title: Construction of lycopene-overproducing Saccharomyces cerevisiae by combining directed evolution and metabolic engineering.
abstract: Improved supply of farnesyl diphosphate (FPP) is often considered as a typical strategy for engineering Saccharomyces cerevisiae 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 S. cerevisiae 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 Xanthophyllomyces dendrorhous was inactivated by deletion of functional domain and directed evolution to obtain mutants with solely phytoene synthase function. Coexpression of the resulting CrtYB11M mutant along with the CrtE and CrtI genes from X. dendrorhous, and the tHMG1 gene from S. cerevisiae led to production of 4.47 mg/g DCW (Dry cell weight) of lycopene and 25.66 mg/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 Crt genes. Finally, fed-batch fermentation was conducted for the diploid strain YXWPD-14 resulting in accumulation of 1.61 g/L (24.41 mg/g DCW) of lycopene, meanwhile, the by-production of squalene was reduced to below 1 mg/g DCW.
authors.affiliation: Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, 310027 Hangzhou, PR China.
authors.forename: Wenping
authors.initials: W
authors.lastname: Xie
authors.affiliation: Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, 310027 Hangzhou, PR China.
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Lv
authors.affiliation: Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, 310027 Hangzhou, PR China; Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Zhejiang University, 310027 Hangzhou, PR China.
authors.forename: Lidan
authors.initials: L
authors.lastname: Ye
authors.affiliation: Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, 310027 Hangzhou, PR China.
authors.forename: Pingping
authors.initials: P
authors.lastname: Zhou
authors.affiliation: Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, 310027 Hangzhou, PR China. Electronic address: yuhongwei@zju.edu.cn.
authors.forename: Hongwei
authors.initials: H
authors.lastname: Yu
mesh_terms: D002338:Carotenoids / Q000096:biosynthesis / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods*; D000077276:Lycopene; D060847:Metabolic Engineering / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
chemical_list: D002338:Carotenoids; D000077276:Lycopene
keywords: Directed evolution; Lycopene; Phytoene synthase; Saccharomyces cerevisiae; Squalene