光滑球拟酵母生产2，3-丁二酮的系统代谢工程策略  

System metabolic engineering strategies for 2,3-butandione production by Torulopsis glabrata

作　　者：高翔[1] 徐楠[1] 李树波[1] 刘立明[1] 

机构地区：[1]江南大学 食品科学与技术国家重点实验室 工业生物技术教育部重点实验室 食品微生物制造工程研究室,江苏无锡214122

出　　处：《微生物学报》2014年第4期398-407,共10页Acta Microbiologica Sinica

基　　金：中组部首批青年拔尖人才支持计划;无锡市科技局项目(CLE01N1111);江苏省杰出青年基金项目(BK2012002)~~

摘　　要：【目的】调控丙酮酸工业生产菌株光滑球拟酵母(Torulopsis glabrata)CCTCC M202019碳代谢流分布促进2,3-丁二酮积累。【方法】过量表达来源于枯草芽孢杆菌(Bacillus subtilis)的乙酰乳酸合成酶(ALS);在此基础上,借助T.glabrata全基因组规模代谢网络模型(GSMM)iNX804解析敲除基因ILV5的必要性;敲除基因BDH以阻断2,3-丁二酮的降解。【结果】过量表达ALS将ALS活性提高了4.6倍,发酵液中2,3-丁二酮浓度从0.01 g/L提高至0.57 g/L。敲除基因ILV5使2,3-丁二酮浓度提高28.1%。敲除基因BDH导致丁二酮还原酶和丁二醇脱氢酶活性分别降低74.4%、76.1%,同时2,3-丁二酮进一步代谢产物3-羟基丁酮和2,3-丁二醇浓度则分别降低52.2%和71.4%,2,3-丁二酮浓度为0.95 g/L。【结论】基于GSMM的系统代谢工程策略能够将碳代谢流从丙酮酸节点导向2,3-丁二酮,实现2,3-丁二酮的有效积累。[ Objective] We regulated the carbon flux distribution of Torulopsis glabrata CCTCC M202019, an efficient pyruvate-producing microorganism, for improved 2, 3-butandione production. [ Methods ] We overexpressed the acetolactate synthase （ALS） from Bacillus subtilis and then used the genome-scale metabolic model （GSMM） for T. glabrata （ named iNX804） to evaluate the importance of deleting the ILV5 gene. In addition, the BDH gene was deleted to restrict the degradation of 2,3-butanedione. [ Results] Overexpression of the ALS resulted in a 4.6-fold increase in ALS activity and increased the extracellular concentration of 2,3-butanedione to 0.57 g/L from 0.01 g/L. The deletion of the ILV5 gene was found to increase the 2,3-butanedione accumulation level by 28.1% , attributed to the disruption of L- valine and L-leucine biosynthetic pathway. With the deletion of the BDH gene, the enzyme activity levels of butanedione reductase and butanediol dehydrogenase were decreased by 74.4% and 76. 1% , respectively. And the accumulations of 3- hydroxybutanone and 2,3-butanediol were decreased by 52.2% and 71.4% , respectively. The final 2,3-butanedione concentration was 0.95 g/L, which was 30. 1% higher than that of the control strain. [ Conclusion] The GSMM based system metabolic engineering can be a functional strategy to redistribute the carbon flux from pyruvate node to 2,3- butanedione andachieve efficient accumulation of 2,3-butanedione.

关 键 词：光滑球拟酵母 2 3-丁二酮 代谢流分布 系统代谢工程 

分 类 号：TS202.3[轻工技术与工程—食品科学]