Gao Xiang, Xu Nan, Li Shubo, Liu Liming
Wei Sheng Wu Xue Bao. 2014 Apr 4;54(4):398-407.
We regulated the carbon flux distribution of Torulopsis glabrata CCTCC M202019, an efficient pyruvate-producing microorganism, for improved 2, 3-butandione production.
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.
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.
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.
我们对光滑球拟酵母CCTCC M202019（一种高效产丙酮酸的微生物）的碳通量分布进行调控，以提高2,3 - 丁二酮的产量。
我们过表达了来自枯草芽孢杆菌的乙酰乳酸合酶（ALS），然后使用光滑球拟酵母的基因组规模代谢模型（GSMM，命名为iNX804）来评估删除ILV5基因的重要性。此外，删除BDH基因以限制2,3 - 丁二酮的降解。
ALS的过表达导致ALS活性增加4.6倍，使2,3 - 丁二酮的胞外浓度从0.01 g/L增加到0.57 g/L。发现删除ILV5基因可使2,3 - 丁二酮积累水平提高28.1%，这归因于L - 缬氨酸和L - 亮氨酸生物合成途径的中断。随着BDH基因的删除，丁二酮还原酶和丁二醇脱氢酶的酶活性水平分别降低了74.4%和76.1%。3 - 羟基丁酮和2,3 - 丁二醇的积累分别减少了52.2%和71.4%。最终的2,3 - 丁二酮浓度为0.95 g/L，比对照菌株高30.1%。
基于GSMM的系统代谢工程可以是一种有效的策略，用于将碳通量从丙酮酸节点重新分配到2,3 - 丁二酮，实现2,3 - 丁二酮的高效积累。
