本文主要研究内容
作者朱康佳（2019）在《产乙醇酸的重组大肠杆菌转录组分析及代谢改造》一文中研究指出：乙醇酸（Glycolate,羟基乙酸,甘醇酸）是极具工业价值的双碳羟基羧酸,具有易降解易吸收等特性,应用于包装、纺织、医疗、化妆品等多个领域。本实验室在前期研究中构建的乙醇酸工程菌株Mgly545,在M9无机盐培养基中,以葡萄糖为碳源合成乙醇酸,最终乙醇酸产量达到16.66 g·L-1。但乙醇酸工程菌株发酵生产乙醇酸的过程中仍存在很多问题:M9培养基适合乙醇酸的生产,但不利于菌体生长,较低的菌体生物量使得乙醇酸生产效率低下,菌株存在菌体生长和乙醇酸产量之间的矛盾。针对以上问题,主要从以下三方面进行研究:（1）为实现工程菌株菌体生长和乙醇酸生产之间的平衡,提高乙醇酸积累效率,探究了不同氮源以及异柠檬酸脱氢酶ICDH敲除对菌体生长和乙醇酸合成的影响。有机氮源可改善Mgly545菌株生长,但会引起乙酸过量积累以及乙醇酸产量降低,不利于工业化生产。在Mgly545菌株基础上缺失ICDH的Mgly625菌株,以蛋白胨和酵母粉为有机氮源时,乙醇酸产量没有受到菌体生长的影响,合成效率为0.80 g·OD-1,是Mgly545在相同氮源中的2.6倍。（2）基于RNA-Seq技术,对Mgly545分别在无机氮源和有机氮源以及Mgly625在有机氮源中发酵的转录组进行比较。发现Mgly625中乙酰辅酶A合成酶acs的显著上调增强了菌株对乙酸的利用,减少了乙酸的积累;磷酸烯醇式丙酮酸羧化激酶pck的显著上调增强了磷酸烯醇式丙酮酸PEP回补途径,使得碳源有效地合成乙醇酸。对这三种转录本胞内NADPH/NADP+进行测定并比较与NADPH合成代谢相关基因的转录水平,发现有机氮源可增强磷酸戊糖途径,为乙醇酸合成提供更多NADPH。ICDH敲除引起了68个基因表达的显著变化,基于此分析了Mgly625菌株中N调节、氨基酸代谢、氧化应激反应和铁转运等与细胞生长及乙醇酸合成的关系。发现添加谷氨酸和丝氨酸可提高Mgly625生物量和生产效率;过量表达精氨酸转运酶基因argT获得的菌株Mgly6252,细胞生长速度提高,生物量增加;培养基中Fe2+的补充可以加速细胞生长并补偿胞内缺乏的还原当量。最终Mgly6252菌株在5L发酵罐中乙醇酸产量达到22.43 g·L-1。（3）为了高通量筛选高产乙醇酸的突变菌株,本研究构建了可实时监测乙醇酸积累水平的生物传感器。在大肠杆菌中,转录调节蛋白GlcC受乙醇酸诱导,从而激活glcD基因的转录。利用PglcD启动子调控绿色荧光蛋白基因（sfGFP）表达,从而构建乙醇酸传感器,将荧光信号与胞内乙醇酸浓度偶联。同时构建了高拷贝传感器质粒pGBS-H和低拷贝传感器质粒pGBS-L。经乙醇酸体外诱导比较,低拷贝传感器对不同浓度乙醇酸的响应较好,检测范围为0-50 mM。在实时检测乙醇酸产量过程中,荧光强度与乙醇酸产量具有较好的线性关系（R2=0.9086）。通过优化PglcD与基因sfGFP之间的RBS,进一步提高了传感器的响应值和检测限。研究还表明全局调控子（ArcA）在微好氧条件下会抑制传感器中荧光蛋白的表达。利用定向进化策略改造了异柠檬酸裂解酶（AceA）,强化了乙醇酸合成。基于乙醇酸生物传感器所建立的定向进化策略极大地简化了筛选流程,为乙醇酸工业微生物快速改造提供了方法借鉴。
Abstract
yi chun suan （Glycolate,qiang ji yi suan ,gan chun suan ）shi ji ju gong ye jia zhi de shuang tan qiang ji suo suan ,ju you yi jiang jie yi xi shou deng te xing ,ying yong yu bao zhuang 、fang zhi 、yi liao 、hua zhuang pin deng duo ge ling yu 。ben shi yan shi zai qian ji yan jiu zhong gou jian de yi chun suan gong cheng jun zhu Mgly545,zai M9mo ji yan pei yang ji zhong ,yi pu tao tang wei tan yuan ge cheng yi chun suan ,zui zhong yi chun suan chan liang da dao 16.66 g·L-1。dan yi chun suan gong cheng jun zhu fa jiao sheng chan yi chun suan de guo cheng zhong reng cun zai hen duo wen ti :M9pei yang ji kuo ge yi chun suan de sheng chan ,dan bu li yu jun ti sheng chang ,jiao di de jun ti sheng wu liang shi de yi chun suan sheng chan xiao lv di xia ,jun zhu cun zai jun ti sheng chang he yi chun suan chan liang zhi jian de mao dun 。zhen dui yi shang wen ti ,zhu yao cong yi xia san fang mian jin hang yan jiu :（1）wei shi xian gong cheng jun zhu jun ti sheng chang he yi chun suan sheng chan zhi jian de ping heng ,di gao yi chun suan ji lei xiao lv ,tan jiu le bu tong dan yuan yi ji yi ning meng suan tuo qing mei ICDHqiao chu dui jun ti sheng chang he yi chun suan ge cheng de ying xiang 。you ji dan yuan ke gai shan Mgly545jun zhu sheng chang ,dan hui yin qi yi suan guo liang ji lei yi ji yi chun suan chan liang jiang di ,bu li yu gong ye hua sheng chan 。zai Mgly545jun zhu ji chu shang que shi ICDHde Mgly625jun zhu ,yi dan bai dong he jiao mu fen wei you ji dan yuan shi ,yi chun suan chan liang mei you shou dao jun ti sheng chang de ying xiang ,ge cheng xiao lv wei 0.80 g·OD-1,shi Mgly545zai xiang tong dan yuan zhong de 2.6bei 。（2）ji yu RNA-Seqji shu ,dui Mgly545fen bie zai mo ji dan yuan he you ji dan yuan yi ji Mgly625zai you ji dan yuan zhong fa jiao de zhuai lu zu jin hang bi jiao 。fa xian Mgly625zhong yi xian fu mei Age cheng mei acsde xian zhe shang diao zeng jiang le jun zhu dui yi suan de li yong ,jian shao le yi suan de ji lei ;lin suan xi chun shi bing tong suan suo hua ji mei pckde xian zhe shang diao zeng jiang le lin suan xi chun shi bing tong suan PEPhui bu tu jing ,shi de tan yuan you xiao de ge cheng yi chun suan 。dui zhe san chong zhuai lu ben bao nei NADPH/NADP+jin hang ce ding bing bi jiao yu NADPHge cheng dai xie xiang guan ji yin de zhuai lu shui ping ,fa xian you ji dan yuan ke zeng jiang lin suan wu tang tu jing ,wei yi chun suan ge cheng di gong geng duo NADPH。ICDHqiao chu yin qi le 68ge ji yin biao da de xian zhe bian hua ,ji yu ci fen xi le Mgly625jun zhu zhong Ndiao jie 、an ji suan dai xie 、yang hua ying ji fan ying he tie zhuai yun deng yu xi bao sheng chang ji yi chun suan ge cheng de guan ji 。fa xian tian jia gu an suan he si an suan ke di gao Mgly625sheng wu liang he sheng chan xiao lv ;guo liang biao da jing an suan zhuai yun mei ji yin argThuo de de jun zhu Mgly6252,xi bao sheng chang su du di gao ,sheng wu liang zeng jia ;pei yang ji zhong Fe2+de bu chong ke yi jia su xi bao sheng chang bing bu chang bao nei que fa de hai yuan dang liang 。zui zhong Mgly6252jun zhu zai 5Lfa jiao guan zhong yi chun suan chan liang da dao 22.43 g·L-1。（3）wei le gao tong liang shai shua gao chan yi chun suan de tu bian jun zhu ,ben yan jiu gou jian le ke shi shi jian ce yi chun suan ji lei shui ping de sheng wu chuan gan qi 。zai da chang gan jun zhong ,zhuai lu diao jie dan bai GlcCshou yi chun suan you dao ,cong er ji huo glcDji yin de zhuai lu 。li yong PglcDqi dong zi diao kong lu se ying guang dan bai ji yin （sfGFP）biao da ,cong er gou jian yi chun suan chuan gan qi ,jiang ying guang xin hao yu bao nei yi chun suan nong du ou lian 。tong shi gou jian le gao kao bei chuan gan qi zhi li pGBS-Hhe di kao bei chuan gan qi zhi li pGBS-L。jing yi chun suan ti wai you dao bi jiao ,di kao bei chuan gan qi dui bu tong nong du yi chun suan de xiang ying jiao hao ,jian ce fan wei wei 0-50 mM。zai shi shi jian ce yi chun suan chan liang guo cheng zhong ,ying guang jiang du yu yi chun suan chan liang ju you jiao hao de xian xing guan ji （R2=0.9086）。tong guo you hua PglcDyu ji yin sfGFPzhi jian de RBS,jin yi bu di gao le chuan gan qi de xiang ying zhi he jian ce xian 。yan jiu hai biao ming quan ju diao kong zi （ArcA）zai wei hao yang tiao jian xia hui yi zhi chuan gan qi zhong ying guang dan bai de biao da 。li yong ding xiang jin hua ce lve gai zao le yi ning meng suan lie jie mei （AceA）,jiang hua le yi chun suan ge cheng 。ji yu yi chun suan sheng wu chuan gan qi suo jian li de ding xiang jin hua ce lve ji da de jian hua le shai shua liu cheng ,wei yi chun suan gong ye wei sheng wu kuai su gai zao di gong le fang fa jie jian 。
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