Engineering peroxisomal biosynthetic pathways for maximization of triterpene production in Yarrowia lipolytica

Ma, Yongshuo; Shang, Yi; Stephanopoulos, Gregory

doi:10.1073/pnas.2314798121

Engineering peroxisomal biosynthetic pathways for maximization of triterpene production in Yarrowia lipolytica

Journal Article · Mon Jan 22 23:00:00 EST 2024 · Proceedings of the National Academy of Sciences of the United States of America

DOI:https://doi.org/10.1073/pnas.2314798121· OSTI ID:2471950

^[1]; Shang, Yi ^[2]; Stephanopoulos, Gregory ^[3]

Massachusetts Institute of Technology (MIT), Cambridge, MA (United States); Massachusetts Institute of Technology, Cambridge, MA (United States)
Yunnan Normal University, Kunming (China)
Massachusetts Institute of Technology (MIT), Cambridge, MA (United States)

Constructing efficient cell factories for product synthesis is frequently hampered by competing pathways and/or insufficient precursor supply. This is particularly evident in the case of triterpenoid biosynthesis in Yarrowia lipolytica, where squalene biosynthesis is tightly coupled to cytosolic biosynthesis of sterols essential for cell viability. Here, we addressed this problem by reconstructing the complete squalene biosynthetic pathway, starting from acetyl-CoA, in the peroxisome, thus harnessing peroxisomal acetyl-CoA pool and sequestering squalene synthesis in this organelle from competing cytosolic reactions. This strategy led to increasing the squalene levels by 1,300-fold relatively to native cytosolic synthesis. Subsequent enhancement of the peroxisomal acetyl-CoA supply by two independent approaches, 1) converting cellular lipid pool to peroxisomal acetyl-CoA and 2) establishing an orthogonal acetyl-CoA shortcut from CO₂-derived acetate in the peroxisome, further significantly improved local squalene accumulation. Using these approaches, we constructed squalene-producing strains capable of yielding 32.8 g/L from glucose, and 31.6 g/L from acetate by employing a cofeeding strategy, in bioreactor fermentations. Our findings provide a feasible strategy for protecting intermediate metabolites that can be claimed by multiple reactions by engineering peroxisomes in Y. lipolytica as microfactories for the production of such intermediates and in particular acetyl-CoA-derived metabolites.

View Accepted Manuscript (DOE)

Research Organization:: Massachusetts Institute of Technology (MIT), Cambridge, MA (United States)

Sponsoring Organization:: USDOE Office of Science (SC); National Key Research and Development Program of China; Yunnan Science Fund

Grant/Contract Number:: SC0022016

OSTI ID:: 2471950

Journal Information:: Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Issue: 5 Vol. 121; ISSN 0027-8424

Publisher:: National Academy of SciencesCopyright Statement

Country of Publication:: United States

Language:: English

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Engineering peroxisomal biosynthetic pathways for maximization of triterpene production in Yarrowia lipolytica

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