代谢工程改造微生物生产芳香族化合物的研究进展

芳香族化合物广泛应用于化学工业。利用代谢工程改造微生物生产各种芳香族化合物越来越受到人们的关注。通过理性改造,微生物可以定向地大量积累人们需要的各种芳香族化合物。此外,通过设计新的反应途径并引入外源基因,可以拓宽微生物生物合成的产物谱,获得某些具有重要应用价值的新的芳香族化合物。这些研究成果对解决化石能源危机和环境可持续发展问题具有积极意义。本文中,笔者主要对近年来微生物生产各种芳香族化合物的最新研究进展及相应的代谢工程改造策略进行综述,为开展相关研究提供参考。     

代谢工程在芳香化合物生物合成研究中的应用

生物技术和代谢工程的发展促进了生物合成研究。概述了近年来利用微生物莽草酸途径进行芳香化合物生物合成研究的现况、代谢工程在提高天然芳香化合物产量和扩大合成非天然产生的芳香化合物范围的应用的进展 ,特别是整体代谢工程对提高第二代工程菌产量的作用。指出了生物合成法是生产氨基酸及其它生物小分子如奎尼酸、维生素和抗生素等的未来趋势 ,在工业化生产中有着广阔的应用前景。     

放线菌次级代谢途径的设计

近来有关放线菌次线产物生物合成的分子遗传学和生物化学方面的进展为我们改造其代谢途径提供了一个明确的方向。近年来,对微生物的初级代谢途径进行基因改造取得了成功,但放线菌的次级代谢工程产物却都没有达到中试或生产规模。     

大肠杆菌细胞工厂的创建技术

大肠杆菌作为一种重要的模式工业微生物,在医药、化工、农业等方面具有广泛的应用。近30年来,多种代谢工程改造的新策略和新技术,被用于设计、构建和优化大肠杆菌化学品细胞工厂,极大地提高了生物法合成化学品的生产速率和产量。文中将从大肠杆菌途径设计、合成途径创建与优化和细胞全局优化三个方面,对大肠杆菌代谢改造起重要推动作用的技术进行综述,并对大肠杆菌代谢工程中关键技术的应用进行了展望。     

嘌呤核苷及其衍生物的代谢工程

嘌呤核苷及其衍生物被广泛应用于食品和医药领域。利用诱变筛选技术可以获得嘌呤核苷类产品的工业生产菌株,但往往耗时,效率低,而且获得的某些高产菌株还存在不稳定的缺陷。菌株代谢调控与生理生化的研究为代谢工程优化嘌呤核苷类产品的合成提供了理论基础,利用代谢工程改造菌株合成嘌呤核苷也引起了研究人员的关注。系统地介绍了微生物嘌呤生物合成途径及其调控机制,综述了嘌呤核苷类产品及其衍生物的代谢工程研究进展,最后讨论了利用代谢工程改造菌株合成这些产品面临的问题及今后的研究方向。     

大环内酯类抗生素代谢工程的研究进展

14-16元环的大环内酯类抗生素(Macrolide antibiotics,MA)是临床上重要的抗感染药物。随着细菌耐药性的不断增加,迫切需要研发出新型MA来应对耐药菌。通过MA与核糖体靶点的相互作用可以指导MA的定向优化,结合快速发展的代谢工程方法可以高效获得所需的MA衍生物。近30年来,代谢工程在改造MA的生物合成获得新衍生物以及在提高其产量方面显示出巨大优势,这些代谢工程的方法包括改造聚酮合酶(Polyketide synthase,PKS)结构域和PKS后修饰酶以及组合生物合成等。另外,文中还对16元环大环内酯类新药可利霉素的研制过程、抗菌活性以及利用代谢工程优化其产生菌进行了详细介绍。     

苄基异喹啉类生物碱生物合成与代谢工程研究进展

苄基异喹啉类生物碱(benzylisoquinoline alkaloids,BIAs)是一类具有重要研究及药用价值的次生代谢产物,目前该类生物碱主要来自于植物。但是BIAs在植物中的含量低、且大部分含有BIAs的植物具有生长周期长、受环境影响大、采集困难和难以大规模种植等问题。为解决这些问题,许多研究人员致力于解析BIAs生物合成途径并利用植物与微生物代谢工程技术提高BIAs的含量与开辟新药源。本研究将从BIAs类型与生物合成途径以及微生物与植物代谢工程几个方面对BIAs的生物合成与代谢工程研究进展进行综述,有利于BIAs的深入研究。     

桉叶素生物合成研究进展

作为桉叶油的主要成分,桉叶素是具有多种生物活性的单萜化合物,被广泛应用于药品、食品及化妆品等领域。桉叶油主要从桉树叶提取,该过程耗费大量人力及自然资源,且容易污染环境。近年来,随着微生物代谢工程与合成生物学的快速发展,加上越来越多萜类生物合成途径得到解析,为桉叶素的绿色生产提供了新的途径。对桉叶素的生物合成途径、桉叶素合酶的结构与功能及近年来桉叶素的微生物合成进行了综述,并对利用微生物代谢工程合成桉叶素等单萜化合物的瓶颈问题及解决方案进行了探讨和归纳,为构建高产桉叶素等单萜微生物工程菌株提供参考。     

芳香族化合物生物降解的研究进展

本文综述了以苯、取代苯、联苯和多环芳烃为代表的芳香族化合物的生物降解途径,其共同之处在于经过两步双加氧酶作用,生成二醇和开环。两步双加氧酶分别为芳环羟基化双加氧酶和芳环断裂双加氧酶。以甲苯途径为代表讨论了芳香族化合物的分子生物学研究情况。代谢工程研究是九十年代兴起的芳香族化合物生物降解的研究内容,通过对甲苯途径的代谢工程研究明确了途径中的关键酶,并通过对关键酶的活性提高使整个途径的代谢流增加。     

生物法合成双乙酰的研究进展

随着对双乙酰生物合成途径的深入研究,利用代谢工程的方法定向改良菌株,优化双乙酰的代谢途径,成为提高双乙酰生产水平的新思路。本文总结了双乙酰的生产菌株和相关代谢途径,综述了改造双乙酰生产菌株的代谢工程策略,提出了未来的研究方向。     

芳香族香料化合物生物合成研究进展

芳香族化合物在香料中占很大的比重,传统生产方式有化学合成和植物提取。化学合成依赖于石油资源,并具有环境不友好、反应条件恶劣等缺点。植物提取方法受限于植物资源,且占用耕地。近年来,随着代谢工程和合成生物学技术的发展,利用可再生原料,微生物合成芳香族香料化合物成为一种新的生产方式。文中介绍了大肠杆菌和酵母菌等模式微生物合成芳香族香料的研究进展,包括利用莽草酸途径合成香兰素等,聚酮途径合成覆盆子酮等。综述重点介绍了生物合成途径解析、人工合成途径创建及代谢调控等,为微生物发酵法生产芳香族香料化合物提供参考。     

链霉菌来源的苯并异色烷醌家族抗生素的生物合成

苯并异色烷醌(benzoisochromanequinones,BIQs)家族抗生素是由链霉菌产生的聚酮类抗生素,其芳香聚酮母核结构中含有并联的两个芳香环和一个吡喃环,具有抗菌、抗肿瘤等多种生物学活性。BIQ抗生素聚酮链的早期生物合成过程代表了芳香聚酮抗生素母核的典型合成机制,而不同的后期修饰则决定了它们结构和生物学活性的多样性。在过去的二十几年中,以放线紫红素和美达霉素为研究重点,BIQ家族抗生素的生物合成机制逐渐得到揭示,但在后期结构修饰方面仍有许多问题有待解决。本文对BIQ家族抗生素的生物合成机制研究进行了综述,比较了不同BIQ家族抗生素结构特点、生物学活性,并重点阐述了它们生物合成中的后期结构修饰和调控过程的研究进展,并对BIQ抗生素在代谢工程方面的研究进行了展望。     

植物多酚的微生物合成

植物多酚属于苯丙烷衍生物,包括酚酸类、茋类、姜黄素类和黄酮类等。它们具有抗氧化、扩血管、抗血凝、抗炎、抗肿瘤、抗病毒等生理药理活性,在医药、食品、化妆品、化工等领域具有巨大的应用市场。微生物具有生长快、培养简单、可工业化等优点,成为异源合成天然产物的重要宿主。近年来,合成生物学的发展促进了植物天然产物的微生物合成,并取得了实质性进展。文中综述了植物多酚代谢途径在工程大肠杆菌、酿酒酵母等微生物中的构建、表达和产物合成现状,讨论了提高产量的前体工程、动态调控、共培养等优化策略,并就未来的多酚途径工程提出展望。     

真菌芳香聚酮化合物的合成生物学研究进展*

真菌芳香聚酮化合物是由真菌非还原聚酮合酶(NR-PKSs)催化形成的具有广泛生物活性的一类天然产物。大部分内源真菌菌株存在难培养、致病性或产率低等问题,从根本上限制了真菌芳香聚酮化合物的开发和应用。随着合成生物学和代谢工程的发展,很多具有生物活性的聚酮产物实现了在工业微生物(如酿酒酵母、构巢曲霉等)中的异源生产,相关研究逐渐成为热点。从合成途径解析与挖掘、底盘细胞的构建与改造等方面综述了近年来真菌芳香聚酮化合物的合成生物学研究进展,为未来真菌芳香聚酮化合物人工代谢途径的高效构建和实现工业化生产奠定基础。     

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.

     

Engineering stilbene metabolic pathways in microbial cells

Numerous in vitro and in vivo studies on biological activities of phytostilbenes have brought to the fore the remarkable properties of these compounds and their derivatives, making them a top storyline in natural product research fields. However, getting stilbenes in sufficient amounts for routine biological activity studies and make them available for pharmaceutical and/or nutraceutical industry applications, is hampered by the difficulty to source them through synthetic chemistry-based pathways or extraction from the native plants. Hence, microbial cell cultures have rapidly became potent workhorse factories for stilbene production. In this review, we present the combined efforts made during the past 15?years to engineer stilbene metabolic pathways in microbial cells, mainly the Saccharomyces cerevisiae baker yeast, the Escherichia coli and the Corynebacterium glutamicum bacteria. Rationalized approaches to the heterologous expression of the partial or the entire stilbene biosynthetic routes are presented to allow the identification and/or bypassing of the major bottlenecks in the endogenous microbial cell metabolism as well as potential regulations of the genes involved in these metabolic pathways. The contributions of bioinformatics to synthetic biology are developed to highlight their tremendous help in predicting which target genes are likely to be up-regulated or deleted for controlling the dynamics of precursor flows in the tailored microbial cells. Further insight is given to the metabolic engineering of microbial cells with “decorating” enzymes, such as methyl and glycosyltransferases or hydroxylases, which can act sequentially on the stilbene core structure. Altogether, the cellular optimization of stilbene biosynthetic pathways integrating more and more complex constructs up to twelve genetic modifications has led to stilbene titers ranging from hundreds of milligrams to the gram-scale yields from various carbon sources. Through this review, the microbial production of stilbenes is analyzed, stressing both the engineering dynamic regulation of biosynthetic pathways and the endogenous control of stilbene precursors.     

Rapid prototyping of microbial production strains for the biomanufacture of potential materials monomers

Bio-based production of industrial chemicals using synthetic biology can provide alternative green routes from renewable resources, allowing for cleaner production processes. To efficiently produce chemicals on-demand through microbial strain engineering, biomanufacturing foundries have developed automated pipelines that are largely compound agnostic in their time to delivery. Here we benchmark the capabilities of a biomanufacturing pipeline to enable rapid prototyping of microbial cell factories for the production of chemically diverse industrially relevant material building blocks. Over 85 days the pipeline was able to produce 17 potential material monomers and key intermediates by combining 160 genetic parts into 115 unique biosynthetic pathways. To explore the scale-up potential of our prototype production strains, we optimized the enantioselective production of mandelic acid and hydroxymandelic acid, achieving gram-scale production in fed-batch fermenters. The high success rate in the rapid design and prototyping of microbially-produced material building blocks reveals the potential role of biofoundries in leading the transition to sustainable materials production.     

Biosynthesis of lactate-containing polyesters by metabolically engineered bacteria

Due to increasing concerns about environmental problems, climate change and limited fossil resources, bio-based production of chemicals and polymers is gaining attention as one of the solutions to these problems. Polyhydroxyalkanoates (PHAs) are polyesters that can be produced by microbial fermentation. PHAs are synthesized using monomer precursors provided from diverse metabolic pathways and are accumulated as distinct granules inside the cells. On the other hand, most so-called bio-based polymers including polybutylene succinate, polytrimethylene terephthalate, and polylactic acid (PLA) are synthesized by a chemical process using monomers produced by fermentation. PLA, an attractive biomass-derived plastic, is currently synthesized by heavy metal-catalyzed ring opening polymerization of L-lactide that is made from fermentation-derived L-lactic acid. Recently, a complete biological process for the production of PLA and PLA copolymers from renewable resources has been developed by direct fermentation of recombinant bacteria employing PHA biosynthetic pathways coupled with a novel metabolic pathway. This could be accomplished by establishing a pathway for generating lactyl-CoA and engineering PHA synthase to accept lactyl-CoA as a substrate combined with systems metabolic engineering. In this article, we review recent advances in the production of lactate-containing homo- and co-polyesters. Challenges remaining to efficiently produce PLA and its copolymers and strategies to overcome these challenges through metabolic engineering combined with enzyme engineering are discussed.     

Biofuel alternatives to ethanol: pumping the microbial well

Engineered microorganisms are currently used for the production of food products, pharmaceuticals, ethanol fuel and more. Even so, the enormous potential of this technology has yet to be fully exploited. The need for sustainable sources of transportation fuels has generated a tremendous interest in technologies that enable biofuel production. Decades of work have produced a considerable knowledge-base for the physiology and pathway engineering of microbes, making microbial engineering an ideal strategy for producing biofuel. Although ethanol currently dominates the biofuel market, some of its inherent physical properties make it a less than ideal product. To highlight additional options, we review advances in microbial engineering for the production of other potential fuel molecules, using a variety of biosynthetic pathways.