Marinobacter: A case study in bioelectrochemical chassis evaluation

The junction of bioelectrochemical systems and synthetic biology opens the door to many potentially groundbreaking technologies. When developing these possibilities, choosing the correct chassis organism can save a great deal of engineering effort and, indeed, can mean the difference between success and failure. Choosing the correct chassis for a specific application requires a knowledge of the metabolic potential of the candidate organisms, as well as a clear delineation of the traits, required in the application. In this review, we will explore the metabolic and electrochemical potential of a single genus, Marinobacter. We will cover its strengths, (salt tolerance, biofilm formation and electrochemical potential) and weaknesses (insufficient characterization of many strains and a less developed toolbox for genetic manipulation) in potential synthetic electromicrobiology applications. In doing so, we will provide a roadmap for choosing a chassis organism for bioelectrochemical systems.     

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。     

Rapid production of l-DOPA by Vibrio natriegens,an emerging next-generation whole-cell catalysis chassis

3, 4-Dihydroxyphenyl-l -alanine (l -DOPA) is a compound of high medical value and is considered effective as a treatment for Parkinson’s disease. Currently, bioproduction of l -DOPA is mainly carried out by whole-cell catalysis mediated by recombinant Escherichia coli carrying heterogeneous tyrosine phenol lyase. Vibrio natriegens is increasingly attracting attention owing to its superiority, including extremely rapid growth and high soluble protein expression capacity. In this study, we attempt to develop an efficient whole-cell catalyst for l -DOPA production using V. natriegens as the chassis. The maximum soluble protein expression by V. natriegens was accomplished in 4 h at 37°C, which was equivalent to that achieved by E. coli in 16 h at 16°C. Furthermore, the maximum productivity reached over 10.0 g l⁻¹ h⁻¹ in the early stage of biocatalysis, nearly two-fold higher than previously reported. Approximately 54.0 g l⁻¹ l -DOPA was obtained with a catechol conversion rate greater than 95%. In conclusion, V. natriegens displays advantages, including rapid protein expression and catalytic rate in the catalysis process for l -DOPA production. These findings strongly suggest that V. natriegens has remarkable potential as a whole-cell catalysis chassis for the production of valuable chemicals.     

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.     

The use of Beneckea natriegens for practical exercises in microbiology and biochemistry

Beneckea natriegens is a rapidly growing, nonpathogenic marine bacterium which can be used with advantage to replace Escherichia coli in many of the microbiology experiments which are performed in undergraduate courses. Some recommendations for the handling and growth of this organism together with some examples of experiments are described. The potential value of this organism as a subject for student research projects is also discussed.     

普鲁兰酶在需钠弧菌中的分泌表达与发酵优化

普鲁兰酶是一种淀粉脱支酶,因其分子量较大,胞外分泌表达难度较高。需钠弧菌(Vibrionatriegens)是一种新型的蛋白表达宿主,拥有高效的蛋白合成效率。本研究使用基因组整合T7 RNA聚合酶表达框的V.natriegens VnDX为宿主,构建了产全长普鲁兰酶PulA及其截短突变体PulN2的重组需钠弧菌,分析了信号肽、发酵温度、诱导剂浓度、甘氨酸浓度及发酵时间等条件对产酶的影响,并且对比了2种普鲁兰酶在V.natriegens VnDX与大肠杆菌(Escherichia coli)BL21(DE3)中的胞外产酶能力。研究结果显示,普鲁兰酶PulA和PulN2在V.natriegens VnDX中的胞外酶活为61.6 U/mL和64.3 U/mL,分别为E.coli BL21(DE3)最大酶活力的110%和62%。上述结果表明V.natriegens VnDX可以分泌表达大分子量的全长普鲁兰酶PulA,本研究可为其他大分子量蛋白在V.natriegens VnDX中的分泌表达提供参考和借鉴。     

Metabolism of the fast-growing bacterium Vibrio natriegens elucidated by 13C metabolic flux analysis

Vibrio natriegens is a fast-growing, non-pathogenic bacterium that is being considered as the next-generation workhorse for the biotechnology industry. However, little is known about the metabolism of this organism which is limiting our ability to apply rational metabolic engineering strategies. To address this critical gap in current knowledge, here we have performed a comprehensive analysis of V. natriegens metabolism. We constructed a detailed model of V. natriegens core metabolism, measured the biomass composition, and performed high-resolution ¹³C metabolic flux analysis (¹³C-MFA) to estimate intracellular fluxes using parallel labeling experiments with the optimal tracers [1,2−¹³C]glucose and [1,6−¹³C]glucose. During exponential growth in glucose minimal medium, V. natriegens had a growth rate of 1.70 1/h (doubling time of 24 min) and a glucose uptake rate of 3.90 g/g/h, which is more than two 2-fold faster than E. coli, although slower than the fast-growing thermophile Geobacillus LC300. ¹³C-MFA revealed that the core metabolism of V. natriegens is similar to that of E. coli, with the main difference being a 33% lower normalized flux through the oxidative pentose phosphate pathway. Quantitative analysis of co-factor balances provided additional insights into the energy and redox metabolism of V. natriegens. Taken together, the results presented in this study provide valuable new information about the physiology of V. natriegens and establish a solid foundation for future metabolic engineering efforts with this promising microorganism.     

Synthetic biology in multicellular organisms: Opportunities in nematodes

Synthetic biology has mainly focused on introducing new or altered functionality in single cell systems: primarily bacteria, yeast, or mammalian cells. Here, we describe the extension of synthetic biology to nematodes, in particular the well-studied model organism Caenorhabditis elegans, as a convenient platform for developing applications in a multicellular setting. We review transgenesis techniques for nematodes, as well as the application of synthetic biology principles to construct nematode gene switches and genetic devices to control motility. Finally, we discuss potential applications of engineered nematodes.     

需钠弧菌外膜囊泡的蛋白质组分析与异源蛋白的递送

[背景] 需钠弧菌（Vibrio natriegens）是一种快速生长的革兰氏阴性菌,作为一种新兴工具在生物技术领域有重要的应用潜力。此前的研究主要集中在开发利用V. natriegens成为体内外重组蛋白生产的工具。然而,许多支持细菌进行快速生长和蛋白质生产的生理活动大部分仍未确定。外膜囊泡（Outer Membrane Vesicle,OMV）是由革兰氏阴性细菌普遍产生的一种球形小泡,其不仅具有重要的功能,而且还可以作为一种应用于疫苗治疗的高效运载工具。[目的] 表征指数生长期OMV的蛋白质组并利用OMV进行异源蛋白的递送。[方法] 使用透射电镜、动态光散射和质谱学的方法,观察OMV的形态及粒径分布并鉴定蛋白组成。以超折叠绿色荧光蛋白（Superfolded Green Fluorescent Protein,sfGFP）作为货物蛋白来确定OMV蛋白载体。[结果] 从细菌培养的指数期中期和末期分别提取的OMV中鉴定到了288个和317个蛋白。这些蛋白分属不同的功能组,包括ABC转运蛋白、鞭毛、双组分系统。相比之下,同时鉴定了全细胞样品,其在指数期中期和末期分别含有1 480个和1 565个蛋白。我们筛选OMV的蛋白作为候选载体发现了一种属于OmpA家族的蛋白（命名为OmpA24）,其能够将sfGFP以融合货物蛋白的形式运载到OMV中。[结论] 首次证实V. natriegens能够在指数生长期产生OMV,并展示了第一个不同生长时期OMV和全细胞的蛋白质组鉴定结果。OmpA24是将外源融合货物蛋白呈递到OMV中的有前景的载体。本研究有助于促进V. natriegens在蛋白表达和OMV介导的分泌中的应用。     

Toolboxes for cyanobacteria: Recent advances and future direction

Photosynthetic cyanobacteria are important primary producers and model organisms for studying photosynthesis and elements cycling on earth. Due to the ability to absorb sunlight and utilize carbon dioxide, cyanobacteria have also been proposed as renewable chassis for carbon-neutral “microbial cell factories”. Recent progresses on cyanobacterial synthetic biology have led to the successful production of more than two dozen of fuels and fine chemicals directly from CO₂, demonstrating their potential for scale-up application in the future. However, compared with popular heterotrophic chassis like Escherichia coli and Saccharomyces cerevisiae, where abundant genetic tools are available for manipulations at levels from single gene, pathway to whole genome, limited genetic tools are accessible to cyanobacteria. Consequently, this significant technical hurdle restricts both the basic biological researches and further development and application of these renewable systems. Though still lagging the heterotrophic chassis, the vital roles of genetic tools in tuning of gene expression, carbon flux re-direction as well as genome-wide manipulations have been increasingly recognized in cyanobacteria. In recent years, significant progresses on developing and introducing new and efficient genetic tools have been made for cyanobacteria, including promoters, riboswitches, ribosome binding site engineering, clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease (CRISPR/Cas) systems, small RNA regulatory tools and genome-scale modeling strategies. In this review, we critically summarize recent advances on development and applications as well as technical limitations and future directions of the genetic tools in cyanobacteria. In addition, toolboxes feasible for using in large-scale cultivation are also briefly discussed.     

Synthetic biology in biofilms: Tools,challenges, and opportunities

The field of synthetic biology seeks to program living cells to perform novel functions with applications ranging from environmental biosensing to smart cell-based therapeutics. Bacteria are an especially attractive chassis organism due to their rapid growth, ease of genetic manipulation, and ability to persist across many environmental niches. Despite significant progress in bacterial synthetic biology, programming bacteria to perform novel functions outside the well-controlled laboratory context remains challenging. In contrast to planktonic laboratory growth, bacteria in nature predominately reside in the context of densely packed communities known as biofilms. While biofilms have historically been considered environmental and biomedical hazards, their physiology and emergent behaviors could be leveraged for synthetic biology to engineer more capable and robust bacteria. Specifically, bacteria within biofilms participate in complex emergent behaviors such as collective organization, cell-to-cell signaling, and division of labor. Understanding and utilizing these properties can enable the effective deployment of engineered bacteria into natural target environments. Toward this goal, this review summarizes the current state of synthetic biology in biofilms by highlighting new molecular tools and remaining biological challenges. Looking to future opportunities, advancing synthetic biology in biofilms will enable the next generation of smart cell-based technologies for use in medicine, biomanufacturing, and environmental remediation.     

10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis

Lactococcus lactis is a Gram-positive lactic acid bacterium that, in addition to its traditional use in food fermentations, is increasingly used in modern biotechnological applications. In the last 25 years great progress has been made in the development of genetic engineering tools and the molecular characterization of this species. A new versatile and tightly controlled gene expression system, based on the auto-regulation mechanism of the bacteriocin nisin, was developed 10 years ago—the NIsin Controlled gene Expression system, called NICE. This system has become one of the most successful and widely used tools for regulated gene expression in Gram-positive bacteria. The review describes, after a brief introduction of the host bacterium L. lactis, the fundaments, components and function of the NICE system. Furthermore, an extensive overview is provided of the different applications in lactococci and other Gram-positive bacteria: (1) over-expression of homologous and heterologous genes for functional studies and to obtain large quantities of specific gene products, (2) metabolic engineering, (3) expression of prokaryotic and eukaryotic membrane proteins, (4) protein secretion and anchoring in the cell envelope, (5) expression of genes with toxic products and analysis of essential genes and (6) large-scale applications. Finally, an overview is given of growth and induction conditions for lab-scale and industrial-scale applications.     

链霉菌底盘细胞的开发现状及其应用

天然产物及其衍生物在现代医疗中扮演着举足轻重的角色,其生物活性多样性以及化学结构的丰富性是新药研发的源泉和动力。利用纯化学方法合成天然产物在技术和成本上有很大的困难,加上许多天然产物的原始产生菌具有培养条件苛刻、产量低下等缺点,而且大量基因簇在原始菌株中是沉默的,这使得利用合成生物学思想来指导天然产物生物合成基因簇的异源表达具有重大意义。作为抗生素、抗肿瘤活性物质、免疫抑制剂等次级代谢产物主要来源的放线菌一直是研究者们关注的焦点,特别是随着基因测序技术的飞速发展,人们发现链霉菌基因组中包含着极为丰富的天然产物生物合成基因簇资源。这意味着开发链霉菌底盘细胞作为异源表达宿主有其得天独厚的优势。本综述从底盘细胞开发的意义入手,重点阐述链霉菌底盘细胞构建的策略及现状,随后通过实例阐述了各种底盘链霉菌的实际应用。     

Wolbachia-induced reproductive anomalies and their future applications

The Wolbachia bacterium is currently one of most prevalent endosymbionts of arthropods. The bacterium has drawn much attention because of its ability to induce reproductive anomalies, such as cytoplasmic incompatibility, feminization, male killing, and parthenogenesis, in various invertebrates. This review focuses on the basic biology of Wolbachia, its infection phenotypes, and its potential future applications.     

Efficient markerless integration of genes in the chromosome of probiotic E. coli Nissle 1917 by bacterial conjugation

The probiotic strain Escherichia coli Nissle 1917 (EcN) is a common bacterial chassis in synthetic biology developments for therapeutic applications given its long track record of safe administration in humans. Chromosomal integration of the genes of interest (GOIs) in the engineered bacterium offers significant advantages in genetic stability and to control gene dose, but common methodologies relying on the transformation of EcN are inefficient. In this work, we implement in EcN the use of bacterial conjugation in combination with markerless genome engineering to efficiently insert multiple GOIs at different loci of EcN chromosome, leaving no antibiotic resistance genes, vector sequences or scars in the modified bacterium. The resolution of cointegrants that leads to markerless insertion of the GOIs requires expression of I-SceI endonuclease and its efficiency is enhanced by λ Red proteins. We show the potential of this strategy by integrating different genes encoding fluorescent and bioluminescent reporters (i.e. GFP, mKate2, luxCDABE) both individually and sequentially. We also demonstrate its application for gene deletions in EcN (ΔflhDC) and to replace the endogenous regulation of chromosomal locus (i.e. flhDC) by heterologous regulatory elements (e.g. tetR-Ptet) in order to have an ectopic control of gene expression in EcN with an external inducer to alter bacterial behaviour (e.g. flagellar motility). Whole-genome sequencing confirmed the introduction of the designed modifications without off-target alterations in the genome. This straightforward approach accelerates the generation of multiple modifications in EcN chromosome for the generation of living bacterial therapeutics.     

合成芳香族化合物的酵母底盘改造策略*

芳香族化合物种类丰富,在多个行业具有广泛的用途,需求量大。通过构建微生物细胞工厂合成芳香族化合物具有独特的优势和工业化应用前景,其中酵母底盘因其清晰的遗传背景、完善的基因操作工具以及成熟的工业发酵体系等优势,常被用于构建细胞工厂。目前改造酵母底盘生产芳香族化合物的研究取得了一系列进展,并针对关键问题提出了一些可行的解决策略。针对酵母合成芳香族化合物的策略与挑战,从芳香族化合物合成路径改造、多样化碳源利用及转运系统改造、基因组多靶点改造、特殊酵母底盘及混菌系统构建、合成生物学高通量技术的应用这五个方面进行系统地梳理和阐述,为生产芳香族化合物的酵母底盘构建与改造提供思路。     

合成生物学方法构建电活性大肠杆菌

电活性微生物具有独特的在细胞内外环境之间传递电子的能力。在对天然电活性微生物电子传递机制充分研究的基础上,通过合成生物学方法异源构建天然电活性微生物电子传递结构基础也可以将遗传背景清晰的非电活性大肠杆菌改造为电活性微生物。构建获得的工程化电活性大肠杆菌可以直接应用于微生物燃料电池和生物传感器等领域,同时也可以作为底盘细胞整合相应的目标产物合成通路实现电能驱动的生物合成。本文以合成生物学方法构建电活性大肠杆菌为主题,详细阐述天然电活性微生物电子传递的机理及结构基础,总结了工程化电活性大肠杆菌的构建策略、成功案例以及应用领域,并对合成生物学方法构建电活性大肠杆菌未来的研究方向进行了展望。