초록
<P><B>Abstract</B></P> <P>Improved supply of farnesyl diphosphate (FPP) is often considered as a typical strategy for engineering <I>Saccharomyces cerevisiae</I> towards efficient terpenoid production. However, in the engineered strains with enhanced precursor supply, the production of the target metabolite is often impeded by insufficient capacity of the heterologous terpenoid pathways, which limits further conversion of FPP. Here, we tried to assemble an unimpeded biosynthesis pathway by combining directed evolution and metabolic engineering in <I>S. cerevisiae</I> for lycopene-overproduction. First, the catalytic ability of phytoene syntheses from different sources was investigated based on lycopene accumulation. Particularly, the lycopene cyclase function of the bifunctional enzyme CrtYB from <I>Xanthophyllomyces dendrorhous</I> was inactivated by deletion of functional domain and directed evolution to obtain mutants with solely phytoene synthase function. Coexpression of the resulting <I>CrtYB11M</I> mutant along with the <I>CrtE</I> and <I>CrtI</I> genes from <I>X. dendrorhous</I>, and the <I>tHMG1</I> gene from <I>S. cerevisiae</I> led to production of 4.47mg/g DCW (Dry cell weight) of lycopene and 25.66mg/g DCW of the by-product squalene. To further increase the FPP competitiveness of the lycopene synthesis pathway, we tried to enhance the catalytic performance of CrtE by directed evolution and created a series of pathway variants by varying the copy number of <I>Crt</I> genes. Finally, fed-batch fermentation was conducted for the diploid strain YXWPD-14 resulting in accumulation of 1.61g/L (24.41mg/g DCW) of lycopene, meanwhile, the by-production of squalene was reduced to below 1mg/g DCW.</P> <P><B>Highlights</B></P> <P> <UL> <LI> CrtYB mutants with solely phytoene synthase function were created. </LI> <LI> Directed evolution of CrtE led to 2.2-time increment of lycopene production. </LI> <LI> Copy number adjustment of <I>Crt</I> genes further enhanced lycopene production. </LI> <LI> Highest lycopene production ever reported for an engineered yeast strain. </LI> </UL> </P>