Tailoring of microbes for the production of high value plant-derived compounds: From pathway engineering to fermentative production

Plant natural products have been an attracting platform for the isolation of various active drugs and other bioactives. However large-scale extraction of these compounds is affected by the difficulty in mass cultivation of these plants and absence of strategies for successful extraction. Even though, synthesis by chemical method is an alternative method; it is less efficient as their chemical structure is highly complex which involve enantio-selectivity. Thus an alternate bio-system for heterologous production of plant natural products using microbes has emerged. Advent of various omics, synthetic and metabolic engineering strategies revolutionised the field of heterologous plant metabolite production. In this context, various engineering methods taken to synthesise plant natural products are described with an additional focus to fermentation strategies.     

Biotechnological production of bio-based long-chain dicarboxylic acids with oleogenious yeasts

Long-chain α,ω-dicarboxylic acids (DCAs) are versatile chemical intermediates of industrial importance used as building blocks for the production of polymers, lubricants, or adhesives. The majority of industrial long-chain DCAs is produced from petro-chemical resources. An alternative is their biotechnological production from renewable materials like plant oil fatty acids by microbial fermentation using oleogenious yeasts. Oleogenious yeasts are natural long-chain DCA producers, which have to be genetically engineered for high-yield DCA production. Although, some commercialized fermentation processes using engineered yeasts are reported, bio-based long-chain DCAs are still far from being a mass product. Further progress in bioprocess engineering and rational strain design is necessary to advance their further commercialization. The present article reviews the basic strategies, as well as novel approaches in the strain design of oleogenious yeasts, such as the combination of traditional metabolic engineering with system biology strategies for high-yield long-chain DCA production. Therefore a detailed overview of the involved metabolic processes for the biochemical long-chain DCA synthesis is given.     

Genetic evidence for natural product-mediated plant-plant allelopathy in rice (Oryza sativa)

? There is controversy as to whether specific natural products play a role in directly mediating antagonistic plant-plant interactions - that is, allelopathy. If proved to exist, such phenomena would hold considerable promise for agronomic improvement of staple food crops such as rice (Oryza sativa). ? However, while substantiated by the presence of phytotoxic compounds at potentially relevant concentrations, demonstrating a direct role for specific natural products in allelopathy has been difficult because of the chemical complexity of root and plant litter exudates. This complexity can be bypassed via selective genetic manipulation to ablate production of putative allelopathic compounds, but such an approach previously has not been applied. ? The rice diterpenoid momilactones provide an example of natural products for which correlative biochemical evidence has been obtained for a role in allelopathy. Here, we apply reverse genetics, using knock-outs of the relevant diterpene synthases (copalyl diphosphate synthase 4 (OsCPS4) and kaurene synthase-like 4 (OsKSL4)), to demonstrate that rice momilactones are involved in allelopathy, including suppressing growth of the widespread rice paddy weed, barnyard grass (Echinochloa crus-galli). ? Thus, our results not only provide novel genetic evidence for natural product-mediated allelopathy, but also furnish a molecular target for breeding and metabolic engineering of this important crop plant.     

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.     

From flavors and pharmaceuticals to advanced biofuels: Production of isoprenoids in Saccharomyces cerevisiae

Isoprenoids denote the largest group of chemicals in the plant kingdom and are employed for a wide range of applications in the food and pharmaceutical industry. In recent years, isoprenoids have additionally been recognized as suitable replacements for petroleum-derived fuels and could thus promote the transition towards a more sustainable society. To realize the biofuel potential of isoprenoids, a very efficient production system is required. While complex chemical structures as well as the low abundance in nature demonstrate the shortcomings of chemical synthesis and plant extraction, isoprenoids can be produced by genetically engineered microorganisms from renewable carbon sources. In this article, we summarize the development of isoprenoid applications from flavors and pharmaceuticals to advanced biofuels and review the strategies to design microbial cell factories, focusing on Saccharomyces cerevisiae for the production of these compounds. While the high complexity of biosynthetic pathways and the toxicity of certain isoprenoids still denote challenges that need to be addressed, metabolic engineering has enabled large-scale production of several terpenoids and thus, the utilization of these compounds is likely to expand in the future.     

Constructing de novo biosynthetic pathways for chemical synthesis inside living cells

Living organisms have evolved a vast array of catalytic functions that make them ideally suited for the production of medicinally and industrially relevant small-molecule targets. Indeed, native metabolic pathways in microbial hosts have long been exploited and optimized for the scalable production of both fine and commodity chemicals. Our increasing capacity for DNA sequencing and synthesis has revealed the molecular basis for the biosynthesis of a variety of complex and useful metabolites and allows the de novo construction of novel metabolic pathways for the production of new and exotic molecular targets in genetically tractable microbes. However, the development of commercially viable processes for these engineered pathways is currently limited by our ability to quickly identify or engineer enzymes with the correct reaction and substrate selectivity as well as the speed by which metabolic bottlenecks can be determined and corrected. Efforts to understand the relationship among sequence, structure, and function in the basic biochemical sciences can advance these goals for synthetic biology applications while also serving as an experimental platform for elucidating the in vivo specificity and function of enzymes and reconstituting complex biochemical traits for study in a living model organism. Furthermore, the continuing discovery of natural mechanisms for the regulation of metabolic pathways has revealed new principles for the design of high-flux pathways with minimized metabolic burden and has inspired the development of new tools and approaches to engineering synthetic pathways in microbial hosts for chemical production.     

代谢工程改造解脂耶氏酵母合成植物萜类化合物的研究进展

萜类化合物是一类广泛存在于植物中的天然产物,其在食品、药品和化工等多个领域中均有广泛的用途,市场潜力巨大。因此,开发生产萜类化合物等植物天然产物可再生的微生物资源来补充甚至代替原有稀少和珍贵的植物资源,具有重要的理论意义和潜在的应用价值。解脂耶氏酵母是目前使用最广泛的非常规酵母底盘细胞之一。近年来,利用代谢工程及合成生物学技术在解脂耶氏酵母底盘细胞中重构与优化萜类化合物的合成途径以实现目标代谢产物的高效合成,已经成为一项研究热点。本文系统总结了有关利用解脂耶氏酵母作为底盘细胞异源生产植物萜类化合物的具体实例和最新进展,包括所涉及的宿主菌株、关键酶、代谢途径及改造策略等,并在最后对该领域的未来发展方向进行了展望。     

Systems biology based metabolic engineering for non-natural chemicals

Production of chemicals in microorganisms is no longer restricted to products arising from native metabolic potential. In this review, we highlight the evolution of metabolic engineering studies, from the production of natural chemicals fermented from biomass hydrolysates, to the engineering of microorganisms for the production of non-natural chemicals. Advances in synthetic biology are accelerating the successful development of microbial cell factories to directly produce value-added chemicals. Here we outline the emergence of novel computational tools for the creation of synthetic pathways, for designing artificial enzymes for non-natural reactions and for re-wiring host metabolism to increase the metabolic flux to products. We also highlight exciting opportunities for applying directed evolution of enzymes, dynamic control of growth and production, growth-coupling strategies as well as decoupled strategies based on orthogonal pathways in the context of non-natural chemicals.     

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.     

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.     

Perspectives and limits of engineering the isoprenoid metabolism in heterologous hosts

Terpenoids belong to the largest class of natural compounds and are produced in all living organisms. The isoprenoid skeleton is based on assembling of C5 building blocks, but the biosynthesis of a great variety of terpenoids ranging from monoterpenoids to polyterpenoids is not fully understood today. Terpenoids play a fundamental role in human nutrition, cosmetics, and medicine. In the past 10 years, many metabolic engineering efforts have been undertaken in plants but also in microorganisms to improve the production of various terpenoids like artemisinin and paclitaxel. Recently, inverse metabolic engineering and combinatorial biosynthesis as main strategies in synthetic biology have been applied to produce high-cost natural products like artemisinin and paclitaxel in heterologous microorganisms. This review describes the recent progresses made in metabolic engineering of the terpenoid pathway with particular focus on fundamental aspects of host selection, vector design, and system biotechnology.     

Heterologous production of polyketides by modular type I polyketide synthases in Escherichia coli

Heterologous production of polyketide compounds, an important class of natural products with complex chemical structures, was first demonstrated with Streptomyces parvulus in 1984. Although Streptomyces strains are good first options for heterologous polyketide biosynthesis, their slow growth kinetics prompt other hosts to also be considered. Escherichia coli provides key elements of an ideal host in terms of the growth rate, culture conditions, and available recombinant DNA tools. Here we review the current status and potential for metabolic engineering of polyketides in E. coli.     

Genomic analysis of carbon monoxide utilization and butanol production by Clostridium carboxidivorans strain P7

Increasing demand for the production of renewable fuels has recently generated a particular interest in microbial production of butanol. Anaerobic bacteria, such as Clostridium spp., can naturally convert carbohydrates into a variety of primary products, including alcohols like butanol. The genetics of microorganisms like Clostridium acetobutylicum have been well studied and their solvent-producing metabolic pathways characterized. In contrast, less is known about the genetics of Clostridium spp. capable of converting syngas or its individual components into solvents. In this study, the type of strain of a new solventogenic Clostridium species, C. carboxidivorans, was genetically characterized by genome sequencing. C. carboxidivorans strain P7(T) possessed a complete Wood-Ljungdahl pathway gene cluster, involving CO and CO(2) fixation and conversion to acetyl-CoA. Moreover, with the exception of an acetone production pathway, all the genetic determinants of canonical ABE metabolic pathways for acetate, butyrate, ethanol and butanol production were present in the P7(T) chromosome. The functionality of these pathways was also confirmed by growth of P7(T) on CO and production of CO(2) as well as volatile fatty acids (acetate and butyrate) and solvents (ethanol and butanol). P7(T) was also found to harbour a 19 Kbp plasmid, which did not include essential or butanol production related genes. This study has generated in depth knowledge of the P7(T) genome, which will be helpful in developing metabolic engineering strategies to improve C. carboxidivorans's natural capacity to produce potential biofuels from syngas.     

Evolutionary and cellular webs in benzylisoquinoline alkaloid biosynthesis

Alkaloids are a group of approximately 12,000 low molecular weight and nitrogenous secondary metabolites found in 20% of plant species. Their potent biological activity suggests that alkaloids function as defense compounds. Benzylisoquinoline alkaloids (BIAs) are derived from tyrosine and are diversified by an intricate biochemical network of intramolecular coupling, reduction, methylation, hydroxylation, and other reactions to generate the estimated 2500 known structures. Several BIAs are used directly as pharmaceuticals or serve as precursors for the synthesis of semi-synthetic drugs. Plants remain the only economical source for the production of compounds such as morphine and codeine owing to their chemical complexity, which makes de novo synthesis challenging and costly. Much research has been directed toward understanding the biosynthesis of the BIAs and manipulating source plants to increase production of key products and pathway intermediates. However, metabolic engineering experiments often yield unexpected results demonstrating the need for an improved perspective on the biochemistry, regulation, and cell biology of BIA pathways. This review summarizes recent advances in the establishment of predictive metabolic engineering within the context of plant alkaloid biosynthesis.     

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.     

Metabolic engineering for plant natural product biosynthesis in microbes

Plant natural products (NPs) not only serve many functions in an organism's survivability but also demonstrate important pharmacological activities. Isolation of NPs from native sources is frequently limited by low abundance and environmental, seasonal, and regional variation while total chemical synthesis of what are often complex structures is typically commercially infeasible. Reconstruction of biosynthetic pathways in heterologous microorganisms offers significant promise for a scalable means to provide sufficient quantities of a desired NP while using inexpensive renewable resources. To this end, metabolic engineering provides the technological platform for enhancing NP production in these engineered heterologous hosts. Recent advancements in the production of isoprenoids, phenylpropanoids, and alkaloids were made possible by utilizing a variety of techniques including combinatorial biosynthesis, codon optimization, expression of regulatory elements, and protein engineering of P450s.     

天然产物类药物的合成生物学研究

结构复杂多样的天然产物是现代药物的重要组成部分和新药发现的重要源泉。建立在基因工程及代谢工程、合成化学、基因组学、系统生物学等学科基础上的合成生物学研究对于结构复杂的天然产物类药物研究有特殊的意义。核心是通过在发酵友好、高效的微生物中设计、构建目标化合物的生物合成途径,经系统地调控和优化由重组微生物发酵生产来源稀缺的天然产物类药物或前体。该方法是不远的将来解决来源、成本与环境、资源协调问题最好的途径之一,也是解决海洋天然产物或特殊生境微生物药物面临的如何持续供应化合物这一个瓶颈问题的最佳选择。该文将对天然产物类药物合成生物学研究涉及的主要策略和重要进展进行阐述。     

Plant 'hairy root' culture

Due to their fast growth rates and biochemical stability, 'hairy root' cultures remain unsurpassed as the choice for model root systems and have promise as a bioprocessing system. Applications are wide-ranging, from the production of natural products and foreign proteins to a model for phytoremediation of organic and metal contaminants. Hairy roots will have a continuing role as an experimental model in plant metabolic engineering.     

萜烯生物合成中关键酶的研究进展*

萜烯类化合物是一类高度多样化的天然产物,具有抗肿瘤、抗氧化及免疫调节等生理活性,因此被广泛应用于医药健康、食品、化妆品领域。然而,直接从自然资源中获取萜烯类化合物效率低、成本高,且往往对生态环境产生不利影响,不能实现绿色可持续生产。微生物合成萜烯类化合物近年来备受关注,研究人员从合成途径的构建与调控、关键酶的理性及半理性改造、发酵工艺优化等多个方面进行了探究,取得了丰硕的成果。其中,合成途径中关键酶的催化效率是影响微生物生产萜烯类化合物的重要因素。针对关键酶的研究对于提高微生物合成萜烯类化合物的能力,推动该类天然产物微生物生产的大规模应用具有重要意义。对萜烯类化合物合成途径中的3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)、1-脱氧-D-木酮糖-5-磷酸合酶(DXS)、异戊二烯基二磷酸合成酶(IDS)和萜烯合酶(TPS)4种关键酶的研究进行了综述,并总结讨论了如何通过代谢工程和蛋白质工程手段以及合成生物学技术调节关键酶的催化活性,提高微生物合成萜烯类化合物的效率,对未来利用微生物合成萜烯类化合物的发展进行了展望。