谷氨酸棒状杆菌pH智能调节系统构建及其适配性研究

pH regulation is a common method for sustaining cell activity in fermentation.Many problems, such as hysteresis of artificial pH regulation,overuse of acid and alkali casing environmental pollution, exist in the prevalent way of pH control in fermentation industry in which online pH detection devices with an acid- or alkali-feed module are used. A new idea for solving this problem is whether the bacteria can achieve real-time sensing and self-regulation of pH.For this purpose, based on the information from previous mining and selection of.regulators responding to acid or alkali and the related genes in acid and alkali metabolism, this program uses Corynebacterium Glutamicum as chassis for the construction of intelligent pH adjustment system with real-time sensing and self-regulation of pH function. The suitability between this system and the chassis will also be studied. Firstly, the genetic parts library of various pH sensing range will be established. Secondly, the intelligent pH adjustment system will be designed and assembled according to the features of the target strains. On the basis of the two steps above and in order to optimize the design of the system, the transcriptome and proteome will be used to study the suitability between this system and the chassis and elucidate the factors which affect the expression of exogenous pH regulators and the function of intracellular acid and alkali metabolism. With optimization of the intelligent pH adjustment system, we can achieve real-time regulation of pH with no artificial feeding of acid or alkali in a particular range. This research will offer new solutions for pH control problems in other engineered strains in fermentation and could facilitate to address the overuse of acid and alkali in fermentation industry.

pH调节是发酵过程中维持细胞活力的常用方法，目前普遍采用的在线监控pH、流加酸/碱的调节方式存在调节滞后，酸/碱过量使用造成环境污染等问题。能否实现微生物自身实时感应并自主调节pH是解决上述问题的新思路。为此，本项目在前期挖掘和筛选可响应酸/碱的调控基因及其代谢相关基因的基础上，以谷氨酸棒状杆菌为底盘宿主，构建可实时感应酸碱并能自主调节的pH智能调节系统，并研究该系统与宿主的适配性机制。首先，构建可响应不同pH的基因元器件库；其次，依据菌株特性设计和组装pH智能调节系统；最后，通过转录组学和蛋白质组学研究该系统与底盘宿主固有生理代谢的适配性，阐明影响外源pH调控元件表达和胞内酸碱代谢功能的因素，优化pH智能调节系统的设计，从而实现一定pH范围内、不流加酸碱便可实现pH实时智能调节。该研究也为其它菌种发酵的pH调节提供了新方法，有利于解决发酵工业中pH调节使用大量酸碱的瓶颈问题。

pH调节是发酵过程中维持细胞活力的常用方法，目前普遍采用的在线监控pH、流加酸/碱的调节方式存在调节滞后，酸/碱过量使用造成环境污染等问题。能否实现微生物自身实时感应并自主调节pH是解决上述问题的新思路。.为此，本项目在前期挖掘和筛选可响应酸/碱的调控基因及其代谢相关基因的基础上，以谷氨酸棒状杆菌为底盘宿主，构建可实时感应酸碱并能自主调节的pH智能调节系统，并研究该系统与宿主的适配性机制。.首先，构建可响应不同pH的基因元器件库，挖掘了来自E.coli和Corynebacterium glutamicum的酸碱相应启动子P-asr和P-atp2,利用报告基因LacZ表征酸碱这两个启动子对不同pH的相应情况。结果为：碱响应型启动子P-atp2在pH10.0时，酶活比较高,酸响应型启动子P-asr在pH6.0时，酶活最高。碱响应型启动子P-atp2在pH6.5时在12h后有响应，产碱基因glsA产碱中和pH,使其恢复正常水平。同时，碱响应型启动子在碱性环境能够响应并起始转录，产酸基因PQO能够产酸使环境中的pH恢复正常水平。.其次，以菌株谷氨酸棒杆菌（Corynebacterium Glutamicum ATCC13032）为出发菌株，经过ARTP诱变后，在2880个单克隆在pH=11.0的96孔板中挑选出18株耐碱菌株，结果表明：在pH=11.0时，耐碱突变菌株iCGTAl-17菌体生生长量达到出发菌株的47.1倍，细胞生长量可达到出发菌株在最适pH值下的93.7%。在2784个耐酸菌株中挑选了25株耐酸菌株，最强耐碱突变菌株iCGTAc-02菌体生长量达到出发菌株的27.3倍，细胞生长量为出发菌株在最适pH值下的108.6%。.最后，通过转录组学测序对诱变得到的耐受性菌株的耐受机制在基因层面进行了解析。.该研究为其它菌种发酵的pH调节提供了新方法，有利于解决发酵工业中pH调节使用大量酸碱的瓶颈问题。