系统代谢工程改造树干毕赤酵母生产琥珀酸

System metabolic engineering of Pichia stipitis for succinic acid production

为提高树干毕赤酵母发酵生产琥珀酸的产量,借助基因组规模代谢网络模型iTL885获得琥珀酸合成的最佳代谢途径为扩增icl1基因和敲除sdh1基因。在此基础上,借助代谢工程策略构建过量表达异柠檬酸裂解酶基因icl1的重组菌株FPLicl、缺失琥珀酸脱氢酶基因sdh1的重组菌株FPLΔsdh和缺失sdh1基因同时过量表达icl1基因的重组菌株FPLΔsdh-icl。结果表明:3株重组菌的异柠檬酸裂解酶活性由0.33 U/mg分别增加为1.6、5.6和6.6U/mg;而琥珀酸脱氢酶活性则从13.8 U/mg分别降为10.7、0.3和0.3 U/mg。在以木糖为C源的培养基中,3株重组菌生产琥珀酸的能力分别是0.30、1.20和1.60 g/L。

To improve the succinic acid production by Pichia stipites with xylose as sole carbon source,an optimum synthesis pathway for succinate production in P. stipites was obtained through aiding by genome-scale metabolic model iTL885.In this optimum pathway,isocitrate lyase gene icl1 was overexpressed but succinate dehydrogenase gene sdh1 should be deleted.Then, the genetic engineering stains FPLicl over-expressing icl1,FPLΔsdh deleting sdh1 and FPLΔsdh-icl with sdh1-deleted and icl1 over-expressed were constructed.The ICL enzyme activity reached at 1.6, 5.6 and 6.6 U/mg, the SDH enzyme activity reduced to 10.7,0.3 and 0.3 U/mg in the three strains,respectively.As a result,0.30,1.20 and 1.60 g/L succinate were produced by the three engineered strains on xylose as carbon source.