微生物代谢工程的研究进展和展望

代谢工程技术是构建微生物细胞工厂的重要方法,其主要目标是通过基因工程等手段将目标代谢产物产量最大化。然而基因工程等操作往往会影响细胞生长速率,导致其生产强度降低。随着合成生物学及相关技术的发展,多种调控策略被应用于代谢工程领域以解决上述问题。通过这些调控可以有效地解决细胞生长与产物合成之间的竞争关系,平衡代谢途径,避免中间代谢产物的过量积累。对这些策略的研究及应用进行了概述和展望。     

微生物细胞工厂的代谢调控

代谢调控是构建微生物细胞工厂的重要技术手段.随着合成生物学技术的不断突破,挖掘和人工设计的高质量调控元件大幅度提升了对细胞代谢网络的改造能力;代谢调控研究也已从单基因的静态调控发展到系统水平上的智能精确动态调控.文中简要综述了近30年来代谢途径表达调控技术在代谢工程领域的研究进展.     

细胞区室化调控萜类化合物微生物合成

萜类化合物具有抗炎、抗氧化、抑制肿瘤细胞增殖等药学活性,在医药行业应用广泛。近年来,利用微生物合成萜类化合物受到广泛关注。在微生物中高效合成萜类化合物离不开代谢途径的调控与优化,其中细胞区室化是常用的调控策略之一,在微生物细胞工厂的构建中发挥着重要作用。代谢途径的细胞区室化具有许多优点,如增加酶和底物的局部浓度,抑制其向副产物转移和减少有毒中间体积累等,可实现萜类化合物的高效合成。近年来利用细胞区室化在微生物中合成萜类化合物的研究逐步展开,但目前对于区室化工程在构建细胞工厂中的应用总结较少。因此,围绕代谢途径区室化的作用,各种细胞器的生理特性及其在调控萜类化合物微生物合成中的应用进行了综述,讨论细胞区室化调控策略的发展、存在的问题及前景,以期为萜类化合物的高效微生物合成提供参考。     

沼泽红假单胞菌作为微生物细胞工厂的应用

沼泽红假单胞菌(Rhodopseudomonas palustris)是一种紫色非硫细菌,具有新陈代谢代谢方式多样、可利用多种碳源、天然产多种化合物等优点,作为微生物细胞工厂具有很大的研究潜力,目前主要应用于废水处理和水产养殖领域。本文中,笔者从三个方面对沼泽红假单胞菌在微生物细胞工厂领域的研究应用进行总结,包括碳源的多样性、基因工程策略以及微生物细胞工厂的应用,如在产燃料燃气和萜烯化合物、微生物燃料电池、微生物电合成和光催化合成等领域。目前沼泽红假单胞菌作为微生物细胞工厂处于发展的初级阶段,笔者对其目前面临的难点进行了总结,并对其进一步的研究方向进行了展望。     

抗逆元件及其在高效微生物细胞工厂构建中的应用进展

微生物作为重要的细胞工厂,其应用时经常面临的各种胁迫条件严重制约细胞活力和生产性能。大量文献证明,微生物的胁迫耐受性是受到胞内多个代谢途径和生理系统调控的复杂表型。那么,挖掘和应用增强菌株胁迫耐受性的抗逆元件是构建高效微生物细胞工厂的有效手段。目前,已报道的微生物抗逆元件主要包括调控细胞壁和细胞膜、DNA修复、氧化应激、相容性溶质、能量产生和信号转导的相关基因以及外排泵、热激蛋白和全局转录因子等。着重介绍了近年来抗逆元件及其在高效微生物细胞工厂构建中的应用实例,同时,讨论了实际应用中可能面临的机遇与挑战。     

基因编码型生物传感器在微生物细胞工厂中的应用进展

合成生物学的迅猛发展推动了微生物细胞工厂中多种复杂化学品的生物合成,但仍存在产量低、生产效率不高等诸多问题。基因编码型生物传感器可以感知细胞内外代谢物浓度及外界环境的波动,产生可测量的信号输出或调控通路中的基因表达水平,具有成本低、操作简单、可再生等优点。目前,基因编码型生物传感器已经成为合成生物学和代谢工程的重要组成部分,是微生物细胞工厂中代谢动态调控及理想表型进化/筛选的强大工具。概述了基因编码型生物传感器的组成及工作原理,重点介绍了基因编码型生物传感器在微生物代谢动态调控及高通量筛选中的最新研究进展,就基因编码型生物传感器设计与构建过程中面临的挑战进行探讨,并展望了其今后的发展方向。     

ATP调控策略及其在微生物代谢产物合成中的应用

辅因子工程是代谢工程的一个新兴分支领域,主要通过直接调控细胞内关键酶的辅因子,如ATP/ADP、NADH/NAD+、NADPH/NADP+等的浓度和形式来实现代谢流的最大化,快速地将物质流导向目标代谢物。ATP作为一种重要辅因子参与微生物细胞内大量的酶催化反应,将物质代谢途径串联或并联成复杂的网络体系,最终使得物质代谢流的分配受到牵制。因此ATP调控策略有望成为微生物菌株改造的有利工具,用于提高目标代谢物的浓度和生产能力,强化微生物对于环境的耐受以及促进底物利用等。文中将重点论述目前常用的有效ATP调控策略以及ATP调控对于细胞代谢的影响,以期为微生物细胞工厂的高效构建提供参考。     

微生物小基因组细胞工厂研究进展

通过合理设计简化微生物基因组,减少细胞内冗余的调控和代谢网络,使得细胞更精简且便于控制,也能更高效地应用于工业生产。微生物小基因组细胞工厂只含有用于工业生产目的的基因,是研究组学的重要工具,也是生物制品工业生产的理想平台。介绍了构建小基因组细胞工厂的整体设计流程,列举了一些模式生物的相关研究进展,对微生物小基因组细胞工厂的应用前景进行了评述。     

基于比较基因组学重构细菌的代谢途径和调控网络

微生物基因组学的迅速发展为从功能基因与蛋白、网络及其调控等不同的角度,全面理解与认识微生物的代谢过程、构建细胞工厂,提供了丰富的背景信息。基于基因组序列进行代谢网络重构,有助于发现新的代谢功能基因、调控元件、甚至新的代谢途径,从而优化设计细胞内从原料到产品的生物合成路线。然而,目前公共数据库平台中代谢途径基因的功能注释,许多是错误或不完整的。以下着重介绍了近年来出现的一些用于代谢途径和调控网络重构的新型比较基因组学技术,并以近期的丙酮丁醇梭菌中木糖代谢途径的重构工作为例来说明其应用。     

多个调控元件调控萜类合成途径基因表达提高β-胡萝卜素的生产

强启动子对于获得目标产物最大代谢流量来说并不一定是最优的;相比之下,使用多个具有不同强度的调控元件对基因表达进行调控更有可能获得最优的表达强度.为了对比使用多个调控元件和使用强启动子调控萜类合成途径基因表达对β-胡萝卜素生产的影响,并通过对关键基因的组合调控提高β-胡萝卜素的生产.文中使用6个强度差异很大的人工调控元件,对萜类合成途径的8个基因进行调控.对于不同的基因,其最适的调控元件强度各不相同.对8个基因的调控使β-胡萝卜素产量提高1.2～3.5倍.和以前报道不一样的是,文中发现用适当强度的调控元件对dxr、ispG和ispH基因进行调控后,也能提高β-胡萝卜素的生产.对dxs和idi基因的组合调控将β-胡萝卜素产量提高了8倍,最终β-胡萝卜素产量达17.59 mg/g干重细胞.结果表明使用多个不同强度的调控元件对基因表达进行调控比仅使用强启动子调控更为有效,为提高目标产品合成能力提供了一种新的基因表达调控方案.     

An expanded role for microbial physiology in metabolic engineering and functional genomics: moving towards systems biology

Microbial physiology has traditionally played a very important role in both fundamental research and in industrial applications of microorganisms. The classical approach in microbial physiology has been to analyze the role of individual components (genes or proteins) in the overall cell function. With the progress in molecular biology it has become possible to optimize industrial fermentations through introduction of directed genetic modification - an approach referred to as metabolic engineering. Furthermore, as a consequence of large sequencing programs the complete genomic sequence has become available for an increasing number of microorganisms. This has resulted in substantial research efforts in assigning function to all identified open reading frames - referred to as functional genomics. In both metabolic engineering and functional genomics there is a trend towards application of a macroscopic view on cell function, and this leads to an expanded role of the classical approach applied in microbial physiology. With the increased understanding of the molecular mechanisms it is envisaged that in the future it will be possible to describe the interaction between all the components in the system (the cell), also at the quantitative level, and this is the goal of systems biology. Clearly this will have a significant impact on microbial physiology as well as on metabolic engineering.     

成像质谱在微生物代谢产物研究中的应用

微生物代谢产物的结构和功能多样,对相邻微生物和环境会产生重要影响。传统的天然产物分离方法不能系统全面地监测单一或混合微生物样品中代谢物的合成和释放模式。成像质谱能够同时可视化观察从单一微生物菌落到复杂微生物群落的多个代谢产物的时空分布,可以用于发现重要的生物活性分子,观察微生物菌落的代谢交流,以及跟踪微生物之间相互竞争过程中代谢物的修饰等方面的研究。本文综述了成像质谱在微生物代谢产物研究中的最新进展,展望了该技术的应用前景。     

Murine gut microbiota is defined by host genetics and modulates variation of metabolic traits

The gastrointestinal tract harbors a complex and diverse microbiota that has an important role in host metabolism. Microbial diversity is influenced by a combination of environmental and host genetic factors and is associated with several polygenic diseases. In this study we combined next-generation sequencing, genetic mapping, and a set of physiological traits of the BXD mouse population to explore genetic factors that explain differences in gut microbiota and its impact on metabolic traits. Molecular profiling of the gut microbiota revealed important quantitative differences in microbial composition among BXD strains. These differences in gut microbial composition are influenced by host-genetics, which is complex and involves many loci. Linkage analysis defined Quantitative Trait Loci (QTLs) restricted to a particular taxon, branch or that influenced the variation of taxa across phyla. Gene expression within the gastrointestinal tract and sequence analysis of the parental genomes in the QTL regions uncovered candidate genes with potential to alter gut immunological profiles and impact the balance between gut microbial communities. A QTL region on Chr 4 that overlaps several interferon genes modulates the population of Bacteroides, and potentially Bacteroidetes and Firmicutes-the predominant BXD gut phyla. Irak4, a signaling molecule in the Toll-like receptor pathways is a candidate for the QTL on Chr15 that modulates Rikenellaceae, whereas Tgfb3, a cytokine modulating the barrier function of the intestine and tolerance to commensal bacteria, overlaps a QTL on Chr 12 that influence Prevotellaceae. Relationships between gut microflora, morphological and metabolic traits were uncovered, some potentially a result of common genetic sources of variation.     

Modular,synthetic chromosomes as new tools for large scale engineering of metabolism

The construction of powerful cell factories requires intensive genetic engineering for the addition of new functionalities and the remodeling of native pathways and processes. The present study demonstrates the feasibility of extensive genome reprogramming using modular, specialized de novo-assembled neochromosomes in yeast. The in vivo assembly of linear and circular neochromosomes, carrying 20 native and 21 heterologous genes, enabled the first de novo production in a microbial cell factory of anthocyanins, plant compounds with a broad range of pharmacological properties. Turned into exclusive expression platforms for heterologous and essential metabolic routes, the neochromosomes mimic native chromosomes regarding mitotic and genetic stability, copy number, harmlessness for the host and editability by CRISPR/Cas9. This study paves the way for future microbial cell factories with modular genomes in which core metabolic networks, localized on satellite, specialized neochromosomes can be swapped for alternative configurations and serve as landing pads for the addition of functionalities.     

Harnessing biodiesel-producing microbes: from genetic engineering of lipase to metabolic engineering of fatty acid biosynthetic pathway

Microbial production routes, notably whole-cell lipase-mediated biotransformation and fatty-acids-derived biosynthesis, offer new opportunities for synthesizing biodiesel. They compare favorably to immobilized lipase and chemically catalyzed processes. Genetically modified whole-cell lipase-mediated in vitro route, together with in vivo and ex vivo microbial biosynthesis routes, constitutes emerging and rapidly developing research areas for effective production of biodiesel. This review presents recent advances in customizing microorganisms for producing biodiesel, via genetic engineering of lipases and metabolic engineering (including system regulation) of fatty-acids-derived pathways. Microbial hosts used include Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris and Aspergillus oryzae. These microbial cells can be genetically modified to produce lipases under different forms: intracellularly expressed, secreted or surface-displayed. They can be metabolically redesigned and systematically regulated to obtain balanced biodiesel-producing cells, as highlighted in this study. Such genetically or metabolically modified microbial cells can support not only in vitro biotransformation of various common oil feedstocks to biodiesel, but also de novo biosynthesis of biodiesel from glucose, glycerol or even cellulosic biomass. We believe that the genetically tractable oleaginous yeast Yarrowia lipolytica could be developed to an effective biodiesel-producing microbial cell factory. For this purpose, we propose several engineered pathways, based on lipase and wax ester synthase, in this promising oleaginous host.     

Bioproduction of L-piperazic acid in gram scale using Aureobasidium melanogenum

Currently, piperazic acid is chemically synthesized using ecologically unfriendly processes. Microbial synthesis from glucose is an attractive alternative to chemical synthesis. In this study, we report the production of L-piperazic acid via microbial fermentation with the first engineered fungal strain of Aureobasidium melanogenum; this strain was constructed by chassis development, genetic element reconstitution and optimization, synthetic rewiring and constitutive genetic circuit reconstitution, to build a robust L-piperazic acid synthetic cascade. These genetic modifications enable A. melanogenum to directly convert glucose to L-piperazic acid without relying on the use of either chemically synthesized precursors or harsh conditions. This bio-based process overcomes the shortcomings of the conventional synthesis routes. The ultimately engineered strain is a very high-efficient cell factory that can excrete 1.12 ± 0.05 g l^-1 of L-piperazic acid after a 120-h 10.0-l fed-batch fermentation; this is the highest titre of L-piperazic acid reported using a microbial cell factory.     

Utilizing elementary mode analysis,pathway thermodynamics,and a genetic algorithm for metabolic flux determination and optimal metabolic network design

Background  

Microbial hosts offer a number of unique advantages when used as production systems for both native and heterologous small-molecules. These advantages include high selectivity and benign environmental impact; however, a principal drawback is low yield and/or productivity, which limits economic viability. Therefore a major challenge in developing a microbial production system is to maximize formation of a specific product while sustaining cell growth. Tools to rationally reconfigure microbial metabolism for these potentially conflicting objectives remain limited. Exhaustively exploring combinations of genetic modifications is both experimentally and computationally inefficient, and can become intractable when multiple gene deletions or insertions need to be considered. Alternatively, the search for desirable gene modifications may be solved heuristically as an evolutionary optimization problem. In this study, we combine a genetic algorithm and elementary mode analysis to develop an optimization framework for evolving metabolic networks with energetically favorable pathways for production of both biomass and a compound of interest.     

国内酿酒酵母分子遗传与育种研究40年

酿酒酵母作为最简单的真核生物,是研究真核生物基本生命规律的重要模式系统,也是生物产业领域非常重要的微生物细胞工厂。在《微生物学通报》创刊40周年之际,本文综述了国内40年来在酿酒酵母分子遗传学与育种研究的重要进展,如分子遗传学研究手段的建立、重要功能基因的分析、重要生命过程的遗传基础和调控机制,以及酵母菌育种技术的建立与应用等。     

Developing Bacillus spp. as a cell factory for production of microbial enzymes and industrially important biochemicals in the context of systems and synthetic biology

Increasing concerns over limited petroleum resources and associated environmental problems are motivating the development of efficient cell factories to produce chemicals, fuels, and materials from renewable resources in an environmentally sustainable economical manner. Bacillus spp., the best characterized Gram-positive bacteria, possesses unique advantages as a host for producing microbial enzymes and industrially important biochemicals. With appropriate modifications to heterologous protein expression and metabolic engineering, Bacillus species are favorable industrial candidates for efficiently converting renewable resources to microbial enzymes, fine chemicals, bulk chemicals, and fuels. Here, we summarize the recent advances in developing Bacillus spp. as a cell factory. We review the available genetic tools, engineering strategies, genome sequence, genome-scale structure models, proteome, and secretion pathways, and we list successful examples of enzymes and industrially important biochemicals produced by Bacillus spp. Furthermore, we highlight the limitations and challenges in developing Bacillus spp. as a robust and efficient production host, and we discuss in the context of systems and synthetic biology the emerging opportunities and future research prospects in developing Bacillus spp. as a microbial cell factory.     

Life-history strategies of soil microbial communities in an arid ecosystem

The overwhelming taxonomic diversity and metabolic complexity of microorganisms can be simplified by a life-history classification; copiotrophs grow faster and rely on resource availability, whereas oligotrophs efficiently exploit resource at the expense of growth rate. Here, we hypothesize that community-level traits inferred from metagenomic data can distinguish copiotrophic and oligotrophic microbial communities. Moreover, we hypothesize that oligotrophic microbial communities harbor more unannotated genes. To test these hypotheses, we conducted metagenomic analyses of soil samples collected from copiotrophic vegetated areas and from oligotrophic bare ground devoid of vegetation in an arid-hyperarid region of the Sonoran Desert, Arizona, USA. Results supported our hypotheses, as we found that multiple ecologically informed life-history traits including average 16S ribosomal RNA gene copy number, codon usage bias in ribosomal genes and predicted maximum growth rate were higher for microbial communities in vegetated than bare soils, and that oligotrophic microbial communities in bare soils harbored a higher proportion of genes that are unavailable in public reference databases. Collectively, our work demonstrates that life-history traits can distill complex microbial communities into ecologically coherent units and highlights that oligotrophic microbial communities serve as a rich source of novel functions.Subject terms: Microbial ecology, Community ecology