工业微生物中NADH的代谢调控

NADH是微生物代谢网络中的一种关键辅因子。调节微生物胞内NADH的形式与浓度是定向改变和优化微生物细胞代谢功能, 实现代谢流最大化、快速化地导向目标代谢产物的重要手段之一。以下在详尽总结了NADH生理功能的基础上, 从生化工程(添加外源电子受体、不同氧化还原态底物及NAD合成前体物, 调节培养环境和氧化还原电势)和代谢工程(过量表达NADH代谢相关酶、缺失NADH竞争途径及引入NADH外源代谢途径)两方面分析、归纳了NADH代谢调控策略, 进而凝练出调控NADH/NAD+比率调节微生物细胞代谢功能研究方面亟待解决的3个科学问题及可能的解决途径。     

氨基酸生物传感器的开发及应用研究进展

氨基酸是蛋白质的基本组成单元,对人和动物的营养健康十分重要,广泛应用于饲料、食品、医药和日化等领域。目前,氨基酸主要通过微生物发酵可再生原料生产,氨基酸产业是我国生物制造的重要支柱产业之一。氨基酸菌株主要通过随机诱变和代谢工程改造结合筛选获得。菌株生产水平进一步提高的核心限制之一是缺乏高效、快速和准确的筛选方法,因此,发展氨基酸菌株的高通量筛选方法对关键功能元件挖掘及高产菌株的创制筛选至关重要。本文综述了氨基酸生物传感器的设计,及其在功能元件、高产菌株的高通量进化筛选和代谢途径动态调控中的应用研究进展,讨论了现有氨基酸生物传感器存在的问题和性能提升改造策略,并展望了开发氨基酸衍生物生物传感器的重要性。     

Replacing Escherichia coli NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a NADP-dependent enzyme from Clostridium acetobutylicum facilitates NADPH dependent pathways

Reactions requiring reducing equivalents, NAD(P)H, are of enormous importance for the synthesis of industrially valuable compounds such as carotenoids, polymers, antibiotics and chiral alcohols among others. The use of whole-cell biocatalysis can reduce process cost by acting as catalyst and cofactor regenerator at the same time; however, product yields might be limited by cofactor availability within the cell. Thus, our study focussed on the genetic manipulation of a whole-cell system by modifying metabolic pathways and enzymes to improve the overall production process. In the present work, we genetically engineered an Escherichia coli strain to increase NADPH availability to improve the productivity of products that require NADPH in its biosynthesis. The approach involved an alteration of the glycolysis step where glyceraldehyde-3-phosphate (GAP) is oxidized to 1,3 bisphophoglycerate (1,3-BPG). This reaction is catalyzed by NAD-dependent endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoded by the gapA gene. We constructed a recombinant E. coli strain by replacing the native NAD-dependent gapA gene with a NADP-dependent GAPDH from Clostridium acetobutylicum, encoded by the gene gapC. The beauty of this approach is that the recombinant E. coli strain produces 2 mol of NADPH, instead of NADH, per mole of glucose consumed. Metabolic flux analysis showed that the flux through the pentose phosphate (PP) pathway, one of the main pathways that produce NADPH, was reduced significantly in the recombinant strain when compared to that of the parent strain. The effectiveness of the NADPH enhancing system was tested using the production of lycopene and epsilon-caprolactone as model systems using two different background strains. The recombinant strains, with increased NADPH availability, consistently showed significant higher productivity than the parent strains.     

Metabolic engineering of microorganisms for the production of multifunctional non-protein amino acids: γ-aminobutyric acid and δ-aminolevulinic acid

Gamma-aminobutyric acid (GABA) and delta-aminolevulinic acid (ALA), playing important roles in agriculture, medicine and other fields, are multifunctional non-protein amino acids with similar and comparable properties and biosynthesis pathways. Recently, microbial synthesis has become an inevitable trend to produce GABA and ALA due to its green and sustainable characteristics. In addition, the development of metabolic engineering and synthetic biology has continuously accelerated and increased the GABA and ALA yield in microorganisms. Here, focusing on the current trends in metabolic engineering strategies for microbial synthesis of GABA and ALA, we analysed and compared the efficiency of various metabolic strategies in detail. Moreover, we provide the insights to meet challenges of realizing industrially competitive strains and highlight the future perspectives of GABA and ALA production.     

Common aspects in the engineering of yeasts for fatty acid- and isoprene-based products

The biosynthetic pathways for most lipophilic metabolites share several common principles. These substances are built almost exclusively from acetyl-CoA as the donor for the carbon scaffold and NADPH is required for the reductive steps during biosynthesis. Due to their hydrophobicity, the end products are sequestered into the same cellular compartment, the lipid droplet. In this review, we will summarize the efforts in the metabolic engineering of yeasts for the production of two major hydrophobic substance classes, fatty acid-based lipids and isoprenoids, with regard to these common aspects. We will compare and discuss the results of genetic engineering strategies to construct strains with enhanced synthesis of the precursor acetyl-CoA and with modified redox metabolism for improved NADPH supply. We will also discuss the role of the lipid droplet in the storage of the hydrophobic product and review the strategies to either optimize this organelle for higher capacity or to achieve excretion of the product into the medium.     

L-valine production in Corynebacterium glutamicum based on systematic metabolic engineering: progress and prospects

L-valine is an essential branched-chain amino acid that cannot be synthesized by the human body and has a wide range of applications in food, medicine and feed. Market demand has stimulated people’s interest in the industrial production of L-valine. At present, the mutagenized or engineered Corynebacterium glutamicum is an effective microbial cell factory for producing L-valine. Because the biosynthetic pathway and metabolic network of L-valine are intricate and strictly regulated by a variety of key enzymes and genes, highly targeted metabolic engineering can no longer meet the demand for efficient biosynthesis of L-valine. In recent years, the development of omics technology has promoted the upgrading of traditional metabolic engineering to systematic metabolic engineering. This whole-cell-scale transformation strategy has become a productive method for developing L-valine producing strains. This review provides an overview of the biosynthesis and regulation mechanism of L-valine, and summarizes the current metabolic engineering techniques and strategies for constructing L-valine high-producing strains. Finally, the opinion of constructing a cell factory for efficiently biosynthesizing L-valine was proposed.

     

Biotechnological production of polyamines by bacteria: recent achievements and future perspectives

In Bacteria, the pathways of polyamine biosynthesis start with the amino acids l-lysine, l-ornithine, l-arginine, or l-aspartic acid. Some of these polyamines are of special interest due to their use in the production of engineering plastics (e.g., polyamides) or as curing agents in polymer applications. At present, the polyamines for industrial use are mainly synthesized on chemical routes. However, since a commercial market for polyamines as well as an industry for the fermentative production of amino acid exist, and since bacterial strains overproducing the polyamine precursors l-lysine, l-ornithine, and l-arginine are known, it was envisioned to engineer these amino acid-producing strains for polyamine production. Only recently, researchers have investigated the potential of amino acid-producing strains of Corynebacterium glutamicum and Escherichia coli for polyamine production. This mini-review illustrates the current knowledge of polyamine metabolism in Bacteria, including anabolism, catabolism, uptake, and excretion. The recent advances in engineering the industrial model bacteria C. glutamicum and E. coli for efficient production of the most promising polyamines, putrescine (1,4-diaminobutane), and cadaverine (1,5-diaminopentane), are discussed in more detail.     

Engineering primary metabolic pathways of industrial micro-organisms

Metabolic engineering is a powerful tool for the optimisation and the introduction of new cellular processes. This is mostly done by genetic engineering. Since the introduction of this multidisciplinary approach, the success stories keep accumulating. The primary metabolism of industrial micro-organisms has been studied for long time and most biochemical pathways and reaction networks have been elucidated. This large pool of biochemical information, together with data from proteomics, metabolomics and genomics underpins the strategies for design of experiments and choice of targets for manipulation by metabolic engineers. These targets are often located in the primary metabolic pathways, such as glycolysis, pentose phosphate pathway, the TCA cycle and amino acid biosynthesis and mostly at major branch points within these pathways. This paper describes approaches taken for metabolic engineering of these pathways in bacteria, yeast and filamentous fungi.     

Metabolic pathways and fermentative production of L-aspartate family amino acids

The L -aspartate family amino acids (AFAAs), L -threonine, L -lysine, L -methionine and L -isoleucine have recently been of much interest due to their wide spectrum of applications including food additives, components of cosmetics and therapeutic agents, and animal feed additives. Among them, L -threonine, L -lysine and L -methionine are three major amino acids produced currently throughout the world. Recent advances in systems metabolic engineering, which combine various high-throughput omics technologies and computational analysis, are now facilitating development of microbial strains efficiently producing AFAAs. Thus, a thorough understanding of the metabolic and regulatory mechanisms of the biosynthesis of these amino acids is urgently needed for designing system-wide metabolic engineering strategies. Here we review the details of AFAA biosynthetic pathways, regulations involved, and export and transport systems, and provide general strategies for successful metabolic engineering along with relevant examples. Finally, perspectives of systems metabolic engineering for developing AFAA overproducers are suggested with selected exemplary studies.     

谷氨酸棒杆菌代谢工程高效生产L-缬氨酸北大核心CSCD

L-缬氨酸作为一种支链氨基酸,广泛应用于医药和饲料等领域。本研究借助多种代谢工程策略相结合的方法,构建了生产L-缬氨酸的微生物细胞工厂,实现了L-缬氨酸的高效生产。首先,通过增强糖酵解途径、减弱副产物代谢途径相结合的方式,强化了L-缬氨酸合成前体丙酮酸的供给;其次,针对L-缬氨酸合成路径关键酶—乙酰羟酸合酶进行定点突变,提高了菌株的抗反馈抑制能力,并利用启动子工程策略,优化了路径关键酶的基因表达水平;最后,利用辅因子工程策略,改变了乙酰羟酸还原异构酶和支链氨基酸转氨酶的辅因子偏好性,由偏好NADPH转变为偏好NADH,从而提高了L-缬氨酸的合成能力。在5L发酵罐中,最优谷氨酸棒杆菌工程菌株Corynebacterium glutamicum K020的L-缬氨酸产量、得率和生产强度分别达到了110g/L、0.51g/g和2.29 g/(L·h)。     

Dynamic control over feedback regulatory mechanisms improves NADPH flux and xylitol biosynthesis in engineered E. coli

We report improved NADPH flux and xylitol biosynthesis in engineered E. coli. Xylitol is produced from xylose via an NADPH dependent reductase. We utilize 2-stage dynamic metabolic control to compare two approaches to optimize xylitol biosynthesis, a stoichiometric approach, wherein competitive fluxes are decreased, and a regulatory approach wherein the levels of key regulatory metabolites are reduced. The stoichiometric and regulatory approaches lead to a 20-fold and 90-fold improvement in xylitol production, respectively. Strains with reduced levels of enoyl-ACP reductase and glucose-6-phosphate dehydrogenase, led to altered metabolite pools resulting in the activation of the membrane bound transhydrogenase and an NADPH generation pathway, consisting of pyruvate ferredoxin oxidoreductase coupled with NADPH dependent ferredoxin reductase, leading to increased NADPH fluxes, despite a reduction in NADPH pools. These strains produced titers of 200 g/L of xylitol from xylose at 86% of theoretical yield in instrumented bioreactors. We expect dynamic control over the regulation of the membrane bound transhydrogenase as well as NADPH production through pyruvate ferredoxin oxidoreductase to broadly enable improved NADPH dependent bioconversions or production via NADPH dependent metabolic pathways.     

Metabolic engineering of edible plant oils]

Plant seed oil is the major source of many fatty acids for human nutrition, and also one of industrial feedstocks. Recent advances in understanding of the basic biochemistry of seed oil biosynthesis, coupled with cloning of the genes encoding the enzymes involved in fatty acid modification and oil accumulation, have set the stage for the metabolic engineering of oilseed crops that produce "designer" plant seed oils with the improved nutritional values for human being. In this review we provide an overview of seed oil biosynthesis/regulation and highlight the key enzymatic steps that are targets for gene manipulation. The strategies of metabolic engineering of fatty acids in oilseeds, including overexpression or suppression of genes encoding single or multi-step biosynthetic pathways and assembling the complete pathway for the synthesis of long-chain polyunsaturated fatty acids (e.g. arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid) are described in detail. The current "bottlenecks" in using common oilseeds as "bioreactors" for commercial production of high-value fatty acids are analyzed. It is also discussed that the future research focuses of oilseed metabolic engineering and the prospects in creating renewable sources and promoting the sustainable development of human society and economy.     

Lactic acid bacterial cell factories for gamma-aminobutyric acid

Gamma-aminobutyric acid is a non-protein amino acid that is widely present in organisms. Several important physiological functions of gamma-aminobutyric acid have been characterized, such as neurotransmission, induction of hypotension, diuretic effects, and tranquilizer effects. Many microorganisms can produce gamma-aminobutyric acid including bacteria, fungi and yeasts. Among them, gamma-aminobutyric acid-producing lactic acid bacteria have been a focus of research in recent years, because lactic acid bacteria possess special physiological activities and are generally regarded as safe. They have been extensively used in food industry. The production of lactic acid bacterial gamma-aminobutyric acid is safe and eco-friendly, and this provides the possibility of production of new naturally fermented health-oriented products enriched in gamma-aminobutyric acid. The gamma-aminobutyric acid-producing species of lactic acid bacteria and their isolation sources, the methods for screening of the strains and increasing their production, the enzymatic properties of glutamate decarboxylases and the relative fundamental research are reviewed in this article. And the potential applications of gamma-aminobutyric acid-producing lactic acid bacteria were also referred to.     

A novel aspect of NADPH production in ageing Penicillium chrysogenum

NADPH is involved in many basically important anabolic processes. For a long time, pentose phosphate pathway (PPS) was regarded as the most important source of NADPH in fungi. Here we present evidence of a metabolic switch to an alternative NADPH-producing pathway in ageing Penicillium chrysogenum cultures, which involves NADP+ -specific isocitrate dehydrogenase (NADP+ -ID) rather than PPS enzymes. Considering the main biochemical functions of NADPH, we propose that NADP+ -ID could have deep impact on many physiological processes switched on glucose deprivation including proteinase production or penicillin biosynthesis. We also demonstrate that although the alternative pathway was inferior to PPS when the fungus was grown on well-utilisable carbon sources yet it could have an important role in fatty acid biosynthesis as well as in the maintenance of high intracellular NADPH/NADP+ ratios.     

Carbon-Flux Distribution within Streptomyces coelicolor Metabolism: A Comparison between the Actinorhodin-Producing Strain M145 and Its Non-Producing Derivative M1146

Metabolic Flux Analysis is now viewed as essential to elucidate the metabolic pattern of cells and to design appropriate genetic engineering strategies to improve strain performance and production processes. Here, we investigated carbon flux distribution in two Streptomyces coelicolor A3 (2) strains: the wild type M145 and its derivative mutant M1146, in which gene clusters encoding the four main antibiotic biosynthetic pathways were deleted. Metabolic Flux Analysis and ¹³C-labeling allowed us to reconstruct a flux map under steady-state conditions for both strains. The mutant strain M1146 showed a higher growth rate, a higher flux through the pentose phosphate pathway and a higher flux through the anaplerotic phosphoenolpyruvate carboxylase. In that strain, glucose uptake and the flux through the Krebs cycle were lower than in M145. The enhanced flux through the pentose phosphate pathway in M1146 is thought to generate NADPH enough to face higher needs for biomass biosynthesis and other processes. In both strains, the production of NADPH was higher than NADPH needs, suggesting a key role for nicotinamide nucleotide transhydrogenase for redox homeostasis. ATP production is also likely to exceed metabolic ATP needs, indicating that ATP consumption for maintenance is substantial.Our results further suggest a possible competition between actinorhodin and triacylglycerol biosynthetic pathways for their common precursor, acetyl-CoA. These findings may be instrumental in developing new strategies exploiting S. coelicolor as a platform for the production of bio-based products of industrial interest.     

Metabolic engineering for higher alcohol production

Engineering microbial hosts for the production of higher alcohols looks to combine the benefits of renewable biological production with the useful chemical properties of larger alcohols. In this review we outline the array of metabolic engineering strategies employed for the efficient diversion of carbon flux from native biosynthetic pathways to the overproduction of a target alcohol. Strategies for pathway design from amino acid biosynthesis through 2-keto acids, from isoprenoid biosynthesis through pyrophosphate intermediates, from fatty acid biosynthesis and degradation by tailoring chain length specificity, and the use and expansion of natural solvent production pathways will be covered.     

Overexpression of O‐methyltransferase leads to improved vanillin production in baker's yeast only when complemented with model‐guided network engineering

Overproduction of a desired metabolite is often achieved via manipulation of the pathway directly leading to the product or through engineering of distant nodes within the metabolic network. Empirical examples illustrating the combined effect of these local and global strategies have been so far limited in eukaryotic systems. In this study, we compared the effects of overexpressing a key gene in de novo vanillin biosynthesis (coding for O‐methyltransferase, hsOMT) in two yeast strains, with and without model‐guided global network modifications. Overexpression of hsOMT resulted in increased vanillin production only in the strain with model‐guided modifications, exemplifying advantage of using a global strategy prior to local pathway manipulation. Biotechnol. Bioeng. 2013; 110: 656–659. © 2012 Wiley Periodicals, Inc.     

Metabolic-flux and network analysis in fourteen hemiascomycetous yeasts

In a quantitative comparative study, we elucidated the glucose metabolism in fourteen hemiascomycetous yeasts from the Genolevures project. The metabolic networks of these different species were first established by (13)C-labeling data and the inventory of the genomes. This information was subsequently used for metabolic-flux ratio analysis to quantify the intracellular carbon flux distributions in these yeast species. Firstly, we found that compartmentation of amino acid biosynthesis in most species was identical to that in Saccharomyces cerevisiae. Exceptions were the mitochondrial origin of aspartate biosynthesis in Yarrowia lipolytica and the cytosolic origin of alanine biosynthesis in S. kluyveri. Secondly, the control of flux through the TCA cycle was inversely correlated with the ethanol production rate, with S. cerevisiae being the yeast with the highest ethanol production capacity. The classification between respiratory and respiro-fermentative metabolism, however, was not qualitatively exclusive but quantitatively gradual. Thirdly, the flux through the pentose phosphate (PP) pathway was correlated to the yield of biomass, suggesting a balanced production and consumption of NADPH. Generally, this implies the lack of active transhydrogenase-like activities in hemiascomycetous yeasts under the tested growth condition, with Pichia angusta as the sole exception. In the latter case, about 40% of the NADPH was produced in the PP pathway in excess of the requirements for biomass production, which strongly suggests the operation of a yet unidentified mechanism for NADPH reoxidation in this species. In most yeasts, the PP pathway activity appears to be driven exclusively by the demand for NADPH.