Engineering antibiotic production and overcoming bacterial resistance

Progress in DNA technology, analytical methods and computational tools is leading to new developments in synthetic biology and metabolic engineering, enabling new ways to produce molecules of industrial and therapeutic interest. Here, we review recent progress in both antibiotic production and strategies to counteract bacterial resistance to antibiotics. Advances in sequencing and cloning are increasingly enabling the characterization of antibiotic biosynthesis pathways, and new systematic methods for de novo biosynthetic pathway prediction are allowing the exploration of the metabolic chemical space beyond metabolic engineering. Moreover, we survey the computer-assisted design of modular assembly lines in polyketide synthases and non-ribosomal peptide synthases for the development of tailor-made antibiotics. Nowadays, production of novel antibiotic can be tranferred into any chosen chassis by optimizing a host factory through specific strain modifications. These advances in metabolic engineering and synthetic biology are leading to novel strategies for engineering antimicrobial agents with desired specificities.     

Computational design and analysis of modular cells for large libraries of exchangeable product synthesis modules

Microbial metabolism can be harnessed to produce a large library of useful chemicals from renewable resources such as plant biomass. However, it is laborious and expensive to create microbial biocatalysts to produce each new product. To tackle this challenge, we have recently developed modular cell (ModCell) design principles that enable rapid generation of production strains by assembling a modular (chassis) cell with exchangeable production modules to achieve overproduction of target molecules. Previous computational ModCell design methods are limited to analyze small libraries of around 20 products. In this study, we developed a new computational method, named ModCell-HPC, that can design modular cells for large libraries with hundreds of products with a highly-parallel and multi-objective evolutionary algorithm and enable us to elucidate modular design properties. We demonstrated ModCell-HPC to design Escherichia coli modular cells towards a library of 161 endogenous production modules. From these simulations, we identified E. coli modular cells with few genetic manipulations that can produce dozens of molecules in a growth-coupled manner with different types of fermentable sugars. These designs revealed key genetic manipulations at the chassis and module levels to accomplish versatile modular cells, involving not only in the removal of major by-products but also modification of branch points in the central metabolism. We further found that the effect of various sugar degradation on redox metabolism results in lower compatibility between a modular cell and production modules for growth on pentoses than hexoses. To better characterize the degree of compatibility, we developed a method to calculate the minimal set cover, identifying that only three modular cells are all needed to couple with all compatible production modules. By determining the unknown compatibility contribution metric, we further elucidated the design features that allow an existing modular cell to be re-purposed towards production of new molecules. Overall, ModCell-HPC is a useful tool for understanding modularity of biological systems and guiding more efficient and generalizable design of modular cells that help reduce research and development cost in biocatalysis.     

Impact of systems biology on metabolic engineering of Saccharomyces cerevisiae

Industrial biotechnology is a rapidly growing field. With the increasing shift towards a bio-based economy, there is rising demand for developing efficient cell factories that can produce fuels, chemicals, pharmaceuticals, materials, nutraceuticals, and even food ingredients. The yeast Saccharomyces cerevisiae is extremely well suited for this objective. As one of the most intensely studied eukaryotic model organisms, a rich density of knowledge detailing its genetics, biochemistry, physiology, and large-scale fermentation performance can be capitalized upon to enable a substantial increase in the industrial application of this yeast. Developments in genomics and high-throughput systems biology tools are enhancing one's ability to rapidly characterize cellular behaviour, which is valuable in the field of metabolic engineering where strain characterization is often the bottleneck in strain development programmes. Here, the impact of systems biology on metabolic engineering is reviewed and perspectives on the role of systems biology in the design of cell factories are given.     

Combinatorial metabolic pathway assembly approaches and toolkits for modular assembly

Synthetic Biology is a rapidly growing interdisciplinary field that is primarily built upon foundational advances in molecular biology combined with engineering design principles such as modularity and interoperability. The field considers living systems as programmable at the genetic level and has been defined by the development of new platform technologies and methodological advances. A key concept driving the field is the Design-Build-Test-Learn cycle which provides a systematic framework for building new biological systems. One major application area for synthetic biology is biosynthetic pathway engineering that requires the modular assembly of different genetic regulatory elements and biosynthetic enzymes. In this review we provide an overview of modular DNA assembly and describe and compare the plethora of in vitro and in vivo assembly methods for combinatorial pathway engineering. Considerations for part design and methods for enzyme balancing are also presented, and we briefly discuss alternatives to intracellular pathway assembly including microbial consortia and cell-free systems for biosynthesis. Finally, we describe computational tools and automation for pathway design and assembly and argue that a deeper understanding of the many different variables of genetic design, pathway regulation and cellular metabolism will allow more predictive pathway design and engineering.     

Road to the future of systems biotechnology: CRISPR-Cas-mediated metabolic engineering for recombinant protein production

The rising potential for CRISPR–Cas-mediated genome editing has revolutionized our strategies in basic and practical bioengineering research. It provides a predictable and precise method for genome modification in a robust and reproducible fashion. Emergence of systems biotechnology and synthetic biology approaches coupled with CRISPR–Cas technology could change the future of cell factories to possess some new features which have not been found naturally. We have discussed the possibility and versatile potentials of CRISPR–Cas technology for metabolic engineering of a recombinant host for heterologous protein production. We describe the mechanisms involved in this metabolic engineering approach and present the diverse features of its application in biotechnology and protein production.     

Yeast systems biotechnology for the production of heterologous proteins

Systems biotechnology has been established as a highly potent tool for bioprocess development in recent years. The applicability to complex metabolic processes such as protein synthesis and secretion, however, is still in its infancy. While yeasts are frequently applied for heterologous protein production, more progress in this field has been achieved for bacterial and mammalian cell culture systems than for yeasts. A critical comparison between different protein production systems, as provided in this review, can aid in assessing the potentials and pitfalls of applying systems biotechnology concepts to heterologous protein producing yeasts. Apart from modelling, the methodological basis of systems biology strongly relies on postgenomic methods. However, this methodology is rapidly moving so that more global data with much higher sensitivity will be achieved in near future. The development of next generation sequencing technology enables an unexpected revival of genomic approaches, providing new potential for evolutionary engineering and inverse metabolic engineering.     

Network design meets in silico evolutionary biology

Cell fate is programmed through gene regulatory networks that perform several calculations to take the appropriate decision. In silico evolutionary optimization mimics the way Nature has designed such gene regulatory networks. In this review we discuss the basic principles of these evolutionary approaches and how they can be applied to engineer synthetic networks. We summarize the basic guidelines to implement an in silico evolutionary design method, the operators for mutation and selection that iteratively drive the network architecture towards a specified dynamical behavior. Interestingly, as it happens in natural evolution, we show the existence of patterns of punctuated evolution. In addition, we highlight several examples of models that have been designed using automated procedures, together with different objective functions to select for the proper behavior. Finally, we briefly discuss the modular designability of gene regulatory networks and its potential application in biotechnology.     

Modular optimization of multi-gene pathways for fumarate production

Microbial fumarate production from renewable feedstock is a promising and sustainable alternative to petroleum-based chemical synthesis. Here, we report a modular engineering approach that systematically removed metabolic pathway bottlenecks and led to significant titer improvements in a multi-gene fumarate metabolic pathway. On the basis of central pathway architecture, yeast fumarate biosynthesis was re-cast into three modules: reduction module, oxidation module, and byproduct module. We targeted reduction module and oxidation module to the cytoplasm and the mitochondria, respectively. Combinatorially tuning pathway efficiency by constructing protein fusions RoMDH-P160A and KGD2-SUCLG2 and optimizing metabolic balance by controlling genes RoPYC, RoMDH-P160A, KGD2-SUCLG2 and SDH1 expression strengths led to significantly improved fumarate production (20.46 g/L). In byproduct module, synthetizing DNA-guided scaffolds and designing sRNA switchs enabled further production improvement up to 33.13 g/L. These results suggest that modular pathway engineering can systematically optimize biosynthesis pathways to enable an efficient production of fumarate.     

微生物模块化共培养工程的应用及控制策略

传统的微生物合成方法通常依赖于单一工程菌株,通过代谢工程改造合成目标产物.由于关键的辅因子、前体和能量被引入到复杂的化合物合成途径中,加重了工程菌株的代谢负担,因而常常会影响目标产物产量.而模块化共培养工程已经成为一种有效进行异源生物合成并有望大大提高产物产量的新方法.在模块化共培养工程中,不同模块菌群之间的相互协调对...     

Systems metabolic engineering: Genome-scale models and beyond

The advent of high throughput genome-scale bioinformatics has led to an exponential increase in available cellular system data. Systems metabolic engineering attempts to use data-driven approaches – based on the data collected with high throughput technologies – to identify gene targets and optimize phenotypical properties on a systems level. Current systems metabolic engineering tools are limited for predicting and defining complex phenotypes such as chemical tolerances and other global, multigenic traits. The most pragmatic systems-based tool for metabolic engineering to arise is the in silico genome-scale metabolic reconstruction. This tool has seen wide adoption for modeling cell growth and predicting beneficial gene knockouts, and we examine here how this approach can be expanded for novel organisms. This review will highlight advances of the systems metabolic engineering approach with a focus on de novo development and use of genome-scale metabolic reconstructions for metabolic engineering applications. We will then discuss the challenges and prospects for this emerging field to enable model-based metabolic engineering. Specifically, we argue that current state-of-the-art systems metabolic engineering techniques represent a viable first step for improving product yield that still must be followed by combinatorial techniques or random strain mutagenesis to achieve optimal cellular systems.     

植物实验生殖生物学与生殖细胞工程:现在与未来

植物实验生殖生物学是植物实验胚胎学发展的新阶段,其主要特征为操作技术水平的提高与多学科综合性研究的加强。花粉原生质体、生殖细胞、精子,胚囊、卵细胞的操作、雌雄配子体外融合、配子-体细胞杂交等,代表了当前的主要研究趋势。实验生殖生物学与基因工程相结合,开辟了植物生殖工程新技术领域的前景。对生殖工程的意义与内容提出了轮廓设想。     

系统生物学与细胞发生系统动力学

系统生物学是系统理论和实验生物技术、计算机数学模型等方法整合的生物系统研究,系统遗传学研究基因组的稳态与进化、功能基因组和生物性状等复杂系统的结构、动态与发生演变等。合成生物学是系统生物学的工程应用,采用工程学方法、基因工程和计算机辅助设计等研究人工生物系统的生物技术。系统与合成生物学的结构理论,序列标志片段显示分析与微流控生物芯片,广泛用于研究细胞代谢、繁殖和应激的自组织进化、生物体形态发生等细胞分子生物系统原理等。     

全细胞网络重建与细胞工厂设计

在各种组学及其相应的网络研究相对成熟的基础上,集成各组学网络的细胞整合型网络或全细胞网络将大大提高对生物表型的预测能力,并成为代谢工程决策的有力武器.本文在阐述了细胞工厂设计中应该考虑细胞整合网络之后,综述了细胞整合网络的重建、分析、设计方法方面的有关问题,并进一步就研究细胞整合网络涉及的数据库、软件平台、并行计算几方面的作用作了介绍.     

Engineering modular ester fermentative pathways in Escherichia coli

Sensation profiles are observed all around us and are made up of many different molecules, such as esters. These profiles can be mimicked in everyday items for their uses in foods, beverages, cosmetics, perfumes, solvents, and biofuels. Here, we developed a systematic ‘natural’ way to derive these products via fermentative biosynthesis. Each ester fermentative pathway was designed as an exchangeable ester production module for generating two precursors− alcohols and acyl-CoAs that were condensed by an alcohol acyltransferase to produce a combinatorial library of unique esters. As a proof-of-principle, we coupled these ester modules with an engineered, modular, Escherichia coli chassis in a plug-and-play fashion to create microbial cell factories for enhanced anaerobic production of a butyrate ester library. We demonstrated tight coupling between the modular chassis and ester modules for enhanced product biosynthesis, an engineered phenotype useful for directed metabolic pathway evolution. Compared to the wildtype, the engineered cell factories yielded up to 48 fold increase in butyrate ester production from glucose.     

海洋生理活性物质的研究及发展趋势

本文概述海洋生理活性物质特有的特异结构和药理作用,对研究治疗心脑血管疾病、癌症和艾滋病等方面的意义及发展前景。阐述我国近年来从海洋生物活性物质中发现如三丙酮胺、喹啉酮、柳珊瑚酸及其衍生物等观种显著生理活性新化合物及１６种海洋生理活性物质;８种已投入市场的海洋药物及保健品。提出对海洋生理活性物质研制成新药的可行途径及应采取的决策和有效措施;提出应用如基因工程、细胞工程、发酵工程和酶学工程等生物技术来研制、生产出纯度高、质量高、成本低的海洋药物资源物质。海洋生物技术领域的迅速发展,对海洋生物活性物质的研究及开发海洋药物具有广阔的应用前景。     

Modular co-culture engineering,a new approach for metabolic engineering

With the development of metabolic engineering, employment of a selected microbial host for accommodation of a designed biosynthetic pathway to produce a target compound has achieved tremendous success in the past several decades. Yet, increasing requirements for sophisticated microbial biosynthesis call for establishment and application of more advanced metabolic engineering methodologies. Recently, important progress has been made towards employing more than one engineered microbial strains to constitute synthetic co-cultures and modularizing the biosynthetic labor between the co-culture members in order to improve bioproduction performance. This emerging approach, referred to as modular co-culture engineering in this review, presents a valuable opportunity for expanding the scope of the broad field of metabolic engineering. We highlight representative research accomplishments using this approach, especially those utilizing metabolic engineering tools for microbial co-culture manipulation. Key benefits and major challenges associated with modular co-culture engineering are also presented and discussed.     

传统与未来的碰撞：食品发酵工程技术与应用进展

随着生物技术的飞速发展,作为食品生物工程的主要组成部分,食品发酵工程技术不断升级,在传统发酵食品的菌种、发酵过程、产品品质得到改善的同时,生物制造的功能食品组分、未来食品等新型产品也应运而生。首先概述了由生物技术和信息技术的进步带来的食品发酵研究手段与生产方式的多层面变革,并重点阐释了利用食品合成生物学设计构建细胞工厂的思路和方法,以及食品生物工程在微生物分析、过程工程和分离工程方面的智能化进程。其次,介绍了现代食品生物工程技术在改善传统发酵食品品质及安全性、生产功能食品组分、添加剂和酶制剂、创制未来食品和开发新型益生食品方面的应用进展。最后,对全球和我国食品发酵产业面临的挑战和未来发展趋势进行了总结和展望,以期为食品发酵的技术革新和工业化应用提供参考。     

The promise of synthetic biology

DNA synthesis has become one of the technological bases of a new concept in biology: synthetic biology. The vision of synthetic biology is a systematic, hierarchical design of artificial, biology-inspired systems using robust, standardized, and well-characterized building blocks. The design concept and examples from four fields of application (genetic circuits, protein design, platform technologies, and pathway engineering) are discussed, which demonstrate the usefulness and the promises of synthetic biology. The vision of synthetic biology is to develop complex systems by simplified solutions using available material and knowledge. Synthetic biology also opens a door toward new biomaterials that do not occur in nature.