遗传改造微生物代谢途径生产新型柴油燃料的研究进展

生物柴油是一种能替代柴油的可再生燃料,然而通过植物油料化学转酯化生产的第一代生物柴油在性能和生产工艺上有很多缺点。近年来随着合成生物学和代谢工程的迅速发展,通过选择合适的微生物并利用各种生物技术改造其代谢合成途径,如脂肪酸合成途径、异戊二烯合成途径,研究人员能利用微生物直接生产性能更加优越、品质更高的新型第二代生物柴油——长链烷烃。文章总结了目前遗传改造微生物代谢途径生产新型柴油的研究进展,并指出目前该领域存在的问题以及今后的发展方向。     

脂肪酸合成系统潜能的挖掘与释放

在全球石油资源不断减少和温室气体不断积累的情况下,急需发展可再生燃料能源及各种生物化工原料和产品。基于该目的,能够生产高能量密度液体生物燃料和高附加值化工品的微生物脂肪酸合成系统备受关注。首先介绍了大肠杆菌脂肪酸代谢系统的组成,然后详细总结了通过改造脂肪酸代谢途径生产脂肪酸以及脂肪酸衍生物的最新研究进展,并介绍了利用体外重建体系来研究脂肪酸合成途径对该系统进行深入挖掘,以及根据得到的信息指导体内脂肪酸途径的改造来释放脂肪酸合成系统的潜能。     

中链化学品微生物制造

中链脂肪酸（C₆-C₁₂）衍生的化学品包括脂肪醇、脂肪烃、中链酯、ω-修饰脂肪酸等,这些化合物是生物燃料、聚合物、日用化学品、特种化学品的重要组分。天然微生物底盘不能合成中链脂肪酸,而通过操纵脂肪酸合成、逆β-氧化等碳链延伸途径,尤其是表达生成游离中链脂肪酸的硫酯酶,可使大肠埃希菌、酿酒酵母等微生物细胞合成超过1 g/L的中链脂肪酸。引入脂肪酸衍生反应,如羧基还原、脱羧、ω-氧化等,可合成许多中链化学品。本文综述了中链化学品合成的酶学基础以及代谢工程策略,为中链化学品高效生物制造提供参考和思路。     

合成微生物群落用于木质纤维素生物质转化的研究进展

木质纤维素在自然界中的储量可观,是生物燃料生产的重要来源。联合生物加工(consolidated bioprocessing)指在不添加酶的情况下,将木质纤维素“一步”转化为生物燃料的过程,在能源危机日益严重的今天具有重要的应用价值。合成微生物群落(synthetic microbial consortia)是由两种或多种纯培养微生物(野生菌株或工程菌株)共同培养而形成的菌群,具有复杂性低、稳定性高等优点,通过协调微生物之间的相互作用以及整个生态系统的稳定,从而实现特定的功能。近年来,合成生物学的快速发展有利于开发新的方法和工具用于合成微生物群落的构建及优化,促进其在联合生物加工方面的应用。本文聚焦于木质纤维素的联合生物加工,综述了合成微生物群落在该领域的研究进展。简单介绍了系统生物学为合成微生物群落的设计提供指导,详细介绍了合成微生物群落的设计原则、优化工具和在实际生产中的应用与挑战,为木质纤维素的联合生物加工提供借鉴意义。     

基于合成生物学的微生物制造研究进展

基于合成生物学的微生物制造在天然产物药物、生物能源、生物基化学品及生物传感器件的研究中发挥越来越重要的作用。本文系统地介绍了合成生物学研究领域的最新技术进展,包括DNA和染色体合成、新生物元件开发与元件库标准化、染色体工程与最小基因组技术、途径装配技术等,并阐述了合成生物学在微生物制造领域内所取得的突破和巨大的应用价值。     

加强研究推动我国生物降解塑料产业化进程一访清华大学生物系教授、长江学者陈国强

陈困强,清华大学教授,博士生导师,教育部长江学者特聘教授。长期从事“工业微生物、生物塑料和生物燃料”的研究。研究领域及方向：工业生物技术和合成生物学;微生物合成高分子材料聚羟基脂肪酸酯（PHA）;PHA在微生物中生理作用的研究;PHA作为组织工程材料的应用研究;     

面向先进生物燃料的合成生物学

先进生物燃料一般指来自于非粮食原料的交通运输用生物燃料。近年来,先进生物燃料的发展引起了众多国家的浓厚兴趣,然而,先进生物燃料正处于关键的技术研发阶段,还需经过大量研发以突破技术障碍和示范生产活动后方能进行商业化部署。过去10年内,合成生物学研究大量兴起并不断取得突破,使人们有可能人工设计构建新的高效生命系统,克服生物燃料发展的技术瓶颈,进行先进生物燃料的生产。在介绍先进生物燃料与合成生物学的发展现状的基础上,分析了合成生物学在先进生物燃料研发中的重要价值与研发进展,探讨了合成生物学的发展潜力。     

合成生物学技术在环境保护中的应用

合成生物学是一个基于生物学和工程学原理的科学领域，其目的是重新设计和重组微生物，以优化或创建具有增强功能的新生物系统。该领域利用分子工具、系统生物学和遗传框架的重编程，从而构建合成途径以获得具有替代功能的微生物。传统上，合成生物学方法通常旨在开发具有成本效益的微生物细胞工厂进而从可再生资源中生产化学物质。然而，近年来合成生物学技术开始在环境保护中发挥着更直接的作用。本综述介绍了基因工程中的合成生物学工具，讨论了基于基因工程的微生物修复策略，强调了合成生物学技术可以通过响应特定污染物进行生物修复来保护环境。其中，规律间隔成簇短回文重复序列（Clustered Regularly Interspersed Short Palindromic Repeats，CRISPR）技术在基因工程细菌和古细菌的生物修复中得到了广泛应用，生物修复领域也出现了很多新的先进技术，包括生物膜工程、人工微生物群落的构建、基因驱动、酶和蛋白质工程等。有了这些新的技术和工具，生物修复将成为当今最好和最有效的污染物去除方式之一。     

合成生物学在微生物细胞工厂构建中的应用

通过微生物发酵的方法生产大宗化学品和天然产物能够部分替代石油化工炼制和植物提取。合成生物学技术的发展极大地提高了构建微生物细胞工厂生产大宗化学品和天然产物的能力。一方面综述了合成生物学在构建细胞工厂时的关键技术,包括最优合成途径的设计、合成途径的创建与优化、细胞性能的优化;另一方面,介绍了应用这些技术构建细胞工厂生产燃料化学品、大宗化学品和天然产物的典型案例。     

高级醇的微生物绿色制造

高级醇是含有两个以上碳原子的醇类,具有比乙醇更优秀的燃料性能,是化石燃料的重要补充与替代品。利用微生物以可再生的生物质为原料进行高级醇的生产可同时缓解当前的能源与环境危机,已成为绿色生物制造的重大发展方向。天然的微生物仅能少量生产个别种类的高级醇,因此,通过代谢工程及合成生物学技术,在模式工业菌株中重构高级醇的合成途径,成为了克服高级醇生产瓶颈的必要手段。该领域自兴起以来已历经数十年发展,取得了多项开创性成果。本文作者有幸亲历了该领域从零到一、直至实现跨越式突破的完整历程。在代谢工程学科创立30周年之际,文中回顾了高级醇异源微生物合成的数个里程碑式工作,从途径创建、途经优化、原料扩展、底盘改造、产业化进程等多方面进行了分析总结。望以此激发学界对高级醇代谢工程的深入关注,促进新技术、新策略的开发应用,推动我国生物能源产业的创新升级。     

A perspective: Photosynthetic production of fatty acid-based biofuels in genetically engineered cyanobacteria

Biofuels are expected to play a key role in the development of a sustainable, economical and environmentally safe source of energy. Microbes offer great potential for applications in technology based biofuel production. Three fundamental questions need to be addressed in order for the development of microbial synthesis of biofuels to be successful. Firstly, what energy resource platform could be used to make biofuels. Secondly, what type of biofuel is the ideal fuel molecule that should be targeted. Finally, what microbial system could be used to transform energy resources into the targeted biofuel molecules. In this perspective, the potential of using photosynthetic microbes (cyanobacteria in particular) in the solar energy driven conversion of carbon dioxide to fatty acid-based biofuels is explored.     

Advanced biofuel production in microbes

The cost-effective production of biofuels from renewable materials will begin to address energy security and climate change concerns. Ethanol, naturally produced by microorganisms, is currently the major biofuel in the transportation sector. However, its low energy content and incompatibility with existing fuel distribution and storage infrastructure limits its economic use in the future. Advanced biofuels, such as long chain alcohols and isoprenoid- and fatty acid-based biofuels, have physical properties that more closely resemble petroleum-derived fuels, and as such are an attractive alternative for the future supplementation or replacement of petroleum-derived fuels. Here, we review recent developments in the engineering of metabolic pathways for the production of known and potential advanced biofuels by microorganisms. We concentrate on the metabolic engineering of genetically tractable organisms such as Escherichia coli and Saccharomyces cerevisiae for the production of these advanced biofuels.     

Calculation of Biodiesel Fuel Characteristics Based on the Fatty Acid Composition of the Lipids of Some Biotechnologically Important Microorganisms

The interdependences between the structure of fatty acid and biofuel characteristics obtained from these fatty acids were briefly reviewed. The fatty acid compositions of the lipids of yeasts and phototrophic microorganisms were analyzed. The main parameters of the biodiesel (iodine value, cetane number, and kinematic viscosity) that can be made from the lipids of these microorganisms were calculated based on the data and compared to the current standards. The lipids of the yeast Rhodosporidium toruloides VKPM Y-3349 were shown to be the most suitable for biofuel production due to the composition and content of fatty acid. The possibilities of a decrease in the prime cost of microbial lipids (along with plant oils) that would make them competitive raw material for biofuel production were considered.     

Algal swimming velocities signal fatty acid accumulation

The use of microalgae for biofuel production will be beneficial to society if we can produce biofuels at large scales with minimal mechanical energy input in the production process. Understanding micro‐algal physiological responses under variable environmental conditions in bioreactors is essential for the optimization of biofuel production. We demonstrate that measuring micro‐algal swimming speed provides information on culture health and total fatty acid accumulation. Three strains of Chlamydomonas reinhardtii were grown heterotrophically on acetate and subjected to various levels of nitrogen starvation. Other nutrient levels were explored to determine their effect on micro‐algal kinetics. Swimming velocities were measured with two‐dimensional micro‐particle tracking velocimetry. The results show an inverse linear relationship between normalized total fatty acid mass versus swimming speed of micro‐algal cells. Analysis of RNA sequencing data confirms these results by demonstrating that the biological processes of cell motion and the generation of energy precursors are significantly down‐regulated. Experiments demonstrate that changes in nutrient concentration in the surrounding media also affect swimming speed. The findings have the potential for the in situ and indirect assessment of lipid content by measuring micro‐algal swimming kinetics. Biotechnol. Bioeng. 2013; 110: 143–152. © 2012 Wiley Periodicals, Inc.     

Analysis of whole-cell fatty acid profiles of verotoxigenic Escherichia coli and Salmonella enteritidis with the microbial identification system.

Differentiation of strains within bacterial species, based on gas chromatographic analysis of whole-cell fatty acid profiles, was assessed with 115 strains of verotoxigenic Escherichia coli and 315 strains of Salmonella enteritidis. Fatty acid-based subgroups within each of the two species were generated. Variability of fatty acid profiles observed in repeat preparations from the same strain approached that observed between subgroups, limiting the usefulness of using fatty acid profiles to subgroup verotoxigenic E. coli and S. enteritidis strains.     

Production of dicarboxylic acids from novel unsaturated fatty acids by laccase-catalyzed oxidative cleavage

The establishment of renewable biofuel and chemical production is desirable because of global warming and the exhaustion of petroleum reserves. Sebacic acid (decanedioic acid), the material of 6,10-nylon, is produced from ricinoleic acid, a carbon-neutral material, but the process is not eco-friendly because of its energy requirements. Laccase-catalyzing oxidative cleavage of fatty acid was applied to the production of dicarboxylic acids using hydroxy and oxo fatty acids involved in the saturation metabolism of unsaturated fatty acids in Lactobacillus plantarum as substrates. Hydroxy or oxo fatty acids with a functional group near the carbon–carbon double bond were cleaved at the carbon–carbon double bond, hydroxy group, or carbonyl group by laccase and transformed into dicarboxylic acids. After 8 h, 0.58 mM of sebacic acid was produced from 1.6 mM of 10-oxo-cis-12,cis-15-octadecadienoic acid (αKetoA) with a conversion rate of 35% (mol/mol). This laccase-catalyzed enzymatic process is a promising method to produce dicarboxylic acids from biomass-derived fatty acids.     

Citric acid cycle biomimic on a carbon electrode

The citric acid cycle is one of the main metabolic pathways living cells utilize to completely oxidize biofuels to carbon dioxide and water. The overall goal of this research is to mimic the citric acid cycle at the carbon surface of an electrode in order to achieve complete oxidation of ethanol at a bioanode to increase biofuel cell energy density. In order to mimic this process, dehydrogenase enzymes (known to be the electron or energy producing enzymes of the citric acid cycle) are immobilized in cascades at an electrode surface along with non-energy producing enzymes necessary for the cycle to progress. Six enzymatic schemes were investigated each containing an additional dehydrogenase enzyme involved in the complete oxidation of ethanol. An increase in current density is observed along with an increase in power density with each additional dehydrogenase immobilized on an electrode, reflecting increased electron production at the bioanode with deeper oxidation of the ethanol biofuel. By mimicking the complete citric acid cycle on a carbon electrode, power density was increased 8.71-fold compared to a single enzyme (alcohol dehydrogenase)-based ethanol/air biofuel cell.     

Structure-based discovery of human L-xylulose reductase inhibitors from database screening and molecular docking

Human L-xylulose reductase (XR) is an enzyme of the glucuronic acid/uronate cycle of glucose metabolism and is a possible target for treatment of the long-term complications of diabetes. In this study we utilised the molecular modelling program DOCK to analyse the 249,071 compounds of the National Cancer Institute Database and retrieved those compounds with high predicted affinity for XR. Several carboxylic acid-based compounds were tested and shown to inhibit XR. These included nicotinic acid (IC50=100 microM), benzoic acid (IC50=29 microM) and their derivatives. These results extend and improve upon the activities of known, commercially available inhibitors of XR such as the aliphatic fatty acid n-butyric acid (IC50=64 microM). To optimise the interaction between the inhibitor and the holoenzyme, the program GRID was used to design de novo compounds based on the inhibitor benzoic acid. The inclusion of a hydroxy-phenyl group and a phosphate to the benzoic acid molecule increased the net binding energy by 1.3- and 2.4-fold, respectively. The resultant compounds may produce inhibitors with improved specificity for XR.