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.     

生物技术生产丁酸的研究进展

丁酸作为一种重要的化工原料,已经广泛应用于食品添加剂与医药等领域。目前,工业上生产丁酸主要是从石油中提取有机化合物进行化学合成。与有机化合物合成法相比,微生物发酵产丁酸的优势有：所用的原料来源非常广,发酵过程低能耗,不污染环境,而且可以持续添加原料发酵生产丁酸。因此,通过生物技术发酵生产丁酸越来越受到人们的重视。介绍了丁酸的性质、产丁酸菌株的特点、微生物发酵产丁酸的细胞代谢途径及其调控、发酵法生产丁酸的工艺运行方式和产丁酸菌株及其代谢产物的生理功能这五部分内容,以期为今后开展发酵法产丁酸的微生物基因工程改造以及生产工艺的优化提供参考。     

高通量技术在萜类合成酶筛选中的应用

萜类化合物种类繁多,生物活性多样,在食品、药品与化妆品等行业中具有广泛的应用。萜类化合物多来源于植物,然而随着合成生物学的快速发展,相较于传统的天然植物提取与化学合成方法,利用工程微生物进行萜类化合物异源合成的方法显得更为经济与环保。萜类合成酶的催化活性及合成产物的结构特性是萜类化合物异源合成的关键。通过蛋白定向进化与理性设计可以有针对性地优化萜类合成酶的催化性能及产物专一性,但该方案需要一个特异的筛选方法来实现蛋白突变体库的高通量筛选。近年来,一系列高通量筛选方法的建立使得萜类合成酶的筛选变得更加灵敏与高效。本文对近期建立的萜类合成酶高通量筛选方法进行了综述,简要概述了各种筛选方法的基本原理与优缺点,并对高通量筛选技术在萜类合成酶改造中的应用做出了展望。     

Sustainable algal biorefineries: capitalizing on many benefits of GABA

We provide physiological and metabolic insights into the complex role of γ-aminobutyric acid (GABA) in fine-tuning algal metabolism to improve productivity. Genetic engineering strategies to improve algal GABA biosynthesis are also discussed. Our aim is to provide an understanding of how GABA can be used for cost-competitive algae-based biofuels and bioproducts.     

Towards systems metabolic engineering of microorganisms for amino acid production

Microorganisms capable of efficient production of amino acids have traditionally been developed by random mutation and selection method, which might cause unwanted physiological changes in cellular metabolism. Rational genome-wide metabolic engineering based on systems and synthetic biology tools, which is termed 'systems metabolic engineering', is rising as an alternative to overcome these problems. Recently, several amino acid producers have been successfully developed by systems metabolic engineering, where the metabolic engineering procedures were performed within a systems biology framework, and entire metabolic networks, including complex regulatory circuits, were engineered in an integrated manner. Here we review the current status of systems metabolic engineering successfully applied for developing amino acid producing strains and discuss future prospects.     

系统生物学和合成生物学研究在生物燃料生产菌株改造中的应用

基于生物质资源生产环境友好的生物燃料,对经济和社会的可持续发展具有重要意义,但其生产成本高的问题十分突出,而高效生产菌株的获得是解决这一问题的根本出路。以下综述了利用系统生物学研究所获得的信息进行菌种改造的过程,重点论述了生产菌株胁迫耐受性方面的研究进展,并讨论了系统生物学、合成生物学和代谢工程技术在改造生物燃料生产菌株中的应用,展望了合成生物学在构建高效生物能源生产菌株方面应用的前景。     

微生物发酵法生产L-色氨酸的代谢工程研究

L-色氨酸作为人体内的一种必需氨基酸,广泛应用于医药、食品与饲料等行业.工业上采用的色氨酸生产方法有化学合成法、转化法及微生物发酵法.近年来,随着代谢工程在色氨酸菌种选育中的成功运用,微生物发酵法逐渐成为主要的色氨酸生产方法.系统综述了微生物发酵法生产色氨酸所涉及的代谢工程策略,包括生物合成色氨酸的代谢调控机制以及途径...     

平台化学品短链支链脂肪酸和短链支链醇的微生物代谢工程

短链支链脂肪酸和短链支链醇均为重要的平台化学品,是合成多种高附加值产品的前体物质,市场需求巨大。目前两者的生产主要是利用基于石化原料的化学合成法。化学合成法存在着严重依赖化石燃料、反应效率低以及极易造成环境污染等缺点。微生物代谢工程的快速发展为这些平台化学品的生产提供了一条极具潜力的生物合成路线。利用微生物代谢工程技术构建生产这些平台化学品的微生物细胞工厂具有绿色清洁、可持续发展和经济效益好等独特优势。本文系统综述了近年来微生物代谢工程技术在短链支链脂肪酸和短链支链醇合成方面的研究进展,包括所涉及的宿主菌株、关键酶、代谢途径及其改造等,并探讨了未来的发展前景。     

3-脱氢莽草酸生物合成的代谢工程

3-脱氢莽草酸,是芳香族氨基酸生物合成代谢途径中一种重要的中间产物,可作为一些化学合成制剂和药物中间原料。这样以无毒可再生物质为起始原料的合成方法与传统的有机合成化学制剂的方法相比,对环境更加有利。此外,它还是一种十分有效的抗氧化剂。工业上一般采用化学合成法和发酵法来生产3-脱氢莽草酸,随着代谢工程的兴起,使得更加理性改造菌株成为可能,这更加促进了发酵法的广泛应用。本文主要介绍了代谢工程在生物合成3-脱氢莽草酸生产菌改造中的应用情况,其中涉及3-脱氢莽草酸生物合成途径中相关基因及其酶的调控、中心代谢途径的改造和3-脱氢莽草酸合成支路的修饰等,并探讨了将来的发展前景。     

微生物发酵生产5-氨基乙酰丙酸研究进展

5-氨基乙酰丙酸是生物体内吡咯生物合成途径的关键中间产物,具有广泛的应用前景。文中从三方面归纳了国内外关于5-氨基乙酰丙酸的最新研究进展:生产5-氨基乙酰丙酸的微生物筛选分离与诱变;基于C4途径的微生物全细胞生物转化合成5-氨基乙酰丙酸;基于微生物代谢工程改造构建高产5-氨基乙酰丙酸的工程菌株。最后,预测了未来5-氨基乙酰丙酸的研究方向和焦点。     

Standardized biosynthesis of flavan-3-ols with effects on pancreatic beta-cell insulin secretion

Flavan-3-ols, such as green tea catechins represent a major group of phenolic compounds with significant medicinal properties. We describe the construction and optimization of Escherichia coli recombinant strains for the production of mono- and dihydroxylated catechins from their flavanone and phenylpropanoid acid precursors. Use of glucose minimal medium, Fe(II), and control of oxygen availability during shake-flask experiments resulted in production yield increases. Additional production improvement resulted from the use of medium rather than high-copy number plasmids and, in the case of mono-hydroxylated compounds, the addition of extracellular cofactors in the culture medium. The established metabolic engineering approach allowed the biosynthesis of natural catechins at high purity for assessing their possible insulinotropic effects in pancreatic beta-cell cultures. We demonstrated that (+)-afzelechin and (+)-catechin modulated the secretion of insulin by pancreatic beta-cells. These results indicate the potential of applying metabolic engineering approaches for the synthesis of natural and non-natural catechin analogues as drug candidates in diabetes treatments.     

Engineering of Corynebacterium glutamicum for high-level γ-aminobutyric acid production from glycerol by dynamic metabolic control

Synthetic biology seeks to reprogram microbial cells for efficient production of value-added compounds from low-cost renewable substrates. A great challenge of chemicals biosynthesis is the competition between cell metabolism and target product synthesis for limited cellular resource. Dynamic regulation provides an effective strategy for fine-tuning metabolic flux to maximize chemicals production. In this work, we created a tunable growth phase-dependent autonomous bifunctional genetic switch (GABS) by coupling growth phase responsive promoters and degrons to dynamically redirect the carbon flux for metabolic state switching from cell growth mode to production mode, and achieved high-level GABA production from low-value glycerol in Corynebacterium glutamicum. A ribosome binding sites (RBS)-library-based pathway optimization strategy was firstly developed to reconstruct and optimize the glycerol utilization pathway in C. glutamicum, and the resulting strain CgGly2 displayed excellent glycerol utilization ability. Then, the initial GABA-producing strain was constructed by deleting the GABA degradation pathway and introducing an exogenous GABA synthetic pathway, which led to 5.26 g/L of GABA production from glycerol. In order to resolve the conflicts of carbon flux between cell growth and GABA production, we used the GABS to reconstruct the GABA synthetic metabolic network, in which the competitive modules of GABA biosynthesis, including the tricarboxylic acid (TCA) cycle module and the arginine biosynthesis module, were dynamically down-regulated while the synthetic modules were dynamically up-regulated after sufficient biomass accumulation. Finally, the resulting strain G7-1 accumulated 45.6 g/L of GABA with a yield of 0.4 g/g glycerol, which was the highest titer of GABA ever reported from low-value glycerol. Therefore, these results provide a promising technology to dynamically balance the metabolic flux for the efficient production of other high value-added chemicals from a low-value substrate in C. glutamicum.     

微生物模块化共培养工程的应用及控制策略

传统的微生物合成方法通常依赖于单一工程菌株,通过代谢工程改造合成目标产物.由于关键的辅因子、前体和能量被引入到复杂的化合物合成途径中,加重了工程菌株的代谢负担,因而常常会影响目标产物产量.而模块化共培养工程已经成为一种有效进行异源生物合成并有望大大提高产物产量的新方法.在模块化共培养工程中,不同模块菌群之间的相互协调对...     

吡咯喹啉醌合成及生产工艺研究进展

吡咯喹啉醌(pyrroloquinoline quinone, PQQ)是继烟酰胺和核黄素之后发现的第三类氧化还原酶辅因子,普遍存在于生物体中参与呼吸链电子传递,具有促进线粒体产生、清除自由基、增强细胞代谢和预防心肌损伤等生理功能,在医药、食品和农业领域具有广泛的应用前景。微生物发酵法是PQQ生产的主要方式,解析PQQ生物合成途径及其调控机制,通过代谢工程选育短周期、高产量的生产菌是PQQ工业化的研究方向之一。本文综述了PQQ的合成途径、高产菌株选育以及微生物发酵生产与分离纯化的研发工作,为深入阐释PQQ的生物合成机制和工业化生产菌株的选育提供参考。     

Genome-scale model for Clostridium acetobutylicum: Part I. Metabolic network resolution and analysis

A genome-scale metabolic network reconstruction for Clostridium acetobutylicum (ATCC 824) was carried out using a new semi-automated reverse engineering algorithm. The network consists of 422 intracellular metabolites involved in 552 reactions and includes 80 membrane transport reactions. The metabolic network illustrates the reliance of clostridia on the urea cycle, intracellular L-glutamate solute pools, and the acetylornithine transaminase for amino acid biosynthesis from the 2-oxoglutarate precursor. The semi-automated reverse engineering algorithm identified discrepancies in reaction network databases that are major obstacles for fully automated network-building algorithms. The proposed semi-automated approach allowed for the conservation of unique clostridial metabolic pathways, such as an incomplete TCA cycle. A thermodynamic analysis was used to determine the physiological conditions under which proposed pathways (e.g., reverse partial TCA cycle and reverse arginine biosynthesis pathway) are feasible. The reconstructed metabolic network was used to create a genome-scale model that correctly characterized the butyrate kinase knock-out and the asolventogenic M5 pSOL1 megaplasmid degenerate strains. Systematic gene knock-out simulations were performed to identify a set of genes encoding clostridial enzymes essential for growth in silico.     

Exopolysaccharides produced by Lactococcus lactis: from genetic engineering to improved rheological properties?

Over the last years, important advances have been made in the study of the production of exopolysaccharides (EPS) by several lactic acid bacteria, including Lactococcus lactis. From different EPS-producing lactococcal strains the specific eps gene clusters have been characterised. They contain eps genes, which are involved in EPS repeating unit synthesis, export, polymerisation, and chain length determination. The function of the glycosyltransferase genes has been established and the availability of these genes opened the way to EPS engineering. In addition to the eps genes, biosynthesis of EPS requires a number of housekeeping genes that are involved in the metabolic pathways leading to the EPS-building blocks, the nucleotide sugars. The identification and characterisation of several of these housekeeping genes (galE, galU, rfbABCD) allows the design of metabolic engineering strategies that should lead to increased EPS production levels by L. lactis. Finally, model developme nt has been initiated in order to predict the physicochemical consequences of the addition of a EPS to a product.     

Metabolic engineering for the optimization of hydrogen production in Escherichia coli: A review

Hydrogen is a potential sustainable energy source and it could become an alternative to fossil fuel combustion, thus helping to reduce greenhouse gas emissions. The biological production of hydrogen, instead of its chemical synthesis, is a promising possibility since this process requires less energy and is more sustainable and eco-friendly. Several microorganisms have been used for this purpose, but Escherichia coli is one of the most widely used in this field. The literature in this area has increased exponentially in the last 10 years and several strategies have been reported in an effort to improve hydrogen production. In this work, the stay of the art of hydrogen biosynthesis by E. coli and metabolic engineering strategies to enhance hydrogen production are reviewed. This work includes a discussion about the hydrogenase complexes responsible for the hydrogen synthesis in this microorganism and the central carbon metabolism pathways connected to this process. The main metabolic engineering strategies applied are discussed, including heterologous gene expression, adaptive evolution and metabolic and protein engineering. On the other hand, culture conditions, including the use of carbon sources such as glycerol, glucose or organic wastes, have also been considered. Yields and productivities of the most relevant engineered strains reported using several carbon sources are also compared.     

A systematic analysis of TCA Escherichia coli mutants reveals suitable genetic backgrounds for enhanced hydrogen and ethanol production using glycerol as main carbon source

Biodiesel has emerged as an environmentally friendly alternative to fossil fuels; however, the low price of glycerol feed‐stocks generated from the biodiesel industry has become a burden to this industry. A feasible alternative is the microbial biotransformation of waste glycerol to hydrogen and ethanol. Escherichia coli, a microorganism commonly used for metabolic engineering, is able to biotransform glycerol into these products. Nevertheless, the wild type strain yields can be improved by rewiring the carbon flux to the desired products by genetic engineering. Due to the importance of the central carbon metabolism in hydrogen and ethanol synthesis, E. coli single null mutant strains for enzymes of the TCA cycle and other related reactions were studied in this work. These strains were grown anaerobically in a glycerol‐based medium and the concentrations of ethanol, glycerol, succinate and hydrogen were analysed by HPLC and GC. It was found that the reductive branch is the more relevant pathway for the aim of this work, with malate playing a central role. It was also found that the putative C4‐transporter dcuD mutant improved the target product yields. These results will contribute to reveal novel metabolic engineering strategies for improving hydrogen and ethanol production by E. coli.     

Aspergillus as a versatile cell factory for organic acid production

Filamentous fungi from the genus Aspergillus are of high importance for biobased organic acid production. So far, a number of Aspergillus strains belonging to phylogenetically distantly related species have been successfully applied in industrial production of organic acids due to their excellent capabilities of secreting high amounts of desired organic acids. For the past decades, numerous efforts have been made to reveal the mechanisms of organic acid biosynthesis in several Aspergillus species and to improve the production of desired organic acids via genetic engineering. This review summarizes the recent breakthroughs in the fundamental understanding of physiological aspects of organic acid accumulation by fungal biocatalysts and highlights the progress in genetic engineering of aspergilli for organic acid production. The challenges for the future applications of aspergilli as commercial cell factories for organic acid production are also discussed.