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

摘要:

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。

Abstract:

Abstract:［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 Lvaline 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 and achieve efficient accumulation of 2，3-butanedione.