Engineering of microorganisms for the production of biofuels and perspectives based on systems metabolic engineering approaches

The increasing oil price and environmental concerns caused by the use of fossil fuel have renewed our interest in utilizing biomass as a sustainable resource for the production of biofuel. It is however essential to develop high performance microbes that are capable of producing biofuels with very high efficiency in order to compete with the fossil fuel. Recently, the strategies for developing microbial strains by systems metabolic engineering, which can be considered as metabolic engineering integrated with systems biology and synthetic biology, have been developed. Systems metabolic engineering allows successful development of microbes that are capable of producing several different biofuels including bioethanol, biobutanol, alkane, and biodiesel, and even hydrogen. In this review, the approaches employed to develop efficient biofuel producers by metabolic engineering and systems metabolic engineering approaches are reviewed with relevant example cases. It is expected that systems metabolic engineering will be employed as an essential strategy for the development of microbial strains for industrial applications.     

Biofuel production in Escherichia coli: the role of metabolic engineering and synthetic biology

The microbial production of biofuels is a promising avenue for the development of viable processes for the generation of fuels from sustainable resources. In order to become cost and energy effective, these processes must utilize organisms that can be optimized to efficiently produce candidate fuels from a variety of feedstocks. Escherichia coli has become a promising host organism for the microbial production of biofuels in part due to the ease at which this organism can be manipulated. Advancements in metabolic engineering and synthetic biology have led to the ability to efficiently engineer E. coli as a biocatalyst for the production of a wide variety of potential biofuels from several biomass constituents. This review focuses on recent efforts devoted to engineering E. coli for the production of biofuels, with emphasis on the key aspects of both the utilization of a variety of substrates as well as the synthesis of several promising biofuels. Strategies for the efficient utilization of carbohydrates, carbohydrate mixtures, and noncarbohydrate carbon sources will be discussed along with engineering efforts for the exploitation of both fermentative and nonfermentative pathways for the production of candidate biofuels such as alcohols and higher carbon biofuels derived from fatty acid and isoprenoid pathways. Continued advancements in metabolic engineering and synthetic biology will help improve not only the titers, yields, and productivities of biofuels discussed herein, but also increase the potential range of compounds that can be produced.     

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。     

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.     

Special focus: Synthetic biology

Special focus: Synthetic biology What is synthetic biology? SynBERC – The Synthetic Biology Engineering Research Center Ars Synthetica iGEM – The International Genetically Engineered Machine competition Some synthetic biology companies Paper watch: Synthetic biology Building blocks for novel functions Knowledge-making distinctions in synthetic biology Scaffold design and manufacturing: From concept to clinic Peptidomimetics – a versatile route to biologically active compounds Metabolic engineering of E. coli E. coli needs safety valves Systems-level metabolic engineering Mammalian synthetic biology Chemical aspects of synthetic biology Synthesis of DNA fragments in yeast Synthetic biology and patentable subject matter Patenting artificial life? Metabolic effects of synthetic rewiring Engineering for biofuels Regulatory elements for synthetic biology Book highlight Systems Biology and Synthetic Biology     

Synthetic Biology Toolkits for Metabolic Engineering of Cyanobacteria

Cyanobacteria are of great importance to Earth's ecology. Due to their capability in photosynthesis and C1 metabolism, they are ideal microbial chassis that can be engineered for direct conversion of carbon dioxide and solar energy into biofuels and biochemicals. Facilitated by the elucidation of the basic biology of the photoautotrophic microbes and rapid advances in synthetic biology, genetic toolkits have been developed to enable implementation of nonnatural functionalities in engineered cyanobacteria. Hence, cyanobacteria are fast becoming an emerging platform in synthetic biology and metabolic engineering. Herein, the progress made in the synthetic biology toolkits for cyanobacteria and their utilization for transforming cyanobacteria into microbial cell factories for sustainable production of biofuels and biochemicals is outlined. Current techniques in heterologous gene expression, strategies in genome editing, and development of programmable regulatory parts and modules for engineering cyanobacteria towards biochemical production are discussed and prospected. As cyanobacteria synthetic biology is still in its infancy, apart from the achievements made, the difficulties and challenges in applying and developing genetic toolkits in cyanobacteria for biochemical production are also evaluated.     

Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels

The ability to generate microorganisms that can produce biofuels similar to petroleum-based transportation fuels would allow the use of existing engines and infrastructure and would save an enormous amount of capital required for replacing the current infrastructure to accommodate biofuels that have properties significantly different from petroleum-based fuels. Several groups have demonstrated the feasibility of manipulating microbes to produce molecules similar to petroleum-derived products, albeit at relatively low productivity (e.g. maximum butanol production is around 20 g/L). For cost-effective production of biofuels, the fuel-producing hosts and pathways must be engineered and optimized. Advances in metabolic engineering and synthetic biology will provide new tools for metabolic engineers to better understand how to rewire the cell in order to create the desired phenotypes for the production of economically viable biofuels.     

Metabolic engineering strategies for acetoin and 2,3-butanediol production: advances and prospects

Acetoin and 2,3-butanediol (2,3-BD) have a large number of industrial applications. The production of acetoin and 2,3-BD has traditionally relied on oil supplies. Microbial production of acetoin and 2,3-BD will alleviate the dependence on oil. Acetoin and 2,3-BD are neighboring metabolites in the 2,3-BD metabolic pathway of bacteria. This review summarizes metabolic engineering strategies for improvement of microbial acetoin and 2,3-BD production. We also propose enhancements to current acetoin and 2,3-BD production strategies, by offering a metabolic engineering approach that is guided by systems biology and synthetic biology.     

Practical application of synthetic biology principles

Synthetic biology can be defined as the “repurposing and redesign of biological systems for novel purposes or applications, ” and the field lies at the interface of several biological research areas. This broad definition can be taken to include a variety of investigative endeavors, and successful design of new biological paradigms requires integration of many scientific disciplines including (but not limited to) protein engineering, metabolic engineering, genomics, structural biology, chemical biology, systems biology, and bioinformatics. This review focuses on recent applications of synthetic biology principles in three areas: (i) the construction of artificial biomolecules and biomaterials; (ii) the synthesis of both fine and bulk chemicals (including biofuels); and (iii) the construction of “smart” biological systems that respond to the surrounding environment.     

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.     

Industrial biotechnology: Tools and applications

Industrial biotechnology involves the use of enzymes and microorganisms to produce value-added chemicals from renewable sources. Because of its association with reduced energy consumption, greenhouse gas emissions, and waste generation, industrial biotechnology is a rapidly growing field. Here we highlight a variety of important tools for industrial biotechnology, including protein engineering, metabolic engineering, synthetic biology, systems biology, and downstream processing. In addition, we show how these tools have been successfully applied in several case studies, including the production of 1, 3-propanediol, lactic acid, and biofuels. It is expected that industrial biotechnology will be increasingly adopted by chemical, pharmaceutical, food, and agricultural industries.     

Engineering microbial factories for synthesis of value-added products

Microorganisms have become an increasingly important platform for the production of drugs, chemicals, and biofuels from renewable resources. Advances in protein engineering, metabolic engineering, and synthetic biology enable redesigning microbial cellular networks and fine-tuning physiological capabilities, thus generating industrially viable strains for the production of natural and unnatural value-added compounds. In this review, we describe the recent progress on engineering microbial factories for synthesis of valued-added products including alkaloids, terpenoids, flavonoids, polyketides, non-ribosomal peptides, biofuels, and chemicals. Related topics on lignocellulose degradation, sugar utilization, and microbial tolerance improvement will also be discussed.     

Engineering biofuel tolerance in non-native producing microorganisms

Large-scale production of renewable biofuels through microbiological processes has drawn significant attention in recent years, mostly due to the increasing concerns on the petroleum fuel shortages and the environmental consequences of the over-utilization of petroleum-based fuels. In addition to native biofuel-producing microbes that have been employed for biofuel production for decades, recent advances in metabolic engineering and synthetic biology have made it possible to produce biofuels in several non-native biofuel-producing microorganisms. Compared to native producers, these non-native systems carry the advantages of fast growth, simple nutrient requirements, readiness for genetic modifications, and even the capability to assimilate CO₂ and solar energy, making them competitive alternative systems to further decrease the biofuel production cost. However, the tolerance of these non-native microorganisms to toxic biofuels is naturally low, which has restricted the potentials of their application for high-efficiency biofuel production. To address the issues, researches have been recently conducted to explore the biofuel tolerance mechanisms and to construct robust high-tolerance strains for non-native biofuel-producing microorganisms. In this review, we critically summarize the recent progress in this area, focusing on three popular non-native biofuel-producing systems, i.e. Escherichia coli, Lactobacillus and photosynthetic cyanobacteria.     

代谢工程30年专刊序言 (2021)

代谢工程利用重组DNA技术、合成生物学、基因组编辑来改变生物体的细胞网络,包括代谢、基因调控和信号网络等。它可以实现加强包括化学品、燃料、化学原料药和其他生物技术产品等代谢物生产的目标,提升生物制造能力与效率。为了梳理和凝练代谢工程30年来的发展状况,《生物工程学报》特组织出版专刊,从代谢工程总体发展、共性技术以及以什么宿主和做什么产品等4个方面展现该领域的发展动态和趋势,并为代谢工程领域的进一步发展提出建设性的意见与展望。     

Systems metabolic engineering of microorganisms for natural and non-natural chemicals

Growing concerns over limited fossil resources and associated environmental problems are motivating the development of sustainable processes for the production of chemicals, fuels and materials from renewable resources. Metabolic engineering is a key enabling technology for transforming microorganisms into efficient cell factories for these compounds. Systems metabolic engineering, which incorporates the concepts and techniques of systems biology, synthetic biology and evolutionary engineering at the systems level, offers a conceptual and technological framework to speed the creation of new metabolic enzymes and pathways or the modification of existing pathways for the optimal production of desired products. Here we discuss the general strategies of systems metabolic engineering and examples of its application and offer insights as to when and how each of the different strategies should be used. Finally, we highlight the limitations and challenges to be overcome for the systems metabolic engineering of microorganisms at more advanced levels.     

Systems metabolic engineering for chemicals and materials

Metabolic engineering has contributed significantly to the enhanced production of various value-added and commodity chemicals and materials from renewable resources in the past two decades. Recently, metabolic engineering has been upgraded to the systems level (thus, systems metabolic engineering) by the integrated use of global technologies of systems biology, fine design capabilities of synthetic biology, and rational-random mutagenesis through evolutionary engineering. By systems metabolic engineering, production of natural and unnatural chemicals and materials can be better optimized in a multiplexed way on a genome scale, with reduced time and effort. Here, we review the recent trends in systems metabolic engineering for the production of chemicals and materials by presenting general strategies and showcasing representative examples.     

Advanced biofuel production in microbes

The cost-effective production of biofuels from renewable materials will begin to address energy security and climate change concerns. Ethanol, naturally produced by microorganisms, is currently the major biofuel in the transportation sector. However, its low energy content and incompatibility with existing fuel distribution and storage infrastructure limits its economic use in the future. Advanced biofuels, such as long chain alcohols and isoprenoid- and fatty acid-based biofuels, have physical properties that more closely resemble petroleum-derived fuels, and as such are an attractive alternative for the future supplementation or replacement of petroleum-derived fuels. Here, we review recent developments in the engineering of metabolic pathways for the production of known and potential advanced biofuels by microorganisms. We concentrate on the metabolic engineering of genetically tractable organisms such as Escherichia coli and Saccharomyces cerevisiae for the production of these advanced biofuels.     

Development of biocatalysts for production of commodity chemicals from lignocellulosic biomass

Lignocellulosic biomass is recognized as potential sustainable source for production of power, biofuels and variety of commodity chemicals which would potentially add economic value to biomass. Recalcitrance nature of biomass is largely responsible for the high cost of its conversion. Therefore, it is necessary to introduce some cost effective pretreatment processes to make the biomass polysaccharides easily amenable to enzymatic attack to release mixed fermentable sugars. Advancement in systemic biology can provide new tools for the development of such biocatalysts for sustainable production of commodity chemicals from biomass. Integration of functional genomics and system biology approaches may generate efficient microbial systems with new metabolic routes for production of commodity chemicals. This paper provides an overview of the challenges that are faced by the processes converting lignocellulosic biomass to commodity chemicals. The critical factors involved in engineering new microbial biocatalysts are also discussed with more emphasis on commodity chemicals.     

Protein design in systems metabolic engineering for industrial strain development

Accelerating the process of industrial bacterial host strain development, aimed at increasing productivity, generating new bio-products or utilizing alternative feedstocks, requires the integration of complementary approaches to manipulate cellular metabolism and regulatory networks. Systems metabolic engineering extends the concept of classical metabolic engineering to the systems level by incorporating the techniques used in systems biology and synthetic biology, and offers a framework for the development of the next generation of industrial strains. As one of the most useful tools of systems metabolic engineering, protein design allows us to design and optimize cellular metabolism at a molecular level. Here, we review the current strategies of protein design for engineering cellular synthetic pathways, metabolic control systems and signaling pathways, and highlight the challenges of this subfield within the context of systems metabolic engineering.