Design and construction of short synthetic terminators for β-amyrin production in Saccharomyces cerevisiae

Abstract

The coupled employment of synthetic biology and metabolic engineering strategies is prolific for the optimized production of value-added compounds. Terminators are the prime components in the “expression cassettes” that influence the net protein production. Herein, β-amyrin is overproduced in Saccharomyces cerevisiae through optimizing the pathway expression by the employment of SST (Short Synthetic Terminators). Initially, the βAS gene was expressed with PTEF1 promoter along-with TCYC1 terminator and produced 2.7 mg L−1β-amyrin. The precursor's supply was enhanced through overexpressing the key regulatory genes of terpenoid pathway i.e. ERG1, ERG9, ERG20, IDI, tHMG1 controlled by constitutive promoters PERG1, PTPI1, PTDH3, PFBA1, PPGK1 that enhanced 3.8-fold β-amyrin production compared with the control strain. Moreover, a set of 23 SST was designed by analyzing the native terminator sequences and characterized, where, SynTER10, SynTER9, SynTER8, SynTER6, SynTER22, and SynTER19, terminators showed 5.57-, 5.04-, 3.55-, 3.40-, 1.89-, and 1.71-fold, enhanced expression of EGFP compared with native TCYC1 terminator. Subsequently, aforesaid strong SST was employed to optimize the expression of the β-amyrin pathway, which increased 3.16-fold production of β-amyrin. However, after optimization of the β-amyrin pathway, the engineered strain enhanced 12.14-fold (32.8 mg L−1) of β-amyrin production. Finally, using the 5 L fermenter, the production of β-amyrin was 108.26 mgL−1 which were 40-fold higher and that of ergosterol was 38.8 mgL−1, 6.80-fold higher comparative to the GSK1 strain.