source.     

The expression of a Pichia stipitis xylose reductase mutant with higher K(M) for NADPH increases ethanol production from xylose in recombinant Saccharomyces cerevisiae

Xylose fermentation by Saccharomyces cerevisiae requires the introduction of a xylose pathway, either similar to that found in the natural xylose-utilizing yeasts Pichia stipitis and Candida shehatae or similar to the bacterial pathway. The use of NAD(P)H-dependent XR and NAD(+)-dependent XDH from P. stipitis creates a cofactor imbalance resulting in xylitol formation. The effect of replacing the native P. stipitis XR with a mutated XR with increased K(M) for NADPH was investigated for xylose fermentation to ethanol by recombinant S. cerevisiae strains. Enhanced ethanol yields accompanied by decreased xylitol yields were obtained in strains carrying the mutated XR. Flux analysis showed that strains harboring the mutated XR utilized a larger fraction of NADH for xylose reduction. The overproduction of the mutated XR resulted in an ethanol yield of 0.40 g per gram of sugar and a xylose consumption rate of 0.16 g per gram of biomass per hour in chemostat culture (0.06/h) with 10 g/L glucose and 10 g/L xylose as carbon source.     

Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle

When xylose metabolism in yeasts proceeds exclusively via NADPH-specific xylose reductase and NAD-specific xylitol dehydrogenase, anaerobic conversion of the pentose to ethanol is intrinsically impossible. When xylose reductase has a dual specificity for both NADPH and NADH, anaerobic alcoholic fermentation is feasible but requires the formation of large amounts of polyols (e.g., xylitol) to maintain a closed redox balance. As a result, the ethanol yield on xylose will be sub-optimal. This paper demonstrates that anaerobic conversion of xylose to ethanol, without substantial by-product formation, is possible in Saccharomyces cerevisiae when a heterologous xylose isomerase (EC 5.3.1.5) is functionally expressed. Transformants expressing the XylA gene from the anaerobic fungus Piromyces sp. E2 (ATCC 76762) grew in synthetic medium in shake-flask cultures on xylose with a specific growth rate of 0.005 h(-1). After prolonged cultivation on xylose, a mutant strain was obtained that grew aerobically and anaerobically on xylose, at specific growth rates of 0.18 and 0.03 h(-1), respectively. The anaerobic ethanol yield was 0.42 g ethanol x g xylose(-1) and also by-product formation was comparable to that of glucose-grown anaerobic cultures. These results illustrate that only minimal genetic engineering is required to recruit a functional xylose metabolic pathway in Saccharomyces cerevisiae. Activities and/or regulatory properties of native S. cerevisiae gene products can subsequently be optimised via evolutionary engineering. These results provide a gateway towards commercially viable ethanol production from xylose with S. cerevisiae.     

Reduced oxidative pentose phosphate pathway flux in recombinant xylose-utilizing Saccharomyces cerevisiae strains improves the ethanol yield from xylose

In recombinant, xylose-fermenting Saccharomyces cerevisiae, about 30% of the consumed xylose is converted to xylitol. Xylitol production results from a cofactor imbalance, since xylose reductase uses both NADPH and NADH, while xylitol dehydrogenase uses only NAD(+). In this study we increased the ethanol yield and decreased the xylitol yield by lowering the flux through the NADPH-producing pentose phosphate pathway. The pentose phosphate pathway was blocked either by disruption of the GND1 gene, one of the isogenes of 6-phosphogluconate dehydrogenase, or by disruption of the ZWF1 gene, which encodes glucose 6-phosphate dehydrogenase. Decreasing the phosphoglucose isomerase activity by 90% also lowered the pentose phosphate pathway flux. These modifications all resulted in lower xylitol yield and higher ethanol yield than in the control strains. TMB3255, carrying a disruption of ZWF1, gave the highest ethanol yield (0.41 g g(-1)) and the lowest xylitol yield (0.05 g g(-1)) reported for a xylose-fermenting recombinant S. cerevisiae strain, but also an 84% lower xylose consumption rate. The low xylose fermentation rate is probably due to limited NADPH-mediated xylose reduction. Metabolic flux modeling of TMB3255 confirmed that the NADPH-producing pentose phosphate pathway was blocked and that xylose reduction was mediated only by NADH, leading to a lower rate of xylose consumption. These results indicate that xylitol production is strongly connected to the flux through the oxidative part of the pentose phosphate pathway.     

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.