Making small molecules in plants: A chassis for synthetic biology-based production of plant natural products

Plant natural products have been extensively exploited in food,medicine,flavor,cosmetic,renewable fuel,and other industrial sectors.Synthetic biology has recently emerged as a promising means for the cost-effective and sustainable production of natural products.Compared with engineering microbes for the production of plant natural products,the potential of plants as chassis for producing these compounds is underestimated,largely due to challenges encountered in engineering plants.Knowledge in pl...     

芳香类天然产物的合成生物学研究进展

植物芳香类天然产物具有重要的药用价值,可制成具有抗菌、抗炎、镇痛、抗氧化、杀虫驱虫、祛痰止咳、安神镇静和抗肿瘤等药效的医药保健用品.然而,由于植物中芳香类天然产物含量较低并且难以提取和纯化,严重限制了其工业化生产及应用.合成生物学和代谢工程技术的发展为天然产物的生产提供了新的思路,可以利用人工微生物细胞工厂来实现多种芳...     

Recent advances in natural products exploitation in Streptomyces via synthetic biology

Natural products of microbial origin have proven to be the wellspring of clinically useful compounds for human therapeutics. Streptomyces species are predominant sources of bioactive compounds, most of which serve as potential drug candidates. While the exploitation of natural products has been severely reduced over the past two decades, the growing crisis of evolution and dissemination of drug resistant pathogens have again attracted great interest in this field. The emerging synthetic biology has been heralded as a new bioengineering platform to discover novel bioactive compounds and expand bioactive natural products diversity and production. Herein, we review recent advances in the natural products exploitation of Streptomyces with the applications of synthetic biology from three major aspects, including recently developed synthetic biology tools, natural products biosynthetic pathway engineering strategies as well as chassis host modifications.     

合成生物学与微生物遗传物质的重构

作为一门新兴学科的合成生物学已经展现出巨大的科学价值和应用前景。近年来已经发表了多篇综述文章,从不同角度对合成生物学进行了总结和论述。文章首次对合成生物学和微生物遗传学之间的关系进行了阐述,同时介绍了合成生物学在微生物遗传物质的重构方面最近的研究进展,包括微生物遗传物质的合成、设计和精简,遗传元件的标准化和遗传线路的模块化。也探讨了合成生物学与微生物遗传工程的关系。     

Engineering of a genome-reduced host: practical application of synthetic biology in the overproduction of desired secondary metabolites

Synthetic biology aims to design and build new biological systems with desirable properties, providing the foundation for the biosynthesis of secondary metabolites. The most prominent representation of synthetic biology has been used in microbial engineering by recombinant DNA technology. However, there are advantages of using a deleted host, and therefore an increasing number of biotechnology studies follow similar strategies to dissect cellular networks and construct genome-reduced microbes. This review will give an overview of the strategies used for constructing and engineering reduced-genome factories by synthetic biology to improve production of secondary metabolites.     

甲基转移酶在微生物合成天然产物中的应用

甲基转移酶(Methyltransferases,MTs)普遍存在于所有生物有机体中,通常以S-腺苷甲硫氨酸作为甲基供体催化底物的甲基化反应,在基因的表达调控和许多天然化合物的合成中起着至关重要的作用。近年来,在微生物中异源表达MTs以实现一些重要天然产物的生物合成取得了巨大的进步,但迄今为止这方面的研究还没有得到详细和全面的总结。文中综述了MTs在微生物合成苯丙烷类化合物、香料类化合物、激素和抗生素等重要天然产物的最新研究进展,重点阐述了应用代谢工程策略高效合成这些甲基化的天然产物,以及利用MTs拓展天然产物分子多样性的研究进展。最后,探讨了MTs应用于微生物合成天然产物所面临的挑战,并对利用MTs进一步高效生产结构和生物活性多样化的天然产物进行了展望。     

Integrated rational and evolutionary engineering of genome-reduced Pseudomonas putida strains promotes synthetic formate assimilation

Formate is a promising, water-soluble C1 feedstock for biotechnology that can be efficiently produced from CO₂—but formatotrophy has been engineered in only a few industrially-relevant microbial hosts. We addressed the challenge of expanding the feedstock range of bacterial hosts by adopting Pseudomonas putida as a robust platform for synthetic formate assimilation. Here, the metabolism of a genome-reduced variant of P. putida was radically rewired to establish synthetic auxotrophies that could be functionally complemented by expressing components of the reductive glycine (rGly) pathway. We adopted a modular engineering approach, dividing C1 assimilation in segments composed of both heterologous activities (sourced from Methylobacterium extorquens) and native biochemical reactions. Modular expression of rGly pathway elements enabled growth on formate as carbon source and acetate (predominantly for energy supply), and adaptive laboratory evolution of two lineages of engineered P. putida formatotrophs lead to doubling times of ca. 15 h. We likewise identified emergent metabolic features for assimilation of C1 units in these evolved P. putida populations. Taken together, our results consolidate the landscape of useful microbial platforms that can be implemented for C1-based biotechnological production towards a formate bioeconomy.     

天然产物的生物合成专刊序言

天然产物是创新药物、食品、香料和日化产品等的重要来源,和人民的健康生活息息相关。近年来,随着现代生物学技术和天然产物化学技术的发展和融合,天然产物生物合成研究得到了迅猛的发展。一批天然产物的生物合成途径被解析,许多天然产物生物合成相关的途径酶与后修饰酶被挖掘和功能表征。进一步,这些参与天然产物生物合成的途径酶编码基因被组装到不同的底盘细胞中,利用合成生物学技术构建细胞工厂,用于天然产物的生物合成。此外,包括基因组编辑等新技术在内的生物技术也被用于天然产物的生物合成。为了进一步促进天然产物生物合成研究的发展,《生物工程学报》特组织出版"天然产物的生物合成"专刊,重点阐述了在天然产物生物合成途径的解析,工具酶的挖掘和功能表征以及生物合成技术制备天然产物三方面所取得的研究进展,并展望未来的发展趋势,为天然产物生物合成的进一步发展提供借鉴和指导。     

基因组工程在医学合成生物学中的应用

合成生物学旨在建立一套完整的工程理论和方法,通过设计和组装基本生物学元件,更为有效地实现复杂生物系统的设计,并使其完成可编程的生物学功能。近年来随着可编程基因组元件的出现,特别是CRISPR和CRISPRi技术平台的建立和完善,使得合成生物学进入了一个全新发展的时期。本文重点综述CRISPR等基因组编辑和调控技术,其在构建可编程生物学元件和复杂基因线路的应用以及合成生物学在医学中(称为医学合成生物学)的发展前景。     

The long-term genetic stability and individual specificity of the human gut microbiome

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Distribution of the tryptophan pathway-derived defensive secondary metabolites gramine and benzoxazinones in Poaceae

The Poaceae is a large taxonomic group consisting of approximately 12,000 species and is classified into 12 subfamilies. Gramine and benzoxazinones (Bxs), which are biosynthesized from the tryptophan pathway, are well-known defensive secondary metabolites in the Poaceae. We analyzed the presence or absence of garamine and Bxs in 64 species in the Poaceae by LC-MS/MS. We found that Hordeum brachyantherum and Hakonechloa macra accumulated gramine, but the presence of gramine was limited to small groups of species. We also detected Bxs in four species in the Pooideae and six species in the Panicoideae. In particular, four species in the Paniceae tribe in Panicoideae accumulaed Bxs, indicating that this tribe is a center of the Bx distribution. Bxs were absent in the subfamilies other than Pooideae and Panicoideae. These findings provide an overview of biased distribution of gramine and Bxs in Poaceae species.     

CRISPR/Cas9在放线菌合成生物学研究中的应用和发展

放线菌是活性天然产物和抗生素药物的重要来源。利用合成生物学高效地开发其中丰富的天然产物资源,将为加速新药开发奠定坚实的基础。CRISPR/Cas9作为一种多功能基因编辑系统,因其便捷高效而被广泛应用于真核生物的遗传操作。但在原核生物尤其是放线菌中的应用仍处于起步阶段,机遇和挑战并存。本综述总结了目前CRISPR/Cas9系统在放线菌基因编辑和调控,以及活性天然产物的产量提升、生物合成机制解析和资源开发等方面的研究进展。同时,也对该系统在应用中面临的包括重组修复效率低,以及靶向切割效率不足等关键挑战进行了分析,并提出了相应的优化解决方法。随着CRISPR/Cas9在放线菌应用中的不断完善和发展,将极大地推动放线菌的合成生物学研究,促进其中天然产物资源的有效挖掘和应用开发。     

大肠杆菌合成启动子的构建及在顺,顺-粘康酸生物合成中的应用

启动子是基因表达调控的重要元件.在代谢工程和合成生物学研究中,经常需要利用不同强度的启动子对代谢途径进行精细调控,来实现代谢平衡,降低中间产物积累,提高目标产物合成.然而目前可获得的启动子难以满足以上要求,而且不同来源的启动子通用性差,缺乏标准化.针对这些问题,设计了1条88个碱基对的启动子,包含典型的-35区、-10区以及核糖体结合区.同时,在转录起始位点上游6个碱基、-35与-10区间隔区14个碱基对中引入简并序列,构建了合成启动子文库.利用合成启动子控制红色荧光蛋白mCherry的表达强度,经过两轮筛选,从5 000多个克隆中获得了720个不同强度的启动子.随机挑选35条不同强度的启动子进行测序分析,结果表明不同强度的启动子具有碱基偏好性.对于强启动子,-13位点嘌呤碱基出现频率高,转录起始区除-4位点外,嘧啶碱基出现的频率高于嘌呤碱基,而-10区与-35区间14个位点的嘌呤碱基与嘧啶碱基出现频率大致相当.最后选取5条不同强度启动子应用于顺,顺-粘康酸合成途径调控优化,结果显示不同强度的启动子可以调节目标产物顺,顺-粘康酸的合成和中间产物儿茶酚的积累.     

Engineering Native and Synthetic Pathways in Pseudomonas putida for the Production of Tailored Polyhydroxyalkanoates

Growing environmental concern sparks renewed interest in the sustainable production of (bio)materials that can replace oil-derived goods. Polyhydroxyalkanoates (PHAs) are isotactic polymers that play a critical role in the central metabolism of producer bacteria, as they act as dynamic reservoirs of carbon and reducing equivalents. PHAs continue to attract industrial attention as a starting point toward renewable, biodegradable, biocompatible, and versatile thermoplastic and elastomeric materials. Pseudomonas species have been known for long as efficient biopolymer producers, especially for medium-chain-length PHAs. The surge of synthetic biology and metabolic engineering approaches in recent years offers the possibility of exploiting the untapped potential of Pseudomonas cell factories for the production of tailored PHAs. In this article, an overview of the metabolic and regulatory circuits that rule PHA accumulation in Pseudomonas putida is provided, and approaches leading to the biosynthesis of novel polymers (e.g., PHAs including nonbiological chemical elements in their structures) are discussed. The potential of novel PHAs to disrupt existing and future market segments is closer to realization than ever before. The review is concluded by pinpointing challenges that currently hinder the wide adoption of bio-based PHAs, and strategies toward programmable polymer biosynthesis from alternative substrates in engineered P. putida strains are proposed.     

Evidence for genetic differentiation and variable recombination rates among Dutch populations of the opportunistic human pathogen Aspergillus fumigatus

As the frequency of antifungal drug resistance continues to increase, understanding the genetic structure of fungal populations, where resistant isolates have emerged and spread, is of major importance. Aspergillus fumigatus is an ubiquitously distributed fungus and the primary causative agent of invasive aspergillosis (IA), a potentially lethal infection in immunocompromised individuals. In the last few years, an increasing number of A. fumigatus isolates has evolved resistance to triazoles, the primary drugs for treating IA infections. In most isolates, this multiple-triazole-resistance (MTR) phenotype is caused by mutations in the cyp51A gene, which encodes the protein targeted by the triazoles. We investigated the genetic differentiation and reproductive mode of A. fumigatus in the Netherlands, the country where the MTR phenotype probably originated, to determine their role in facilitating the emergence and distribution of resistance genotypes. Using 20 genome-wide neutral markers, we genotyped 255 Dutch isolates including 25 isolates with the MTR phenotype. In contrast to previous reports, our results show that Dutch A. fumigatus genotypes are genetically differentiated into five distinct populations. Four of the five populations show significant linkage disequilibrium, indicative of an asexual reproductive mode, whereas the fifth population is in linkage equilibrium, indicative of a sexual reproductive mode. Notably, the observed genetic differentiation among Dutch isolates does not correlate with geography, although all isolates with the MTR phenotype nest within a single, predominantly asexual, population. These results suggest that both reproductive mode and genetic differentiation contribute to the structure of Dutch A. fumigatus populations and are probably shaping the evolutionary dynamics of drug resistance in this potentially deadly pathogen.     

Engineering biosynthesis of high-value compounds in photosynthetic organisms

The photosynthetic, autotrophic lifestyle of plants and algae position them as ideal platform organisms for sustainable production of biomolecules. However, their use in industrial biotechnology is limited in comparison to heterotrophic organisms, such as bacteria and yeast. This usage gap is in part due to the challenges in generating genetically modified plants and algae and in part due to the difficulty in the development of synthetic biology tools for manipulating gene expression in these systems. Plant and algal metabolism, pre-installed with multiple biosynthetic modules for precursor compounds, bypasses the requirement to install these pathways in conventional production organisms, and creates new opportunities for the industrial production of complex molecules. This review provides a broad overview of the successes, challenges and future prospects for genetic engineering in plants and algae for enhanced or de novo production of biomolecules. The toolbox of technologies and strategies that have been used to engineer metabolism are discussed, and the potential use of engineered plants for industrial manufacturing of large quantities of high-value compounds is explored. This review also discusses the routes that have been taken to modify the profiles of primary metabolites for increasing the nutritional quality of foods as well as the production of specialized metabolites, cosmetics, pharmaceuticals and industrial chemicals. As the universe of high-value biosynthetic pathways continues to expand, and the tools to engineer these pathways continue to develop, it is likely plants and algae will become increasingly valuable for the biomanufacturing of high-value compounds.     

Computational discovery of soybean promoter cis‐regulatory elements for the construction of soybean cyst nematode‐inducible synthetic promoters

Computational methods offer great hope but limited accuracy in the prediction of functional cis‐regulatory elements; improvements are needed to enable synthetic promoter design. We applied an ensemble strategy for de novo soybean cyst nematode (SCN)‐inducible motif discovery among promoters of 18 co‐expressed soybean genes that were selected from six reported microarray studies involving a compatible soybean–SCN interaction. A total of 116 overlapping motif regions (OMRs) were discovered bioinformatically that were identified by at least four out of seven bioinformatic tools. Using synthetic promoters, the inducibility of each OMR or motif itself was evaluated by co‐localization of gain of function of an orange fluorescent protein reporter and the presence of SCN in transgenic soybean hairy roots. Among 16 OMRs detected from two experimentally confirmed SCN‐inducible promoters, 11 OMRs (i.e. 68.75%) were experimentally confirmed to be SCN‐inducible, leading to the discovery of 23 core motifs of 5‐ to 7‐bp length, of which 14 are novel in plants. We found that a combination of the three best tools (i.e. SCOPE, W‐AlignACE and Weeder) could detect all 23 core motifs. Thus, this strategy is a high‐throughput approach for de novo motif discovery in soybean and offers great potential for novel motif discovery and synthetic promoter engineering for any plant and trait in crop biotechnology.     

基因工程微生物合成鼠李糖脂表面活性剂的研究进展

鼠李糖脂是一类重要的生物表面活性剂。相比于化学合成的表面活性剂,其具有更优秀的理化性质及环境友好等特点,被广泛应用于微生物采油、环境污染修复等工程中。目前,鼠李糖脂的工业生产主要采用铜绿假单胞菌这一具有致病性的天然合成菌株,与此同时,受菌株遗传背景的限制,优化发酵过程等方法在产量提升方面遇到了一些瓶颈问题。利用基因工程方法对菌株进行改良有望进一步提高鼠李糖脂生产的安全性、产量、产物性能等多项指标,因此受到了越来越广泛的关注。本文综述了近年来利用基因工程方法优化鼠李糖脂生物合成的最新进展,讨论了异源合成、代谢通路改造、基因表达优化、蛋白质工程、底盘工程等多种策略的应用,并展望了一系列可行的研究方向。     

合成生物学时代基于非模式细菌的工业底盘细胞研究现状与展望

工业微生物底盘细胞的开发将为工业生物技术的发展提供优良的细胞工厂,有利于实现环境保护及经济可持续发展.基于合成生物学"设计-构建-测试-学习"(Design-Build-Test-Learn,DBTL)策略,对底盘细胞进行多维度的理性或半理性改造是实现"建物致知"以及"建物致用"目标的重要手段.文中简述了合成生物学DB...     

Design and expression of a synthetic phyC gene encoding the neutral phytase in Pichia pastoris

The 1074-bp phyCs gene (optimized phyC gene) encoding neutral phytase was designed andsynthesized according to the methylotrophic yeast Pichia pastoris codon usage bias without altering theprotein sequence.The expression vector,pP9K-phyCs,was linearized and transformed in P.pastoris.Theyield of total extracellular phytase activity was 17.6 U/ml induced in Buffered Methanol-complex Medium(BMMY) and 18.5 U/ml in Wheat Bran Extract Induction (WBEI) medium at the flask scale,respectively,improving over 90 folds compared with the wild-type isolate.Purified enzyme showed temperature optimumof 70℃ and pH optimum of 7.5.The enzyme activity retained 97% of the relative activity afterincubation at 80℃ for 5 min.Because of the heavy glycosylation the expressed phytase had a molecularsize of approximately 51 kDa.After deglycosylation by endoglycosylase H (EndoH_f),the enzyme had anapparent molecular size of 42 kDa.Its property and thermostability was affected by the glycosylation.