Yao Zhen, Zhou Pingping, Su Bingmei, Su Sisi, Ye Lidan, Yu Hongwei
Institute of Bioengineering, College of Chemical and Biological Engineering , Zhejiang University , Hangzhou 310027 , PR China.
Fujian Key Laboratory of Marine Enzyme Engineering, College of Biological Science and Engineering , Fuzhou University , Fuzhou 350116 , PR China.
ACS Synth Biol. 2018 Sep 21;7(9):2308-2316. doi: 10.1021/acssynbio.8b00289. Epub 2018 Aug 31.
Isoprene, as a versatile bulk chemical, has wide industrial applications. Here, we attempted to improve isoprene biosynthesis in Saccharomyces cerevisiae by simultaneous strengthening of precursor supply and conversion via a combination of pathway compartmentation and protein engineering. At first, a superior isoprene synthase mutant ISPSLN was created by saturation mutagenesis, leading to almost 4-fold improvement in isoprene production. Subsequent introduction of ISPSLN to strains with strengthened precursor supply in either cytoplasm or mitochondria implied an imperfect match between the synthesis and conversion of the isopentenyl pyrophosphate (IPP)/dimethylallyl diphosphate (DMAPP) pool. To reconstruct metabolic balance between the upstream and downstream flux, additional copies of diphosphomevalonate decarboxylase gene ( MVD1) and isopentenyl-diphosphate delta-isomerase gene ( IDI1) were introduced into the cytoplasmic and mitochondrial engineered strains. Finally, the diploid strain created by mating the above haploid strains produced 11.9 g/L of isoprene, the highest ever reported in eukaryotic cells.
异戊二烯作为一种用途广泛的大宗化学品，具有广泛的工业应用。在此，我们试图通过途径区室化和蛋白质工程相结合的方式，同时加强前体供应和转化，来提高酿酒酵母中异戊二烯的生物合成。首先，通过饱和诱变创建了一个优良的异戊二烯合酶突变体ISPSLN，使异戊二烯产量提高了近4倍。随后将ISPSLN引入细胞质或线粒体中前体供应增强的菌株，这意味着异戊烯基焦磷酸（IPP）/二甲基烯丙基焦磷酸（DMAPP）库的合成与转化之间存在不完全匹配。为了重建上下游通量之间的代谢平衡，将二磷酸甲羟戊酸脱羧酶基因（MVD1）和异戊烯基二磷酸δ-异构酶基因（IDI1）的额外拷贝引入细胞质和线粒体工程菌株中。最后，通过将上述单倍体菌株进行交配产生的二倍体菌株产生了11.9 g/L的异戊二烯，这是真核细胞中报道的最高产量。
