本文主要研究内容
作者罗正山（2019）在《光滑球拟酵母积累丙酮酸关键生理调控机制解析》一文中研究指出：丙酮酸（pyruvic acid）是细胞代谢的关键中间产物,也是一种重要的有机酸,被广泛应用于食品工业、农业、医药工业等领域。近年来,发酵法生产丙酮酸受到国内外学者的密切关注。在众多已发现的丙酮酸生产菌株中,光滑球拟酵母（Candida glabrata）因具有高葡萄糖和酸耐受性,被认为是最具应用前景的丙酮酸生产菌株。然而,该菌株的丙酮酸发酵过程存在副产物形成机制不明确、胞内中心化合物调控复杂和溶氧依赖性强等问题影响了其大规模生产。本研究以C.glabrata CCTCC M202019为出发菌株,借助于生化工程和代谢工程策略,鉴定该酵母丙酮酸发酵过程中的主要副产物及其合成途径,并在此基础上提出代谢工程改造C.glabrata中心代谢网络的策略来强化丙酮酸合成及改善菌株对低氧环境的适应性。主要研究结果如下:（1）高通量筛选丙酮酸发酵过程差异明显的C.glabrata突变菌株为副产物鉴定提供基础。结合ARTP随机诱变和硝酸铁多孔板检测技术建立了一种高通量筛选丙酮酸发酵过程差异明显突变菌株的方法。进一步结合QPix 420全自动挑菌仪和全自动移液工作站等自动化仪器,提高了该方法的筛选通量和精度,使得整个高通量筛选过程更加高效。利用该方法从30000个突变子中经过几轮的高通量筛选,初筛获得了9株高产丙酮酸的突变菌株。通过摇瓶发酵复筛实验,最终获得了一株丙酮酸摇瓶产量提高32.3%的C.glabrata H6菌株和一株能使发酵液自发凝固的丙酮酸生产菌株C.glabrata 4-C10。这些丙酮酸发酵过程差异明显的突变菌株的获得为进一步鉴定C.glabrata丙酮酸发酵过程中的副产物提供基础。（2）鉴定C.glabrata发酵生产丙酮酸过程中的主要副产物及其代谢途径。通过对获得的C.glabrata 4-C10突变菌株和出发菌株的发酵液进行分离纯化,并结合酸水解、光谱分析及合成单体组分分析实验,鉴定了C.glabrata发酵生产丙酮酸过程中的主要副产物为多聚糖。对C.glabrata的副产物合成途径中相关基因进行转录水平分析,发现CAGL0H02695g和CAGL0K10626g基因是造成菌株胞外多聚糖积累的重要基因。在C.glabrata出发菌株中敲除这两个重要基因,使得菌株的副产物多聚糖积累降低了15.6%,而丙酮酸摇瓶产量提高了11.8%。实验结果也表明,虽然降低多聚糖合成会一定程度的改善丙酮酸胞外积累,但也会严重影响菌体生长。因此,完全阻断多聚糖合成来改善丙酮酸积累的策略似乎不可行。而调控与多聚糖合成密切相关的中心化合物来协调菌株生长与多聚糖合成似乎是进一步改善丙酮酸积累的高效策略。（3）调控C.glabrata发酵过程中丙酮酸亚细胞分布来强化丙酮酸的胞外积累。已有大量报道表明副产物多聚糖积累与胞内丙酮酸和ATP的亚细胞分布密切相关。借助载体工程策略研究丙酮酸的亚细胞分布对C.glabrata的中心碳代谢途径和丙酮酸胞外积累的影响,找到有利于丙酮酸胞外积累的最佳丙酮酸胞内分布趋势。强化丙酮酸进入线粒体的过程,改善了C.glabrata的中心碳代谢途径和丙酮酸胞外积累速率。在此过程中,发现和验证了不断积累的胞质丙酮酸趋势是影响C.glabrata中心碳代谢和丙酮酸胞外积累的关键因素。通过细胞膜定位表达线粒体丙酮酸载体蛋白调控丙酮酸向胞外转运,使C.glabrata中胞质丙酮酸分布趋势由上升趋势转变为下降趋势,达到了改善C.glabrata菌株的中心碳代谢途径和丙酮酸积累的目的,使得丙酮酸的积累量达到29.0g/L。同时,构建了一种结合sortase A介导连接和流式细胞检测技术的快速膜定位检测方法。（4）构建高效ATP无效循环系统缓解ATP对丙酮酸合成途径的反馈抑制。已有报道表明高胞内ATP水平不仅有利于菌株高效合成多聚糖等副产物,而且还会对糖酵解途径产生严重的反馈抑制,进而影响目标产物的合成。利用代谢工程策略构建了一种能高效降解C.glabrata胞内ATP的无效循环系统（ATP-FCS）,并进一步优化了该系统与中心代谢途径的关系改善了C.glabrata菌株的丙酮酸积累过程。通过对C.glabrata菌株生理特性的分析,将两种酶促反应过程（丙酮酸羧化酶（PYC2p）和磷酸烯醇式丙酮酸羧激酶（PCK1p））引入C.glabrata的细胞质中,结合菌株本身的部分代谢途径构建ATP无效循环系统。对该系统进行评估,发现ATP-FCS能高效维持C.glabrata丙酮酸发酵过程中的胞内ATP和ROS处于较低水平,并有效改善了中心碳代谢流流向丙酮酸的效率,使得丙酮酸的胞外积累量提高了49.7%。另外,进一步优化ATP-FCS并协调ATP-FCS与其他代谢途径的关系,使得C.glabrata丙酮酸的最大摇瓶产量达到40.2 g/L。（5）设计低氧全局诱导因子缓解C.glabrata丙酮酸发酵过程对高溶氧的依赖性。高的胞外溶氧会加速菌株胞内丙酮酸进入TCA循环和氧化磷酸化途径,导致大量丙酮酸氧化分解成CO2,同时产生大量ATP。借助于代谢工程策略研究了低氧诱导因子（HIF-1）在低氧条件下对C.glabrata丙酮酸发酵过程的影响,改善C.glabrata低氧条件下的丙酮酸积累过程。通过文献挖掘和数据库分析手段,发现C.glabrata葡萄糖转运和糖酵解途径中的大部分酶基因启动子区域存在HIF-1因子特异性识别的A/GCGTC基序。通过引入HIF-1因子于C.glabrata中,改善了菌株在低氧条件下的葡萄糖转运和糖酵解途径中一些关键酶基因的转录水平,并使低氧条件下的丙酮酸产量提高了32.4%。进一步优化菌株的溶氧控制条件、敲除C.glabrata中的脯氨酸羟基化酶来改善HIF-1在菌株胞内的稳定性,使得丙酮酸在低氧条件下的积累效率得到了进一步提高。最后,维持菌株在发酵罐上的溶氧水平为10%,使得菌株的最大丙酮酸产量达53.1 g/L。
Abstract
bing tong suan （pyruvic acid）shi xi bao dai xie de guan jian zhong jian chan wu ,ye shi yi chong chong yao de you ji suan ,bei an fan ying yong yu shi pin gong ye 、nong ye 、yi yao gong ye deng ling yu 。jin nian lai ,fa jiao fa sheng chan bing tong suan shou dao guo nei wai xue zhe de mi qie guan zhu 。zai zhong duo yi fa xian de bing tong suan sheng chan jun zhu zhong ,guang hua qiu ni jiao mu （Candida glabrata）yin ju you gao pu tao tang he suan nai shou xing ,bei ren wei shi zui ju ying yong qian jing de bing tong suan sheng chan jun zhu 。ran er ,gai jun zhu de bing tong suan fa jiao guo cheng cun zai fu chan wu xing cheng ji zhi bu ming que 、bao nei zhong xin hua ge wu diao kong fu za he rong yang yi lai xing jiang deng wen ti ying xiang le ji da gui mo sheng chan 。ben yan jiu yi C.glabrata CCTCC M202019wei chu fa jun zhu ,jie zhu yu sheng hua gong cheng he dai xie gong cheng ce lve ,jian ding gai jiao mu bing tong suan fa jiao guo cheng zhong de zhu yao fu chan wu ji ji ge cheng tu jing ,bing zai ci ji chu shang di chu dai xie gong cheng gai zao C.glabratazhong xin dai xie wang lao de ce lve lai jiang hua bing tong suan ge cheng ji gai shan jun zhu dui di yang huan jing de kuo ying xing 。zhu yao yan jiu jie guo ru xia :（1）gao tong liang shai shua bing tong suan fa jiao guo cheng cha yi ming xian de C.glabratatu bian jun zhu wei fu chan wu jian ding di gong ji chu 。jie ge ARTPsui ji you bian he xiao suan tie duo kong ban jian ce ji shu jian li le yi chong gao tong liang shai shua bing tong suan fa jiao guo cheng cha yi ming xian tu bian jun zhu de fang fa 。jin yi bu jie ge QPix 420quan zi dong tiao jun yi he quan zi dong yi ye gong zuo zhan deng zi dong hua yi qi ,di gao le gai fang fa de shai shua tong liang he jing du ,shi de zheng ge gao tong liang shai shua guo cheng geng jia gao xiao 。li yong gai fang fa cong 30000ge tu bian zi zhong jing guo ji lun de gao tong liang shai shua ,chu shai huo de le 9zhu gao chan bing tong suan de tu bian jun zhu 。tong guo yao ping fa jiao fu shai shi yan ,zui zhong huo de le yi zhu bing tong suan yao ping chan liang di gao 32.3%de C.glabrata H6jun zhu he yi zhu neng shi fa jiao ye zi fa ning gu de bing tong suan sheng chan jun zhu C.glabrata 4-C10。zhe xie bing tong suan fa jiao guo cheng cha yi ming xian de tu bian jun zhu de huo de wei jin yi bu jian ding C.glabratabing tong suan fa jiao guo cheng zhong de fu chan wu di gong ji chu 。（2）jian ding C.glabratafa jiao sheng chan bing tong suan guo cheng zhong de zhu yao fu chan wu ji ji dai xie tu jing 。tong guo dui huo de de C.glabrata 4-C10tu bian jun zhu he chu fa jun zhu de fa jiao ye jin hang fen li chun hua ,bing jie ge suan shui jie 、guang pu fen xi ji ge cheng chan ti zu fen fen xi shi yan ,jian ding le C.glabratafa jiao sheng chan bing tong suan guo cheng zhong de zhu yao fu chan wu wei duo ju tang 。dui C.glabratade fu chan wu ge cheng tu jing zhong xiang guan ji yin jin hang zhuai lu shui ping fen xi ,fa xian CAGL0H02695ghe CAGL0K10626gji yin shi zao cheng jun zhu bao wai duo ju tang ji lei de chong yao ji yin 。zai C.glabratachu fa jun zhu zhong qiao chu zhe liang ge chong yao ji yin ,shi de jun zhu de fu chan wu duo ju tang ji lei jiang di le 15.6%,er bing tong suan yao ping chan liang di gao le 11.8%。shi yan jie guo ye biao ming ,sui ran jiang di duo ju tang ge cheng hui yi ding cheng du de gai shan bing tong suan bao wai ji lei ,dan ye hui yan chong ying xiang jun ti sheng chang 。yin ci ,wan quan zu duan duo ju tang ge cheng lai gai shan bing tong suan ji lei de ce lve shi hu bu ke hang 。er diao kong yu duo ju tang ge cheng mi qie xiang guan de zhong xin hua ge wu lai xie diao jun zhu sheng chang yu duo ju tang ge cheng shi hu shi jin yi bu gai shan bing tong suan ji lei de gao xiao ce lve 。（3）diao kong C.glabratafa jiao guo cheng zhong bing tong suan ya xi bao fen bu lai jiang hua bing tong suan de bao wai ji lei 。yi you da liang bao dao biao ming fu chan wu duo ju tang ji lei yu bao nei bing tong suan he ATPde ya xi bao fen bu mi qie xiang guan 。jie zhu zai ti gong cheng ce lve yan jiu bing tong suan de ya xi bao fen bu dui C.glabratade zhong xin tan dai xie tu jing he bing tong suan bao wai ji lei de ying xiang ,zhao dao you li yu bing tong suan bao wai ji lei de zui jia bing tong suan bao nei fen bu qu shi 。jiang hua bing tong suan jin ru xian li ti de guo cheng ,gai shan le C.glabratade zhong xin tan dai xie tu jing he bing tong suan bao wai ji lei su lv 。zai ci guo cheng zhong ,fa xian he yan zheng le bu duan ji lei de bao zhi bing tong suan qu shi shi ying xiang C.glabratazhong xin tan dai xie he bing tong suan bao wai ji lei de guan jian yin su 。tong guo xi bao mo ding wei biao da xian li ti bing tong suan zai ti dan bai diao kong bing tong suan xiang bao wai zhuai yun ,shi C.glabratazhong bao zhi bing tong suan fen bu qu shi you shang sheng qu shi zhuai bian wei xia jiang qu shi ,da dao le gai shan C.glabratajun zhu de zhong xin tan dai xie tu jing he bing tong suan ji lei de mu de ,shi de bing tong suan de ji lei liang da dao 29.0g/L。tong shi ,gou jian le yi chong jie ge sortase Ajie dao lian jie he liu shi xi bao jian ce ji shu de kuai su mo ding wei jian ce fang fa 。（4）gou jian gao xiao ATPmo xiao xun huan ji tong huan jie ATPdui bing tong suan ge cheng tu jing de fan kui yi zhi 。yi you bao dao biao ming gao bao nei ATPshui ping bu jin you li yu jun zhu gao xiao ge cheng duo ju tang deng fu chan wu ,er ju hai hui dui tang jiao jie tu jing chan sheng yan chong de fan kui yi zhi ,jin er ying xiang mu biao chan wu de ge cheng 。li yong dai xie gong cheng ce lve gou jian le yi chong neng gao xiao jiang jie C.glabratabao nei ATPde mo xiao xun huan ji tong （ATP-FCS）,bing jin yi bu you hua le gai ji tong yu zhong xin dai xie tu jing de guan ji gai shan le C.glabratajun zhu de bing tong suan ji lei guo cheng 。tong guo dui C.glabratajun zhu sheng li te xing de fen xi ,jiang liang chong mei cu fan ying guo cheng （bing tong suan suo hua mei （PYC2p）he lin suan xi chun shi bing tong suan suo ji mei （PCK1p））yin ru C.glabratade xi bao zhi zhong ,jie ge jun zhu ben shen de bu fen dai xie tu jing gou jian ATPmo xiao xun huan ji tong 。dui gai ji tong jin hang ping gu ,fa xian ATP-FCSneng gao xiao wei chi C.glabratabing tong suan fa jiao guo cheng zhong de bao nei ATPhe ROSchu yu jiao di shui ping ,bing you xiao gai shan le zhong xin tan dai xie liu liu xiang bing tong suan de xiao lv ,shi de bing tong suan de bao wai ji lei liang di gao le 49.7%。ling wai ,jin yi bu you hua ATP-FCSbing xie diao ATP-FCSyu ji ta dai xie tu jing de guan ji ,shi de C.glabratabing tong suan de zui da yao ping chan liang da dao 40.2 g/L。（5）she ji di yang quan ju you dao yin zi huan jie C.glabratabing tong suan fa jiao guo cheng dui gao rong yang de yi lai xing 。gao de bao wai rong yang hui jia su jun zhu bao nei bing tong suan jin ru TCAxun huan he yang hua lin suan hua tu jing ,dao zhi da liang bing tong suan yang hua fen jie cheng CO2,tong shi chan sheng da liang ATP。jie zhu yu dai xie gong cheng ce lve yan jiu le di yang you dao yin zi （HIF-1）zai di yang tiao jian xia dui C.glabratabing tong suan fa jiao guo cheng de ying xiang ,gai shan C.glabratadi yang tiao jian xia de bing tong suan ji lei guo cheng 。tong guo wen suo wa jue he shu ju ku fen xi shou duan ,fa xian C.glabratapu tao tang zhuai yun he tang jiao jie tu jing zhong de da bu fen mei ji yin qi dong zi ou yu cun zai HIF-1yin zi te yi xing shi bie de A/GCGTCji xu 。tong guo yin ru HIF-1yin zi yu C.glabratazhong ,gai shan le jun zhu zai di yang tiao jian xia de pu tao tang zhuai yun he tang jiao jie tu jing zhong yi xie guan jian mei ji yin de zhuai lu shui ping ,bing shi di yang tiao jian xia de bing tong suan chan liang di gao le 32.4%。jin yi bu you hua jun zhu de rong yang kong zhi tiao jian 、qiao chu C.glabratazhong de fu an suan qiang ji hua mei lai gai shan HIF-1zai jun zhu bao nei de wen ding xing ,shi de bing tong suan zai di yang tiao jian xia de ji lei xiao lv de dao le jin yi bu di gao 。zui hou ,wei chi jun zhu zai fa jiao guan shang de rong yang shui ping wei 10%,shi de jun zhu de zui da bing tong suan chan liang da 53.1 g/L。
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论文作者分别是来自江南大学的罗正山，发表于刊物江南大学2019-10-21论文，是一篇关于光滑球拟酵母论文,丙酮酸论文,副产物鉴定论文,代谢工程论文,低氧适应性论文,江南大学2019-10-21论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自江南大学2019-10-21论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。
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