Xenobiology: A Journey towards Parallel Life Forms

Xenobiology is the science of estranged life forms. More specifically, this is an emergent technoscience that combines advances in genetic engineering with the design of biological systems based on unusual biochemistries delivered by chemical compounds of mostly anthropogenic origin. Xenobiology enables us to create and study strange new life forms, “aliens”, not in the way science fiction books do it, but in terms of enlightened science, design, and engineering.     

The Quest for Xenobiotic Enzymes: From New Enzymes for Chemistry to a Novel Chemistry of Life

Enzyme engineering has made impressive progress in the past decades, paving the way for the widespread use of enzymes for various purposes. In contrast to “classical” enzyme engineering, which focuses on optimizing specific properties of natural enzymes, a more recent trend towards the creation of artificial enzymes that catalyze fundamentally distinct, new-to-nature reactions is observable. While approaches for creating such enzymes differ significantly, they share the common goal of enabling biocatalytic novelty to broaden the range of applications for enzymes. Although most artificial enzymes reported to date are only moderately active and barely function in vivo, they have the potential to endow cells with capabilities that were previously out of reach and thus herald a new wave of “functional xenobiology”. Herein, we highlight recent developments in the field of artificial enzymes with a particular focus on challenges and opportunities for their use in xenobiology.     

Courses Based on iGEM/BIOMOD Competitions Are the Ideal Format for Research-Based Learning of Xenobiology

Synthetic biology and especially xenobiology, as emerging new fields of science, have reached an intellectual and experimental maturity that makes them suitable for integration into the university curricula of chemical and biological disciplines. Novel scientific fields that include laboratory work are perfect playgrounds for developing highly motivating research-based teaching modules. We believe that research-based learning enriched by digital tools is the best approach for teaching new emerging essentials of academic education. This is especially true when the scientific field as such is still not canonized with text books and best-practice examples. Our experience shows that iGEM/BIOMOD competitions represent an excellent basis for designing research-based courses in xenobiology. Therefore, we present a report on “iGEM–Synthetic Biology” offered at the Technische Universität Berlin as an example.     

多元模块工程在代谢工程中的应用与研究进展

随着代谢工程理论体系的发展,代谢工程的研究方法目前已从对单一途径的调控转变为对整个代谢网络的全局调控。同时,为了在工业微生物领域实现与化学工业生产规模相当的生物炼制过程,代谢工程需要一套通用的菌株优化策略。其中关键问题之一,是解决代谢通量的不平衡。本文介绍了基于传统的理性代谢工程与近年来兴起的组合工程中存在的问题,研究者提出了一种模块化的代谢网络优化策略--多元模块工程（multivariate modular metabolic engineering,MMME）。阐述了多元模块工程的原理和方法,列举了其常用的调控技术和手段,在此基础上综述了近年来模块化策略在代谢工程领域的应用进展,提出了该策略面临的主要问题并展望了其未来的发展方向。     

PR10/Bet v1-like Proteins as Novel Contributors to Plant Biochemical Diversity

Pathogenesis-related (PR) proteins constitute a broad class of plant proteins with analogues found throughout nature from bacteria to higher eukaryotes. PR proteins were first noted in plants as part of the hypersensitive response, but have since been assigned an array of biological roles. The PR10/Bet v1-like proteins are a subset of PR proteins characterized by an ability to bind a wide range of lipophilic ligands, uniquely positioning them as contributors to specialized biosynthetic pathways. PR10/Bet v1-like proteins participate in the production of plant alkaloids and phenolics including flavonoids, both as general binding proteins and in special cases as catalysts. Owing initially to the perceived allergenic properties of PR10/Bet v1-like proteins, many were studied at the structural level to elucidate the basis for ligand binding. These studies provided a foundation for more recent efforts to understand higher-level structural order and how PR10/Bet v1-like proteins catalyse key reactions in plant pathways. Synthetic biology aimed at reconstituting plant-specialized metabolism in microorganisms uses knowledge of these proteins to fine-tune performance in new systems.     

Isobiology: A Variational Principle for Exploring Synthetic Life

Most developments in synthetic biology try to depart from life as we know it, attempting to create orthogonal constructions. Here, following a variational principle, I try to explore how slight changes in the buildup of cells reveal critical features of life's physics. In a first section, I suggest that we use stable isotopes of the atoms of life to see how living cells fare, beginning with life in heavy water. Subsequently, isotopes of the other main biogenic atoms are suggested as an extension of the variational principle, despite their likely very small influence on the course of biological activity. Finally, two atoms of the second row of Mendeleev's table, boron and fluorine are explored as a further extension of the principle. The use of the former is still in its infancy, whereas the latter, based on existing fluorinases, could open the door to a more general use of halogens in synthetic biology.     

他克莫司微生物合成的研究进展

介绍了他克莫司的理化特征和免疫抑制活性,重点阐述了他克莫司发酵法生产的研究现状,着重介绍了他克莫司的合成基因簇及代谢途径研究进展,对他克莫司代谢工程改造进行综述并对今后的研究趋势进行了展望--结合系统生物学为已有的他克莫司菌株进一步进行代谢工程改造提供指导;通过合成生物学构建他克莫司合成途径全新高效的前体关键酶。     

Engineering Bacterial Cellulose by Synthetic Biology

Synthetic biology is an advanced form of genetic manipulation that applies the principles of modularity and engineering design to reprogram cells by changing their DNA. Over the last decade, synthetic biology has begun to be applied to bacteria that naturally produce biomaterials, in order to boost material production, change material properties and to add new functionalities to the resulting material. Recent work has used synthetic biology to engineer several Komagataeibacter strains; bacteria that naturally secrete large amounts of the versatile and promising material bacterial cellulose (BC). In this review, we summarize how genetic engineering, metabolic engineering and now synthetic biology have been used in Komagataeibacter strains to alter BC, improve its production and begin to add new functionalities into this easy-to-grow material. As well as describing the milestone advances, we also look forward to what will come next from engineering bacterial cellulose by synthetic biology.     

Construction of microbial cell factories for industrial bioprocesses

The development of industrial microbial processes is gaining unprecedented momentum. Increased concern for environmental issues and the prospect of declining petroleum resources has shifted the industrial focus increasingly to microorganisms as biocatalysts. At the same time systems biology and synthetic biology supply industry and academia with new tools to design optimal microbial cell factories. Among the tools are systems biology approaches allowing the modelling of cellular networks for rational strain design, single cell analyses methods for gaining insight into population hetereogeneity, and an exciting combination of tools from structural biology and synthetic biology, permitting the catalysis of new (unnatural) enzymatic reactions or the production of new (unnatural) chemicals. This perspective article outlines recent advances and new developments within the field of microbial cell factory design. Copyright © 2012 Society of Chemical Industry     

Alteration of the Route to Menaquinone towards Isochorismate-Derived Metabolites

Chorismate and isochorismate constitute branch-point intermediates in the biosynthesis of many aromatic metabolites in microorganisms and plants. To obtain unnatural compounds, we modified the route to menaquinone in Escherichia coli. We propose a model for the binding of isochorismate to the active site of MenD ((1R,2S, 5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxycyclohex-3-ene-1-carboxylate (SEPHCHC) synthase) that explains the outcome of the native reaction with α-ketoglutarate. We have rationally designed variants of MenD for the conversion of several isochorismate analogues. The double-variant Asn117Arg–Leu478Thr preferentially converts (5S,6S)-5,6-dihydroxycyclohexa-1,3-diene-1-carboxylate (2,3-trans-CHD), the hydrolysis product of isochorismate, with a >70-fold higher ratio than that for the wild type. The single-variant Arg107Ile uses (5S,6S)-6-amino-5-hydroxycyclohexa-1,3-diene-1-carboxylate (2,3-trans-CHA) as substrate with >6-fold conversion compared to wild-type MenD. The novel compounds have been made accessible in vivo (up to 5.3 g L⁻¹). Unexpectedly, as the identified residues such as Arg107 are highly conserved (>94 %), some of the designed variations can be found in wild-type SEPHCHC synthases from other bacteria (Arg107Lys, 0.3 %). This raises the question for the possible natural occurrence of as yet unexplored branches of the shikimate pathway.     

产蒎烯人工酵母细胞的构建

蒎烯可衍生为高能量密度燃料,但在酿酒酵母中的全生物合成却未见报道。酿酒酵母由于拥有强大的蛋白表达和翻译后修饰系统以及完整的内膜系统,相比于大肠杆菌等原核生物更适于P450等蛋白的表达,因此将酿酒酵母作为宿主细胞,对于蒎烯或者其他物质实现如“疯狂碳环”的高能量化是至关重要的。本研究在酿酒酵母底盘中表达内源焦磷酸香叶酯合成酶（ERG20）的突变体ERG20^ww和火炬松来源的蒎烯合酶（PtPS）构建了蒎烯的合成路径。通过截短PtPS N端2～51位氨基酸残基（tPtPS）,蒎烯产量较初始产量（0.329 mg·L^-1）提高了2.23倍。在过表达异戊二烯焦磷酸异构酶（IDI1）和RNA聚合酶Ш负调控因子（MAF1）的基础上,表达ERG20^ww和tPtPS的融合蛋白,蒎烯产量进一步提高了5.16倍。通过将内源基因ERG20启动子原位替换为弱启动子HXT1,下调ERG20的转录,蒎烯的产量提高了26.0%。最终通过调节发酵过程中的培养基pH使蒎烯产量达11.7 mg·L^-1,较初始产量提高了34.5倍。本研究在酿酒酵母中实现蒎烯的从头合成,并获得已知蒎烯摇瓶水平的最高产量。     

Application of synthetic biology in manufacture of bio-based plastics

Synthetic biology is a new discipline that uses engineering ideas as a guide to transform and reconstruct natural biological genomes, synthesize new biological components, construct new metabolic routes, and produce novel products or obtain new phenotypes. Bio-based plastics are plastics produced under the action of microorganisms or the chemical reactions using natural materials as raw materials. The usage of synthetic biology to construct engineered strains to produce bio-based plastics has become a hot topic in academia and industry. This paper reviews the development of synthetic biology and important techniques in the field of synthetic biology, focusing on the research progress in the field of metabolic pathways and engineering optimization for the construction of bio-based plastic polymer monomers and derivatives such as polyhydroxyalkanoate, nylon, polylactic acid, and butylene glycol succinate using synthetic biological techniques.     

合成生物学在生物基塑料制造中的应用

合成生物学是以工程学思想为指导,对天然生物基因组进行改造和重构,合成新的生物元件,构建新的代谢途径,生产新产品或获得新表型的新兴学科。生物基塑料是以天然物质为原料在微生物作用或化学反应下生成的塑料。利用合成生物学改造工程菌株的方法制备合成生物基塑料已经成为学术界和产业界关注的热点。本文综述了合成生物学的发展和重要的合成生物学技术,重点综述了利用合成生物学技术构建聚羟基烷酸酯、尼龙、聚乳酸和丁二酸丁二醇酯等生物基塑料聚合物单体及其衍生物的代谢途径和工程优化领域的研究进展。     

代谢工程改造解脂耶氏酵母生产脂肪酸及其衍生物

微生物来源的脂肪酸及其衍生物广泛应用于能源、材料和营养化学品等领域,可用于生产航空燃油、聚合物、增塑剂、润滑剂和食品添加剂等。解脂耶氏酵母是一种研究最为透彻的产油脂酵母,具有高产各种脂肪酸及其衍生物的潜力。本文综述了近年来解脂耶氏酵母遗传操作工具的发展,并介绍了通过代谢工程技术改造解脂耶氏酵母生产脂肪酸及其衍生物的进展,在此基础上,展望了通过构建解脂耶氏酵母细胞工厂合成特定脂肪酸及其衍生物的未来发展方向。     

微生物细胞工厂碳流调控进展

随着代谢工程技术的进步,越来越多微生物细胞工厂可用于化学品发酵生产。微生物细胞生产化学品具有生产条件温和、环境友好等优势,是实现化学品绿色可持续生产的重要手段。为了提高微生物细胞工厂的产量、得率和生产强度,传统代谢工程手段主要采用基因过表达或基因敲除方式增大目标代谢路径碳代谢流。然而由于代谢流调控精度不足,易导致细胞生产能力下降。本文主要针对微生物细胞工厂碳流调控中存在的瓶颈问题,从代谢流改造靶点选择、细胞生长与产物合成碳流平衡、副产物路径与产物合成竞争、产物合成效率强化四个角度,系统综述微生物细胞工厂碳代谢流调控的最新进展。并从高精度、仿生学、智能化、多任务、快响应调控工具的设计出发,对未来微生物细胞工厂的发展趋势进行展望。     

Taming the Reactivity of Monoterpene Synthases To Guide Regioselective Product Hydroxylation

Monoterpenoids are industrially important natural products with applications in the flavours, fragrances, fuels and pharmaceutical industries. Most monoterpenoids are produced by plants, but recently two bacterial monoterpene synthases have been identified, including a cineole synthase (bCinS). Unlike plant cineole synthases, bCinS is capable of producing nearly pure cineole from geranyl diphosphate in a complex cyclisation cascade that is tightly controlled. Here we have used a multidisciplinary approach to show that Asn305 controls water attack on the α-terpinyl cation and subsequent cyclisation and deprotonation of the α-terpineol intermediate, key steps in the cyclisation cascade which direct product formation towards cineole. Mutation of Asn305 results in variants that no longer produce α-terpineol or cineole. Molecular dynamics simulations revealed that water coordination is disrupted in all variants tested. Quantum mechanics calculations indicate that Asn305 is most likely a (transient) proton acceptor for the final deprotonation step. Our synergistic approach gives unique insight into how a single residue, Asn305, tames the promiscuous chemistry of monoterpene synthase cyclisation cascades. It does this by tightly controlling the final steps in cineole formation catalysed by bCinS to form a single hydroxylated monoterpene product.     

酿酒酵母高效合成萜类化合物的组合调控策略

萜类化合物具有广泛的生理活性与重要的经济价值,利用酿酒酵母进行萜类合成具有低价、高效等优势。然而部分植物源合成萜类的关键酶在酿酒酵母中难表达、产量低,难以工业应用,因此有效的调控策略显得至关重要。本文从萜类化合物在酿酒酵母中的合成途径入手,介绍了关键酶、代谢途径、CRISPR基因编辑系统和人工合成染色体技术4个方面的调控策略在酿酒酵母合成萜类化合物中的应用。阐述了关键酶的筛选、改造,理性与非理性设计,MVA途径、乙酰辅酶A合成途径与亚细胞结构的代谢途径改造的优势。指出了多重调控策略组合调控的方式是实现酿酒酵母高效合成萜类化合物的有效方法。此外,CRISPR基因编辑系统与人工合成染色体技术的快速发展将为酿酒酵母细胞工厂的深入开发与利用提供有力工具。     

动态调控元件及其在微生物代谢工程中的应用

代谢工程是通过对代谢途径的设计、构建与优化,进行营养品、药品、生物燃料以及化工产品等各种生物基产品合成的关键技术。传统的改造策略如基因的敲除、弱化与过表达会造成代谢流的失衡,而利用微生物自身的调控方式和调控元件,构建合成调控元件,对代谢途径进行动态调控,可以平衡细胞生长与产物合成,从而实现高产量、高底物转化率与高生产强度的统一。利用微生物在转录水平对于外界环境以及胞内代谢物浓度的变化的响应机制,以及在转录后水平通过顺式及反式作用元件的调控,和在蛋白质水平通过途径酶的别构调节以及对蛋白质降解速率的调节,都能开发出相应的动态调控元件并对微生物的代谢进行动态调控。本文分别从转录水平、转录后水平及蛋白质水平3个层次总结了目前常见的一些动态调控元件,并对其在微生物代谢工程中的应用进行了介绍。     

Expanding the repertoire of aromatic chemicals by microbial production

Microbial production of aromatic chemicals would greatly contribute to solving the problems with fossil resource supply and environmentally sustainable development. Engineering and extending the shikimate/aromatic amino acid biosynthetic pathways are important routes for microbial production of various aromatic chemicals. With advances in metabolic engineering and synthetic biology, we can broaden the product spectrum and obtain several valuable and novel aromatic chemicals from renewable feedstocks. Here, in this review, the latest research progress on microbial production of various aromatic chemicals, and recent metabolic engineering and synthetic biology strategies targeting the central carbon metabolism, the shikimate and aromatic amino acid biosynthetic pathways are summarized and discussed. This work aims to provide some valuable tips for the construction of cost‐effective engineered strains for producing various aromatic chemicals. © 2018 Society of Chemical Industry