Abstract:

In the research works of constructing taxadiene aritficaial synthetic cell, S. cerevisiae is a commonly used chassis. The highest taxadiene yield in S.cerevisiae that has been reported was 8.7 mg/L. In this work, the fitness of different taxadiene biosynthetic modules with different S. cerevisiae chassis was studied to elevate the taxadiene yield to a higher level. We chose the S.cerevisiae strains BY4742 and the single knoc-kout strains of BY4742 as chassis, constructed the multi-plasmids taxadiene biosynthetic module by optimizing the mevalonic acid(MVA) pathway with tHMGR, importing the synthetic genes GGPPSsa and tTS, up-regulating and co-expressing the genes BTS1 and ERG20 which are correlated with the synthesis of geranylgeranyl diphosphate(GGPP). Then we redesigned and reconstructed the module inserted into a single CEN plasmid by the method of DNA assembler. Through fitting the different modules with different chassis, we acquired strains with diffe-rent taxadiene yields, the highest yield was 74.84 mg/L in the strain with the single-plasmid module and the chassis of YNL280C. The results indicated that the single-plasmid taxadiene biosynthetic module was more stable in the chassis than the multi-plasmids one, and the regulation of the pathways correlated with the MVA pathway will also influent the taxadiene yield.

Key words: Taxadiene, Synthetic biology, Artificial synthetic cell, Fitness