细胞筛选平台在人类功能基因组研究中的应用

人类基因组全序列的精细图已完成,当前生命科学面临的重要任务就是如何将基因组序列信息转化为基因的功能信息,了解生命活动的分子机理,改善人类健康,为生物技术发展提供动力 . 在一系列功能基因组研究新技术中,高通量 (high-throughput) 和高内涵 (high-content) 的细胞筛选技术平台已经显示出巨大潜力,发挥着越来越重要的作用 . 通过在体外培养的哺乳动物细胞中基因过表达或抑制基因表达,分析所产生信号传导通路和 / 或细胞表型改变,可以直接发现基因功能 . 近年来一些技术上的进展,使细胞筛选平台具有微量、自动、高效、高通量,以及可以系统研究的特点,已经成为功能基因组研究的核心方法之一 . 近 2~3 年来已经出现一批成功应用细胞筛选平台进行大规模功能基因组研究的报道 . 我国在这一领域的研究也开始起步,将对我国生物技术的源头创新研究产生深远的影响 .     

前体代谢工程提高红霉素产量的研究进展

红霉素是十四元大环内酯类抗生素,具有广泛的医药价值和巨大的新药开发潜力。红霉素的主要成分红霉素A由丙酰辅酶A和甲基丙二酰辅酶A作为前体通过聚酮合酶合成大环内酯骨架,再经羟基化、糖基化、甲基化等一系列修饰合成。根据红霉素A的生物合成路线,我们从前体喂养途径、糖基化和甲基化优化等方面,简要综述近年来利用前体代谢工程手段提高红霉素产量的研究进展。     

代谢工程

代谢工程,也称途径工程,是基因工程一个重要分支,一般是多基因的基因工程,与细胞的基因调控、代谢调控和生化工程密切相关。讨论了代谢工程的应用,包括通过改变代谢流和代谢途径提高产量,改善生产过程,构建新的代谢途径和产生新的代谢产物等。     

啤酒酵母代谢工程研究进展

啤酒工业上应用的啤酒酵母菌株在生产中都会存在着某些方面的缺陷。通过分析啤酒酵母某些代谢产物的代谢途径,寻找改变其代谢流量的方法,然后用分子生物学手段对其代谢流量加以改变,来调节啤酒酵母某些产物的代谢水平已经成为啤酒酵母育种的新方式。对酵母的底物利用、可操作性、控制有害副产物的产量及改善啤酒风味等方面的研究成果进行了综述。     

代谢网络刚性与代谢工程

本文介绍了有关代谢网络,网络刚性和代谢工程的基本观点,特别是关于网络刚性和载流途径的工作假设,还讨论了代谢工程的应用与面临的难题。     

聚酮类化合物生物合成的代谢工程研究进展

聚酮化合物是一类重要的具有生物活性的次级代谢物。本文讨论了以聚酮生物合成酶为核心的聚酮化合物生物合成途径,以及近年来有关代谢工程在聚酮类化合物生物合成方面的研究工作进展,主要包括将聚酮生物合成途径引入新的宿主、代谢流量分析在提高聚酮化合物中的应用及合成新的聚酮化舍物等。     

全局转录调控及其在代谢工程中的应用

全局转录调控是一种全新的改进细胞表型的定向进化方法,通过error-prone PCR、DNA shuffling等技术对细胞中的σ因子和其他转录元件进行多轮突变修饰,改变RNA聚合酶的转录效率和对启动子的亲和能力,使细胞的转录在整体水平上发生改变,导致许多由多种基因控制的细胞表型得以改进。全局转录调控可以对代谢途径快速优化,在代谢工程中已被成功地应用于各种代谢产物的生物合成中。随着全局转录调控理论的不断完善,其应用前景也将越来越广阔。     

Metabolic Engineering of Plant L-Ascorbic Acid Biosynthesis: Recent Trends and Applications

ABSTRACT

Vitamin C (L-ascorbic acid; AsA) is the major soluble antioxidant found in plants and is also an essential component of human nutrition. Although numerous biotechnological methods have been exploited to increase its yield, pressures such as commercial competition and environmental concerns make it urgent to find a new way for industrial production of plant-derived AsA. Engineering plant AsA has now become feasible because of our increased understanding of its biosynthetic pathway. Several possible strategies could be followed to increase AsA production, such as overcoming the rate limiting steps in the biosynthetic pathway, promoting recycling, and reducing catabolism. For these purposes, genes of plant, microbial and animal origins have been successfully used. Several examples will be given to illustrate these various approaches. The existing and potential achievements in increasing AsA production would provide the opportunity for enhancing nutritional quality and stress tolerance of crop plants.     

天冬氨酸家族主要氨基酸高产菌株的选育策略

L-苏氨酸与L-赖氨酸是L-天冬氨酸家族氨基酸(AFAAs)中的重要成员,近年来由于其在食品、化妆品、动物饲料添加剂等方面的广泛应用而备受关注,市场需求逐年上升。运用代谢工程手段构建高产菌,可有效地提高L-苏氨酸和L-赖氨酸的生产水平。本文详述了L-苏氨酸与L-赖氨酸的合成途径、调控机制以及两种氨基酸高产菌株的构建策略。     

代谢工程应用研究进展

代谢工程发展已有二十多年的时间,其利用重组DNA技术,调控细胞生理功能,在微生物、植物和动物细胞中得到了广泛的应用。综述了代谢工程在微生物、植物和动物细胞中应用研究的最新进展,并对其今后发展方向做出展望。     

阿维菌素的生物合成及代谢工程研究进展

阿维菌素(avermectin)是由除虫链霉菌(Streptomycesavermitilis)产生的一种具有杀虫活性的大环内酯类抗生素,在农业和畜牧业中应用广泛。本文综述了有关除虫链霉菌基因组序列分析、阿维菌素的生物合成以及阿维菌素育种和代谢工程的研究进展。     

Thiolase engineering for enhanced butanol production in Clostridium acetobutylicum

Biosynthetic thiolases catalyze the condensation of two molecules acetyl‐CoA to acetoacetyl‐CoA and represent key enzymes for carbon–carbon bond forming metabolic pathways. An important biotechnological example of such a pathway is the clostridial n‐butanol production, comprising various natural constraints that limit titer, yield, and productivity. In this study, the thiolase of Clostridium acetobutylicum, the model organism for solventogenic clostridia, was specifically engineered for reduced sensitivity towards its physiological inhibitor coenzyme A (CoA‐SH). A high‐throughput screening assay in 96‐well microtiter plates was developed employing Escherichia coli as host cells for expression of a mutant thiolase gene library. Screening of this library resulted in the identification of a thiolase derivative with significantly increased activity in the presence of free CoA‐SH. This optimized thiolase comprised three amino acid substitutions (R133G, H156N, G222V) and its gene was expressed in C. acetobutylicum ATCC 824 to assess the effect of reduced CoA‐SH sensitivity on solvent production. In addition to a clearly delayed ethanol and acetone formation, the ethanol and butanol titers were increased by 46% and 18%, respectively, while the final acetone concentrations were similar to the vector control strain. These results demonstrate that thiolase engineering constitutes a suitable methodology applicable to improve clostridial butanol production, but other biosynthetic pathways involving thiolase‐mediated carbon flux limitations might also be subjected to this new metabolic engineering approach. Biotechnol. Bioeng. 2013; 110: 887–897. © 2012 Wiley Periodicals, Inc.     

植物糖基转移酶及其在代谢工程中的应用

糖基转移酶催化植物次生代谢物合成,在植物的生长发育过程以及代谢工程应用方面起着重要作用。该文介绍糖基转移酶的基本特性,总结近年来研究植物糖基转移酶基因的克隆与功能分析的方法,详细阐述了微生物中3个C-糖基转移酶基因（UrdGT2、gilGT和iroA）的克隆和功能研究,概括糖基转移酶在代谢工程的研究进展,并展望今后的研究趋势,为植物C-糖基转移酶的生物学功能研究和代谢应用方面提供有益帮助。     

代谢工程在芳香化合物生物合成研究中的应用

生物技术和代谢工程的发展促进了生物合成研究。概述了近年来利用微生物莽草酸途径进行芳香化合物生物合成研究的现况、代谢工程在提高天然芳香化合物产量和扩大合成非天然产生的芳香化合物范围的应用的进展 ,特别是整体代谢工程对提高第二代工程菌产量的作用。指出了生物合成法是生产氨基酸及其它生物小分子如奎尼酸、维生素和抗生素等的未来趋势 ,在工业化生产中有着广阔的应用前景。     

植物萜类代谢工程

植物萜类化合物不仅在植物生命活动中起重要作用,而且具有重要商业价值。随着近年来萜类代谢途径和调控机理研究的深入,代谢工程已成为提高萜类产量最有潜力的途径之一。对萜类代谢工程领域具代表性的研究结果进行了全面回顾,然后讨论了萜类代谢工程的研究方法和策略,其中重点探讨了功能基因组学方法在萜类代谢途径及调控机理研究方面的应用。     

Metabolic engineering of Cyanobacteria and microalgae for enhanced production of biofuels and high‐value products

A lot of research has been performed on Cyanobacteria and microalgae with the aim to produce numerous biotechnological products. However, native strains have a few shortcomings, like limitations in cultivation, harvesting and product extraction, which prevents reaching optimal production value at lowest costs. Such limitations require the intervention of genetic engineering to produce strains with superior properties. Promising advancements in the cultivation of Cyanobacteria and microalgae have been achieved by improving photosynthetic efficiency through increasing RuBisCO activity and truncation of light‐harvesting antennae. Genetic engineering has also contributed to final product extraction by inducing autolysis and product secretory systems, to enable direct product recovery without going through costly extraction steps. In this review, we summarize the different enzymes and pathways that have been targeted thus far for improving cultivation aspects, harvesting and product extraction in Cyanobacteria and microalgae. With synthetic biology advancements, genetically engineered strains can be generated to resolve demanding process issues and achieve economic practicality. This comprehensive overview of gene modifications will be useful to researchers in the field to employ on their strains to increase their yields and improve the economic feasibility of the production process.