Metabolic pathway engineering in lactic acid bacteria

Lactic acid bacteria (LAB) display a relatively simple carbon and energy metabolism where the sugar source is converted mainly to lactic acid. In Lactococcus lactis metabolic engineering has been very successful in the re-routing of lactococcal pyruvate metabolism to products other than lactic acid. Current metabolic engineering approaches tend to focus on more complex, biosynthetic pathways leading to end-products that generate a health benefit for the consumer (nutraceuticals). Several examples of research on these minor pathways in L. lactis have illustrated the potential of LAB as producers of these metabolites. Whole genome sequencing efforts and corresponding global technologies will have an impact on metabolic engineering in the future.     

Therapeutic peptide production in Drosophila

Bioactive peptides are important therapeutic drugs, yet conventional methods of peptide synthesis are challenged to meet increasing demand. We developed a novel and efficient means of metabolic engineering: therapeutic peptide production in Drosophila and as a proof of concept, we demonstrate production of fully matured human insulin. This in vivo system offers an innovative means to produce valuable bioactive peptides for therapies, its inherent flexibility facilitates drug development, and its ease of producing fully processed peptides simplifies metabolic engineering of new peptide products.     

历久弥新：进化中的代谢工程

20世纪90年代,Bailey及Stephanopoulos等提出了经典代谢工程的理念,旨在利用DNA重组技术对代谢网络进行改造,以达到细胞性能改善,目标产物增加的目的。自代谢工程诞生以来的30年,生命科学蓬勃发展,基因组学、系统生物学、合成生物学等新学科不断涌现,为代谢工程的发展注入了新的内涵与活力。经典代谢工程研究已进入到前所未有的系统代谢工程阶段。组学技术、基因组代谢模型、元件组装、回路设计、动态控制、基因组编辑等合成生物学工具与策略的应用,大大提升了复杂代谢的设计与合成能力;机器学习的介入以及进化工程与代谢工程的结合,为系统代谢工程的未来开辟了新的方向。文中对过去30年代谢工程的发展趋势作了梳理,介绍了代谢工程在发展中不断创新的理论与方法及其应用。     

生理代谢参数RQ在指导发酵过程优化中的应用

过程在线参数检测是进行发酵过程工程优化控制的基础。呼吸熵RQ是微生物胞内微观代谢流在宏观代谢参数上的响应,反映了微生物培养过程中底物的利用情况、产物和副产物的合成情况、及微观代谢途径通量的变化。结合发酵葡萄糖酸、2,3-丁二醇谷氨酸、柠檬酸、头孢菌素C、毕赤酵母α-干扰素产品的工业生产过程,分析了以微生物胞内微观代谢与宏观的生理参数RQ为指导的发酵工艺优化策略。RQ值在发酵过程中可以根据尾气数据进行在线采集,对指导通过宏观代谢参数的调控来最优化微生物胞内的代谢途经通量,提高目的产物的产率具有非常重要的意义。     

Metabolic and evolutionary engineering research in Turkey and beyond

This review discusses metabolic engineering research with an emphasis on evolutionary (whole cell and protein) engineering, which is an inverse metabolic engineering approach. For each section on metabolic, inverse metabolic and evolutionary engineering research, a general review of the major global studies in the literature is made and research examples from Turkey are given and discussed. It is expected that with the rapid development in systems biology and the novel powerful analytical technologies to identify the genetic basis of cellular phenotypes, metabolic and evolutionary engineering research will become widespread and increasingly important in Turkey, following global scientific trends.     

Production and engineering of terpenoids in plant cell culture

Terpenoids are a diverse class of natural products that have many functions in the plant kingdom and in human health and nutrition. Their chemical diversity has led to the discovery of over 40,000 different structures, with several classes serving as important pharmaceutical agents, including the anticancer agents paclitaxel (Taxol) and terpenoid-derived indole alkaloids. Many terpenoid compounds are found in low yield from natural sources, so plant cell cultures have been investigated as an alternate production strategy. Metabolic engineering of whole plants and plant cell cultures is an effective tool to both increase terpenoid yield and alter terpenoid distribution for desired properties such as enhanced flavor, fragrance or color. Recent advances in defining terpenoid metabolic pathways, particularly in secondary metabolism, enhanced knowledge concerning regulation of terpenoid accumulation, and application of emerging plant systems biology approaches, have enabled metabolic engineering of terpenoid production. This paper reviews the current state of knowledge of terpenoid metabolism, with a special focus on production of important pharmaceutically active secondary metabolic terpenoids in plant cell cultures. Strategies for defining pathways and uncovering rate-influencing steps in global metabolism, and applying this information for successful terpenoid metabolic engineering, are emphasized.     

Protein-based biorefining: metabolic engineering for production of chemicals and fuel with regeneration of nitrogen fertilizers

Threats to stable oil supplies and concerns over environmental emissions have pushed for renewable biofuel developments to minimize dependence on fossil resources. Recent biofuel progress has moved towards fossil resource-independent carbon cycles, but environmental issues regarding use of nitrogen fertilizers have not been addressed on a global scale. The recently demonstrated conversion of waste protein biomass into advanced biofuels and renewable chemicals, while recycling nitrogen fertilizers, offers a glimpse of the efforts needed to balance the nitrogen cycle at scale. In general, the catabolism of protein into biofuels is challenging because of physiological regulation and thermodynamic limitations. This conversion became possible with metabolic engineering around ammonia assimilation, intracellular nitrogen flux, and quorum sensing. This review highlights the metabolic engineering solutions in transforming those cellular processes into driving forces for the high yield of chemical products from protein.     

Structure-driven protein engineering for production of valuable natural products

Proteins are the most frequently used biocatalysts, and their structures determine their functions. Modifying the functions of proteins on the basis of their structures lies at the heart of protein engineering, opening a new horizon for metabolic engineering by efficiently generating stable enzymes. Many attempts at classical metabolic engineering have focused on improving specific metabolic fluxes and producing more valuable natural products by increasing gene expression levels and enzyme concentrations. However, most naturally occurring enzymes show limitations, and such limitations have hindered practical applications. Here we review recent advances in protein engineering in synthetic biology, chemoenzymatic synthesis, and plant metabolic engineering and describe opportunities for designing and constructing novel enzymes or proteins with desirable properties to obtain more active natural products.     

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。     

A systems-level approach for metabolic engineering of yeast cell factories

The generation of novel yeast cell factories for production of high-value industrial biotechnological products relies on three metabolic engineering principles: design, construction, and analysis. In the last two decades, strong efforts have been put on developing faster and more efficient strategies and/or technologies for each one of these principles. For design and construction, three major strategies are described in this review: (1) rational metabolic engineering; (2) inverse metabolic engineering; and (3) evolutionary strategies. Independent of the selected strategy, the process of designing yeast strains involves five decision points: (1) choice of product, (2) choice of chassis, (3) identification of target genes, (4) regulating the expression level of target genes, and (5) network balancing of the target genes. At the construction level, several molecular biology tools have been developed through the concept of synthetic biology and applied for the generation of novel, engineered yeast strains. For comprehensive and quantitative analysis of constructed strains, systems biology tools are commonly used and using a multi-omics approach. Key information about the biological system can be revealed, for example, identification of genetic regulatory mechanisms and competitive pathways, thereby assisting the in silico design of metabolic engineering strategies for improving strain performance. Examples on how systems and synthetic biology brought yeast metabolic engineering closer to industrial biotechnology are described in this review, and these examples should demonstrate the potential of a systems-level approach for fast and efficient generation of yeast cell factories.     

Synthetic biology and metabolic engineering of actinomycetes for natural product discovery

Actinomycetes are one of the most valuable sources of natural products with industrial and medicinal importance. After more than half a century of exploitation, it has become increasingly challenging to find novel natural products with useful properties as the same known compounds are often repeatedly re-discovered when using traditional approaches. Modern genome mining approaches have led to the discovery of new biosynthetic gene clusters, thus indicating that actinomycetes still harbor a huge unexploited potential to produce novel natural products. In recent years, innovative synthetic biology and metabolic engineering tools have greatly accelerated the discovery of new natural products and the engineering of actinomycetes. In the first part of this review, we outline the successful application of metabolic engineering to optimize natural product production, focusing on the use of multi-omics data, genome-scale metabolic models, rational approaches to balance precursor pools, and the engineering of regulatory genes and regulatory elements. In the second part, we summarize the recent advances of synthetic biology for actinomycetal metabolic engineering including cluster assembly, cloning and expression, CRISPR/Cas9 technologies, and chassis strain development for natural product overproduction and discovery. Finally, we describe new advances in reprogramming biosynthetic pathways through polyketide synthase and non-ribosomal peptide synthetase engineering. These new developments are expected to revitalize discovery and development of new natural products with medicinal and other industrial applications.     

Medicinally important secondary metabolites in recombinant microorganisms or plants: Progress in alkaloid biosynthesis

Plants produce a high diversity of natural products or secondary metabolites which are important for the communication of plants with other organisms. A prominent function is the protection against herbivores and/or microbial pathogens. Some natural products are also involved in defence against abiotic stress, e.g. UV-B exposure. Many of the secondary metabolites have interesting biological properties and quite a number are of medicinal importance. Because the production of the valuable natural products, such as the anticancer drugs paclitaxel, vinblastine or camptothecin in plants is a costly process, biotechnological alternatives to produce these alkaloids more economically become increasingly important. This review provides an overview of the state of art to produce alkaloids in recombinant microorganisms, such as bacteria or yeast. Some progress has been made in metabolic engineering usually employing a single recombinant alkaloid gene. More importantly, for benzylisoquinoline, monoterpene indole and diterpene alkaloids (taxanes) as well as some terpenoids and phenolics the proof of concept for production of complex alkaloids in recombinant Escherichia coli and yeast has already been achieved. In a long-term perspective, it will probably be possible to generate gene cassettes for complete pathways, which could then be used for production of valuable natural products in bioreactors or for metabolic engineering of crop plants. This will improve their resistance against herbivores and/or microbial pathogens.     

碳一气体生物转化中的产乙酸菌改造与发酵工艺优化

当前的线性经济发展模式依赖化石能源且增加二氧化碳的排放,加剧全球变暖和环境污染。因此,亟需开发碳捕获和利用的技术,建立循环经济。利用产乙酸菌进行碳一气体(一氧化碳和二氧化碳)转化是一项前景广阔的技术,具有较高的碳源灵活性和产物选择性,能够合成多种化学品和燃料。本文聚焦产乙酸菌在碳一气体转化过程中的生理代谢机制、遗传和代谢工程改造、发酵工艺优化以及提升碳原子经济性等方面的研究进展,以期为产乙酸菌气体发酵的工业规模放大及“负碳”生产提供参考。     

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.     

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.     

乳酸乳球菌NZ9000基因组规模代谢网络模型的构建与验证

随着后基因组时代的到来,工业微生物的代谢工程改造在工业生产上发挥着越来越重要的作用。而基因组规模代谢网络模型(Genome-scalemetabolicmodel,GSMM)将生物体体内所有已知代谢信息进行整合,为全局理解生物体的代谢状态、理性指导代谢工程改造提供了最佳的平台。乳酸乳球菌NZ9000(Lactococcuslactis NZ9000)作为工业发酵领域的重要菌株之一,由于其遗传背景清晰且几乎不分泌蛋白,是基因工程改造和外源蛋白表达的理想模式菌株。文中基于基因组功能注释和比较基因组学构建了L.lactisNZ9000的首个基因组规模代谢网络模型iWK557,包含557个基因、668个代谢物、840个反应,并进一步在定性和定量两个层次验证了iWK557的准确性,以期为理性指导L. lactis NZ9000代谢工程改造提供良好工具。     

Metabolic engineering of Bacillus subtilis fueled by systems biology: Recent advances and future directions

By combining advanced omics technology and computational modeling, systems biologists have identified and inferred thousands of regulatory events and system-wide interactions of the bacterium Bacillus subtilis, which is commonly used both in the laboratory and in industry. This dissection of the multiple layers of regulatory networks and their interactions has provided invaluable information for unraveling regulatory mechanisms and guiding metabolic engineering. In this review, we discuss recent advances in the systems biology and metabolic engineering of B. subtilis and highlight current gaps in our understanding of global metabolism and global pathway engineering in this organism. We also propose future perspectives in the systems biology of B. subtilis and suggest ways that this approach can be used to guide metabolic engineering. Specifically, although hundreds of regulatory events have been identified or inferred via systems biology approaches, systematic investigation of the functionality of these events in vivo has lagged, thereby preventing the elucidation of regulatory mechanisms and further rational pathway engineering. In metabolic engineering, ignoring the engineering of multilayer regulation hinders metabolic flux redistribution. Post-translational engineering, allosteric engineering, and dynamic pathway analyses and control will also contribute to the modulation and control of the metabolism of engineered B. subtilis, ultimately producing the desired cellular traits. We hope this review will aid metabolic engineers in making full use of available systems biology datasets and approaches for the design and perfection of microbial cell factories through global metabolism optimization.     

Systems metabolic engineering: Genome-scale models and beyond

The advent of high throughput genome-scale bioinformatics has led to an exponential increase in available cellular system data. Systems metabolic engineering attempts to use data-driven approaches – based on the data collected with high throughput technologies – to identify gene targets and optimize phenotypical properties on a systems level. Current systems metabolic engineering tools are limited for predicting and defining complex phenotypes such as chemical tolerances and other global, multigenic traits. The most pragmatic systems-based tool for metabolic engineering to arise is the in silico genome-scale metabolic reconstruction. This tool has seen wide adoption for modeling cell growth and predicting beneficial gene knockouts, and we examine here how this approach can be expanded for novel organisms. This review will highlight advances of the systems metabolic engineering approach with a focus on de novo development and use of genome-scale metabolic reconstructions for metabolic engineering applications. We will then discuss the challenges and prospects for this emerging field to enable model-based metabolic engineering. Specifically, we argue that current state-of-the-art systems metabolic engineering techniques represent a viable first step for improving product yield that still must be followed by combinatorial techniques or random strain mutagenesis to achieve optimal cellular systems.     

Opposition to transgenic technologies: ideology, interests and collective action frames

Genetic engineering has enabled significant, accepted innovations in medicine and other fields. In agriculture, however, a global cognitive divide around 'genetically modified organisms' (GMOs) has limited the diffusion and scope of this technology. The framing of agricultural products of recombinant DNA technology as GMOs lacks biological coherence, but has proved to be a powerful frame for opposition. Disaggregating the concept of the 'GMO' is a necessary condition for confronting misconceptions that constrain the use of biotechnology in addressing imperatives of development and escalating challenges from nature, especially in less-industrialized nations.