Toward systems metabolic engineering of Aspergillus and Pichia species for the production of chemicals and biofuels

Recently genome sequence data have become available for Aspergillus and Pichia species of industrial interest. This has stimulated the use of systems biology approaches for large-scale analysis of the molecular and metabolic responses of Aspergillus and Pichia under defined conditions, which has resulted in much new biological information. Case-specific contextualization of this information has been performed using comparative and functional genomic tools. Genomics data are also the basis for constructing genome-scale metabolic models, and these models have helped in the contextualization of knowledge on the fundamental biology of Aspergillus and Pichia species. Furthermore, with the availability of these models, the engineering of Aspergillus and Pichia is moving from traditional approaches, such as random mutagenesis, to a systems metabolic engineering approach. Here we review the recent trends in systems biology of Aspergillus and Pichia species, highlighting the relevance of these developments for systems metabolic engineering of these organisms for the production of hydrolytic enzymes, biofuels and chemicals from biomass.     

Systems metabolic engineering design: Fatty acid production as an emerging case study

Increasing demand for petroleum has stimulated industry to develop sustainable production of chemicals and biofuels using microbial cell factories. Fatty acids of chain lengths from C₆ to C₁₆ are propitious intermediates for the catalytic synthesis of industrial chemicals and diesel‐like biofuels. The abundance of genetic information available for Escherichia coli and specifically, fatty acid metabolism in E. coli, supports this bacterium as a promising host for engineering a biocatalyst for the microbial production of fatty acids. Recent successes rooted in different features of systems metabolic engineering in the strain design of high‐yielding medium chain fatty acid producing E. coli strains provide an emerging case study of design methods for effective strain design. Classical metabolic engineering and synthetic biology approaches enabled different and distinct design paths towards a high‐yielding strain. Here we highlight a rational strain design process in systems biology, an integrated computational and experimental approach for carboxylic acid production, as an alternative method. Additional challenges inherent in achieving an optimal strain for commercialization of medium chain‐length fatty acids will likely require a collection of strategies from systems metabolic engineering. Not only will the continued advancement in systems metabolic engineering result in these highly productive strains more quickly, this knowledge will extend more rapidly the carboxylic acid platform to the microbial production of carboxylic acids with alternate chain‐lengths and functionalities. Biotechnol. Biotechnol. Bioeng. 2014;111: 849–857. © 2014 Wiley Periodicals, Inc.     

非常规酵母的分子遗传学及合成生物学研究进展

先进的合成生物学技术与传统的分子遗传学技术的结合更有助于实现酵母底盘细胞的快速改造和优化。酵母合成生物学研究最早开始于常规酵母——酿酒酵母(Saccharomyces cerevisiae),近些年来又迅速扩展至一些非常规酵母,包括巴斯德毕赤酵母(Pichiapastoris)、解脂耶氏酵母(Yarrowialipolytica)、乳酸克鲁维酵母(Kluyveromyces lactis)和多形汉逊酵母(Hansenula polymorpha)等。借助合成生物学技术与工具,目前科学家们已经成功开发出了能够高效生产生物材料、生物燃料、生物基化学品、蛋白质制剂、食品添加剂和药物等工业产品的重组非常规酵母工程菌株。本文系统总结了合成生物学工具(主要是基因组编辑工具)、合成生物学组件(主要是启动子和终止子)和相关分子遗传学方法在上述非常规酵母系统(底盘细胞)中的最新研究进展和应用情况,并讨论了其他合成生物学技术在这些非常规酵母表达系统中的潜在适用性和应用前景。这为研究人员利用合成生物学方法在这一新型非模式微生物底盘细胞中设计和构建各种高附加值工业产品的异源合成模块并最终实现目标化合物的高效生物合成提供了科学的理论指导。     

Quorum sensing-based metabolic engineering of the precursor supply in Streptomyces coelicolor to improve heterologous production of neoaureothin

Streptomyces are important industrial bacteria that produce pharmaceutically valuable polyketides. However, mass production on an industrial scale is limited by low productivity, which can be overcome through metabolic engineering and the synthetic biology of the host strain. Recently, the introduction of an auto-inducible expression system depending on microbial physiological state has been suggested as an important tool for the industrial-scale production of polyketides. In this study, titer improvement by enhancing the pool of CoA-derived precursors required for polyketide production was driven in a quorum sensing (QS)-dependent manner. A self-sustaining and inducer-independent regulatory system, named the QS-based metabolic engineering of precursor pool (QMP) system, was constructed, wherein the expression of genes involved in precursor biosynthesis was regulated by the QS-responsive promoter, scbAp. The QMP system was applied for neoaureothin production in a heterologous host, Streptomyces coelicolor M1152, and productivity increased by up to 4-fold. In particular, the engineered hyperproducers produced high levels of neoaureothin without adversely affecting cell growth. Overall, this study showed that self-regulated metabolic engineering mediated by QS has the potential to engineer strains for polyketide titer improvement.     

Constraints-based genome-scale metabolic simulation for systems metabolic engineering

Random mutagenesis and selection approaches used traditionally for the development of industrial strains have largely been complemented by metabolic engineering, which allows purposeful modification of metabolic and cellular characteristics by using recombinant DNA and other molecular biological techniques. As systems biology advances as a new paradigm of research thanks to the development of genome-scale computational tools and high-throughput experimental technologies including omics, systems metabolic engineering allowing modification of metabolic, regulatory and signaling networks of the cell at the systems-level is becoming possible. In silico genome-scale metabolic model and its simulation play increasingly important role in providing systematic strategies for metabolic engineering. The in silico genome-scale metabolic model is developed using genomic annotation, metabolic reactions, literature information, and experimental data. The advent of in silico genome-scale metabolic model brought about the development of various algorithms to simulate the metabolic status of the cell as a whole. In this paper, we review the algorithms developed for the system-wide simulation and perturbation of cellular metabolism, discuss the characteristics of these algorithms, and suggest future research direction.     

The path to next generation biofuels: successes and challenges in the era of synthetic biology

Volatility of oil prices along with major concerns about climate change, oil supply security and depleting reserves have sparked renewed interest in the production of fuels from renewable resources. Recent advances in synthetic biology provide new tools for metabolic engineers to direct their strategies and construct optimal biocatalysts for the sustainable production of biofuels. Metabolic engineering and synthetic biology efforts entailing the engineering of native and de novo pathways for conversion of biomass constituents to short-chain alcohols and advanced biofuels are herewith reviewed. In the foreseeable future, formal integration of functional genomics and systems biology with synthetic biology and metabolic engineering will undoubtedly support the discovery, characterization, and engineering of new metabolic routes and more efficient microbial systems for the production of biofuels.     

丙酮丁醇梭菌的基因编辑工具及代谢工程改造

丙酮丁醇梭菌作为极具潜力的新型生物燃料丁醇的生产菌,受到各国研究学者的广泛关注。通过丙酮丁醇梭菌(ABE)发酵生产丁醇,由于生产成本高,限制了其工业化应用。随着基因组学和分子生物学的快速发展,适用于丙酮丁醇的基因编辑工具不断发展并应用于提高菌株的发酵性能。本文对丙酮丁醇梭菌基因编辑工具和代谢工程改造取得的进展进行综述。     

Genome-scale analysis of the metabolic networks of oleaginous Zygomycete fungi

Microbial lipids are becoming an attractive option for the industrial production of foods and oleochemicals. To investigate the lipid physiology of the oleaginous microorganisms, at the system level, genome-scale metabolic networks of Mortierella alpina and Mucor circinelloides were constructed using bioinformatics and systems biology. As scaffolds for integrated data analysis focusing on lipid production, consensus metabolic routes governing fatty acid synthesis, and lipid storage and mobilisation were identified by comparative analysis of developed metabolic networks. Unique metabolic features were identified in individual fungi, particularly in NADPH metabolism and sterol biosynthesis, which might be related to differences in fungal lipid phenotypes. The frameworks detailing the metabolic relationship between M. alpina and M. circinelloides generated in this study is useful for further elucidation of the microbial oleaginicity, which might lead to the production improvement of microbial oils as alternative feedstocks for oleochemical industry.     

Metabolic engineering of Escherichia coli: A sustainable industrial platform for bio-based chemical production

In order to decrease carbon emissions and negative environmental impacts of various pollutants, more bulk and/or fine chemicals are produced by bioprocesses, replacing the traditional energy and fossil based intensive route. The Gram-negative rod-shaped bacterium, Escherichia coli has been studied extensively on a fundamental and applied level and has become a predominant host microorganism for industrial applications. Furthermore, metabolic engineering of E. coli for the enhanced biochemical production has been significantly promoted by the integrated use of recent developments in systems biology, synthetic biology and evolutionary engineering. In this review, we focus on recent efforts devoted to the use of genetically engineered E. coli as a sustainable platform for the production of industrially important biochemicals such as biofuels, organic acids, amino acids, sugar alcohols and biopolymers. In addition, representative secondary metabolites produced by E. coli will be systematically discussed and the successful strategies for strain improvements will be highlighted. Moreover, this review presents guidelines for future developments in the bio-based chemical production using E. coli as an industrial platform.     

Systems approaches to succinic acid-producing microorganisms

Succinic acid is a cellular metabolite belonging to the C4-dicarboxylic acid family, and the fermentative production of succinic acid via the use of recombinant microorganisms has recently become the focus of an increasing amount of attention. Considering the difficulty inherent to the direct application of natural succinic acid producers to the industrial process, a variety of systems biology studies have been conducted regarding the development of enhanced succinic acid production systems. This review shows how the metabolic processes of microorganisms, includingEscherichia coli andMannheimia succiniciproducens, have been optimized in order to achieve enhanced succinic acid production. First, their metabolic networks were constructed on the basis of complete genome sequences, after which their metabolic characteristics were estimated viain silico computer modeling. Metabolic engineering strategies were designed in accordance with the results ofin silico modeling and metabolically engineered versions of bothE. coli andM. succiniciproducens have been constructed. The succinic acid productivity and yield obtained using metabolically engineered bacteria was significantly higher than that obtained using wild-type bacteria.     

High-efficiency production of bisabolene from waste cooking oil by metabolically engineered Yarrowia lipolytica

The natural plant product bisabolene serves as a precursor for the production of a wide range of industrially relevant chemicals. However, the low abundance of bisabolene in plants renders its isolation from plant sources non-economically viable. Therefore, creation of microbial cell factories for bisabolene production supported by synthetic biology and metabolic engineering strategies presents a more competitive and environmentally sustainable method for industrial production of bisabolene. In this proof-of-principle study, for the first time, we engineered the oleaginous yeast Yarrowia lipolytica to produce α-bisabolene, β-bisabolene and γ-bisabolene through heterologous expression of the α-bisabolene synthase from Abies grandis, the β-bisabolene synthase gene from Zingiber officinale and the γ-bisabolene synthase gene from Helianthus annuus respectively. Subsequently, two metabolic engineering approaches, including overexpression of the endogenous mevalonate pathway genes and introduction of heterologous multidrug efflux transporters, were employed in order to improve bisabolene production. Furthermore, the fermentation conditions were optimized to maximize bisabolene production by the engineered Y. lipolytica strains from glucose. Finally, we explored the potential of the engineered Y. lipolytica strains for bisabolene production from the waste cooking oil. To our knowledge, this is the first report of bisabolene production in Y. lipolytica using metabolic engineering strategies. These findings provide valuable insights into the engineering of Y. lipolytica for a higher-level production of bisabolene and its utilization in converting waste cooking oil into various industrially valuable products.     

Biotechnological production of feed nucleotides by microbial strain improvement

Sodium salts of inosine monophosphate (IMP) and guanosine monophosphate (GMP) are potent flavour enhancers. They are widely used as food additives in combination with monosodium glutamate (MSG) to synergistically increase umami flavour. In recent years, both inosine and guanosine derivatives have gained further importance because of their beneficial effects, related to their antioxidant, neuroprotective, cardiotonic and immunomodulatory properties. The industrial production of both IMP and GMP is mainly achieved either by RNA breakdown and nucleotide extraction or by microbial fermentation using different microorganisms such as Corynebacterium, Bacillus, or Escherichia coli. This work reviews the metabolic pathways and regulatory networks of purine synthesis, including both IMP and GMP, and the biotechnological processes applied to the production of these compounds, ranging from classical random mutagenesis to rational design by metabolic engineering. Recent advances of systems biology approaches, along with the rapid development of synthetic biology, may offer a basis for future manipulations to further increase the productivity of the fermentation processes.     

四乙酰基植物鞘氨醇生物合成的研究进展

四乙酰基植物鞘氨醇(tetraacetyl phytosphingosine, TAPS)是一种性能卓越的天然护肤品原料,经去乙酰化后生成的植物鞘氨醇可作为前体合成保湿护肤品神经酰胺,因此广泛应用于护肤化妆品行业。非常规酵母威克汉姆西弗酵母(Wickerhamomyces ciferrii)是已知的唯一可天然分泌四乙酰基植物鞘氨醇的微生物,目前已成为四乙酰基植物鞘氨醇工业生产的宿主。本文介绍了四乙酰基植物鞘氨醇的发现、功能及其生物合成途径,综述了近年来利用单倍体筛选、诱变育种和代谢工程改造威克汉姆西弗酵母高产四乙酰基植物鞘氨醇的研究进展,并展望了实现四乙酰基植物鞘氨醇工业生产的未来发展方向。     

Recent advances in metabolic engineering of Corynebacterium glutamicum for bioproduction of value-added aromatic chemicals and natural products

Recent progress in synthetic and systems metabolic engineering technologies has explored the potential of microbial cell factories for the production of industrially relevant bulk and fine chemicals from renewable biomass resources in an eco-friendly manner. Corynebacterium glutamicum, a workhorse for industrial amino acid production, has currently evolved into a promising microbial platform for bioproduction of various natural and non-natural chemicals from renewable feedstocks. Notably, it has been recently demonstrated that metabolically engineered C. glutamicum can overproduce several commercially valuable aromatic and related chemicals such as shikimate, 4-hydroxybenzoate, and 4-aminobenzoate from sugars at remarkably high titer suitable to commercial application. On the other hand, overexpression and/or extension of its endogenous metabolic pathways by integrating heterologous metabolic pathways enabled production of structurally intricate and valuable natural chemicals like plant polyphenols, carotenoids, and fatty acids. In this review, we summarize recent advances in metabolic engineering of C. glutamicum for production of those value-added aromatics and other natural products, which highlights high potential and the versatility of this microbe for bioproduction of diverse chemicals.

     

代谢工程改造酿酒酵母合成植物萜类D-柠檬烯的策略

植物萜类化合物是以异戊二烯为结构单位的一大类植物天然的次生代谢产物。D-柠檬烯属于单萜类化合物,由于它具有抑菌、增香、抗癌、止咳、平喘等多种功能,已被广泛应用于食品、香料、医疗等行业。目前D-柠檬烯的工业生产主要是从植物的果皮或者果肉中提取的,但提取方法存在着分离纯化复杂、产率低、能耗大等缺点。而本世纪初合成生物学技术的兴起,为微生物异源合成天然活性化合物带来了全新的理念与工具,打破了物种间的界限,使微生物异源合成D-柠檬烯成为现实。构建定向、高效的异源合成D-柠檬烯的微生物细胞工厂,实现微生物发酵法替换传统的植物提取法,具有重要的经济与社会效益。本文主要回顾了近几年利用代谢工程改造酿酒酵母异源合成萜类化合物取得的成就,阐述了以酿酒酵母作为底盘微生物,利用代谢工程和合成生物学的手段构建高产D-柠檬烯的合成策略。     

Opportunities for yeast metabolic engineering: Lessons from synthetic biology

Constant progress in genetic engineering has given rise to a number of promising areas of research that facilitated the expansion of industrial biotechnology. The field of metabolic engineering, which utilizes genetic tools to manipulate microbial metabolism to enhance the production of compounds of interest, has had a particularly strong impact by providing new platforms for chemical production. Recent developments in synthetic biology promise to expand the metabolic engineering toolbox further by creating novel biological components for pathway design. The present review addresses some of the recent advances in synthetic biology and how these have the potential to affect metabolic engineering in the yeast Saccharomyces cerevisiae. While S. cerevisiae for years has been a robust industrial organism and the target of multiple metabolic engineering trials, its potential for synthetic biology has remained relatively unexplored and further research in this field could strongly contribute to industrial biotechnology. This review also addresses are general considerations for pathway design, ranging from individual components to regulatory systems, overall pathway considerations and whole-organism engineering, with an emphasis on potential contributions of synthetic biology to these areas. Some examples of applications for yeast synthetic biology and metabolic engineering are also discussed.     

Next‐Generation Industrial Biotechnology‐Transforming the Current Industrial Biotechnology into Competitive Processes

The chemical industry has made a contribution to modern society by providing cost‐competitive products for our daily use. However, it now faces a serious challenge regarding environmental pollutions and greenhouse gas emission. With the rapid development of molecular biology, biochemistry, and synthetic biology, industrial biotechnology has evolved to become more efficient for production of chemicals and materials. However, in contrast to chemical industries, current industrial biotechnology (CIB) is still not competitive for production of chemicals, materials, and biofuels due to their low efficiency and complicated sterilization processes as well as high‐energy consumption. It must be further developed into “next‐generation industrial biotechnology” (NGIB), which is low‐cost mixed substrates based on less freshwater consumption, energy‐saving, and long‐lasting open continuous intelligent processing, overcoming the shortcomings of CIB and transforming the CIB into competitive processes. Contamination‐resistant microorganism as chassis is the key to a successful NGIB, which requires resistance to microbial or phage contaminations, and available tools and methods for metabolic or synthetic biology engineering. This review proposes a list of contamination‐resistant bacteria and takes Halomonas spp. as an example for the production of a variety of products, including polyhydroxyalkanoates under open‐ and continuous‐processing conditions proposed for NGIB.     

Recent Advances in the Physiology and Genetics of Amino Acid-Producing Bacteria

Abstract

Corynebacterium glutamicum and its close relatives, C. flavum and C. lactofennentum, have been used for over 3 decades in the industrial production of amino acids by fermentation. Since 1984, several research groups have started programs to develop metabolic engineering principles for amino acid-producing Corynebacferium strains. Initially, the programs concentrated on the isolation of genes encoding (deregulated) biosynthetic enzymes and the development of general molecular biology tools such as cloning vectors and DNA transfer methods. With most of the genes and tools now available, recombinant DNA technology can be applied in strain improvement. To accomplish these improvements, it is critical and advantageous to understand the mechanisms of gene expression and regulation as well as the biochemistry and physiology of the species being engineered. This review explores the advances made in the understanding and application of amino acid-producing bacteria in the early 1990s.     

Pyruvate production in Candida glabrata: manipulation and optimization of physiological function

Candida glabrata, a multi-vitamin auxotrophic yeast, can accumulate a large amount of pyruvate extracellularly using glucose as the carbon source, a characteristic that has facilitated the cost-effective biotechnological production of pyruvate on an industrial scale. In this review, we describe the current advances in further improving the performance of C. glabrata for efficient pyruvate production, which includes: optimization of the vitamin and dissolved oxygen concentrations, regulation of intracellular cofactor levels and improvement of the environmental robustness of C. glabrata. We also discuss the current efforts using systems biology to understand the metabolism of C. glabrata. Finally, perspectives on engineering and exploiting C. glabrata as a cell factory for efficiently producing various chemicals and materials are discussed.