s12934-022-01915-0.pdf (3.63 MB)
Download file

Enhanced production of amyrin in Yarrowia lipolytica using a combinatorial protein and metabolic engineering approach

Download (3.63 MB)
journal contribution
posted on 25.09.2022, 04:57 authored by Jing Kong, Lin Miao, Zhihui Lu, Shuhui Wang, Baixiang Zhao, Cuiying Zhang, Dongguang Xiao, Desmond TeoDesmond Teo, Su Jan Susanna LeongSu Jan Susanna Leong, Adison WongAdison Wong, Aiqun Yu

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.

Funding

Ministry of Education, Singapore (R-MOE-A401-F028)

Lee Foundation, Singapore (T-LEE-T201-A001)

History

Journal/Conference/Book title

Microbial Cell Factories

Publication date

2022-09-09

Version

Published

Corresponding author

Adison Wong; Aiqun Yu

Project ID

  • 7343 (R-IND-A401-0047; R-IND-A401-0057; R-MOE-A401-F028) Development of Asian-centric Sustainable Foods