초록
<P><B>Abstract</B></P> <P>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, <I>β</I>-amyrin is overproduced in <I>Saccharomyces cerevisiae</I> through optimizing the pathway expression by the employment of SST (Short Synthetic Terminators). Initially, the <I>β</I>AS gene was expressed with P<SUB>TEF1</SUB> promoter along-with T<SUB>CYC1</SUB> terminator and produced 2.7 mg L<SUP>−1</SUP> <I>β</I>-amyrin. The precursor's supply was enhanced through overexpressing the key regulatory genes of terpenoid pathway i.e. <I>ERG1, ERG9, ERG20, IDI, tHMG1</I> controlled by constitutive promoters P<SUB>ERG1</SUB>, P<SUB>TPI1</SUB>, P<SUB>TDH3</SUB>, P<SUB>FBA1</SUB>, P<SUB>PGK1</SUB> that enhanced 3.8-fold <I>β</I>-amyrin production compared with the control strain. Moreover, a set of 23 SST was designed by analyzing the native terminator sequences and characterized, where, Syn<SUP>TER10</SUP>, Syn<SUP>TER9</SUP>, Syn<SUP>TER8</SUP>, Syn<SUP>TER6</SUP>, Syn<SUP>TER22</SUP>, and Syn<SUP>TER19</SUP>, terminators showed 5.57-, 5.04-, 3.55-, 3.40-, 1.89-, and 1.71-fold, enhanced expression of <I>EGFP</I> compared with native T<SUB>CYC1</SUB> terminator. Subsequently, aforesaid strong SST was employed to optimize the expression of the <I>β</I>-amyrin pathway, which increased 3.16-fold production of <I>β</I>-amyrin. However, after optimization of the <I>β</I>-amyrin pathway, the engineered strain enhanced 12.14-fold (32.8 mg L<SUP>−1</SUP>) of <I>β</I>-amyrin production. Finally, using the 5 L fermenter, the production of <I>β</I>-amyrin was 108.26 mgL<SUP>−1</SUP> which were 40-fold higher and that of ergosterol was 38.8 mgL<SUP>−1</SUP>, 6.80-fold higher comparative to the GSK1 strain.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Metabolic Engineering & Synthetic Biology approaches were used to produce β-amyrin. </LI> <LI> β-amyrin production was enhanced 3.8-fold via expression of Key MVA pathway genes. </LI> <LI> 23 SST were designed and tested in engineering of β-amyrin pathway. </LI> <LI> Employment of 6 strongest SST enhanced the production of β-amyrin by 12.14-fold. </LI> <LI> Via Fed-batch fermentation the production of β-amyrin was enhanced by 40-fold. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>