Paramasivan Kalaivani, Mutturi Sarma
Microbiology and Fermentation Technology Department, CSIR-Central Food Technological Research Institute , Mysore, India.
Academy of Scientific and Innovative Research , Mysore, New Delhi, India.
J Agric Food Chem. 2017 Sep 20;65(37):8162-8170. doi: 10.1021/acs.jafc.7b02945. Epub 2017 Sep 6.
Although overexpression of the tHMG1 gene is a well-known strategy for terpene synthesis in Saccharomyces cerevisiae, the optimal level for tHMG1p has not been established. In the present study, it was observed that two copies of the tHMG1 gene on a dual gene expression cassette improved squalene synthesis in laboratory strain by 16.8-fold in comparison to single-copy expression. It was also observed that tHMG1p is limited by its cofactor (NADPH), as the overexpression of NADPH regenerating genes', viz., ZWF1 and POS5 (full length and without mitochondrial presequence), has led to its increased enzyme activity. Further, it was demonstrated that overexpression of full-length POS5 has improved squalene synthesis in cytosol. Finally, when tHMG1 and full-length POS5 were co-overexpressed there was a net 27.5-fold increase in squalene when compared to control strain. These results suggest novel strategies to increase squalene accumulation in S. cerevisiae.
尽管tHMG1基因的过表达是酿酒酵母中萜类合成的一种众所周知的策略，但tHMG1p的最佳水平尚未确定。在本研究中，观察到双基因表达盒上的两个tHMG1基因拷贝与单拷贝表达相比，使实验室菌株中的角鲨烯合成提高了16.8倍。还观察到tHMG1p受其辅因子（NADPH）的限制，因为NADPH再生基因ZWF1和POS5（全长且无线粒体前导序列）的过表达导致其酶活性增加。此外，证明全长POS5的过表达改善了细胞质中的角鲨烯合成。最后，当tHMG1和全长POS5共过表达时，与对照菌株相比，角鲨烯净增加了27.5倍。这些结果提示了增加酿酒酵母中角鲨烯积累的新策略。
