Author:
Kong Jing,Miao Lin,Lu Zhihui,Wang Shuhui,Zhao Baixiang,Zhang Cuiying,Xiao Dongguang,Teo Desmond,Leong Susanna Su Jan,Wong Adison,Yu Aiqun
Abstract
Abstract
Background
Amyrin is an important triterpenoid and precursor to a wide range of cosmetic, pharmaceutical and nutraceutical products. In this study, we metabolically engineered the oleaginous yeast, Yarrowia lipolytica to produce α- and β-amyrin on simple sugar and waste cooking oil.
Results
We first validated the in vivo enzymatic activity of a multi-functional amyrin synthase (CrMAS) from Catharanthus roseus, by expressing its codon-optimized gene in Y. lipolytica and assayed for amyrins. To increase yield, prevailing genes in the mevalonate pathway, namely HMG1, ERG20, ERG9 and ERG1, were overexpressed singly and in combination to direct flux towards amyrin biosynthesis. By means of a semi-rational protein engineering approach, we augmented the catalytic activity of CrMAS and attained ~ 10-folds higher production level on glucose. When applied together, protein engineering with enhanced precursor supplies resulted in more than 20-folds increase in total amyrins. We also investigated the effects of different fermentation conditions in flask cultures, including temperature, volumetric oxygen mass transfer coefficient and carbon source types. The optimized fermentation condition attained titers of at least 100 mg/L α-amyrin and 20 mg/L β-amyrin.
Conclusions
The design workflow demonstrated herein is simple and remarkably effective in amplifying triterpenoid biosynthesis in the yeast Y. lipolytica.
Funder
the Ministry of Education, Singapore
Lee Foundation, Singapore
Natural Science Foundation of Tianjin, China
Research Foundation of Tianjin Municipal Education Commission, China
Innovative Research Team of Tianjin Municipal Education Commission, China
Tianjin Municipal Science and Technology Project
Publisher
Springer Science and Business Media LLC
Subject
Applied Microbiology and Biotechnology,Bioengineering,Biotechnology
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