Boosting the biosynthesis of betulinic acid and related triterpenoids in Yarrowia lipolytica via multimodular metabolic engineering

Abstract

Background: Betulinic acid is a pentacyclic lupane-type triterpenoid and a potential antiviral and antitumor drug, but the amount of betulinic acid in plants is low and cannot meet the demand for this compound. Yarrowia lipolytica, as an oleaginous yeast, is a promising microbial cell factory for the production of highly hydrophobic compounds due to the ability of this organism to accumulate large amounts of lipids that can store hydrophobic products and supply sufficient precursors for terpene synthesis. However, engineering for the heterologous production of betulinic acid and related triterpenoids has not developed as systematically as that for the production of other terpenoids, thus the production of betulinic acid in microbes remains unsatisfactory. Results: In this study, we applied a multimodular strategy to systematically improve the biosynthesis of betulinic acid and related triterpenoids in Y. lipolytica by engineering four functional modules, namely, the heterogenous CYP/CPR, MVA, acetyl-CoA generation, and redox cofactor supply modules. First, by screening 25 combinations of cytochrome P450 monooxygenases (CYPs) and NADPH-cytochrome P450 reductases (CPRs), each of which originated from 5 different sources, we selected two optimal betulinic acid-producing strains. Then, ERG1, ERG9, and HMG1 in the MVA module were overexpressed in the two strains, which dramatically increased betulinic acid production and resulted in a strain (YLJCC56) that exhibited the highest betulinic acid yield of 51.87 ± 2.77 mg/L. Then, we engineered the redox cofactor supply module by introducing NADPH-or NADH-generating enzymes and the acetyl-CoA generation module by directly overexpressing acetyl-CoA synthases or reinforcing the β-oxidation pathway, which further increased the total triterpenoid yield (the sum of the betulin, betulinic acid, betulinic aldehyde yields). Finally, we engineered these modules in combination, and the total triterpenoid yield reached 204.89 ± 11.56 mg/L (composed of 65.44% betulin, 23.71% betulinic acid and 10.85% betulinic aldehyde) in shake flask cultures. Conclusions: Here, we systematically engineered Y. lipolytica and achieved, to the best of our knowledge, the highest betulinic acid and total triterpenoid yields reported in microbes. Our study provides a suitable reference for studies on heterologous exploitation of P450 enzymes and manipulation of triterpenoid production in Y. lipolytica.

High production of triterpenoids in Yarrowia lipolytica through manipulation of lipid components

“…In order to improve lupeol production, we first up-regulated the mevalonate pathway by overexpressing the rate-limiting enzyme HMG1 (3-hydroxy-3-methylglutaryl coenzyme A reductase) [29] in strain LU-9, generating strain LU-10. It has been reported that synchronous overexpression of ERG1 (squalene synthase) and ERG9 (squalene monooxygenase) [30] exhibited cooperativity towards enhancing terpenes synthesis [22]. Hence, we further performed this combination based on the HMG1 overexpression to generate strain LU-11.…”

Coupling metabolic addiction with negative autoregulation to improve strain stability and pathway yield

“…The lipogenic acetyl-CoA and malonyl-CoA flux have been repurposed for production of β-carotenoids (Gao, Tong et al 2017, Larroude, Celinska et al 2018, polyketides (Markham, Palmer et al 2018, Liu, Marsafari et al 2019, Lv, Marsafari et al 2019, Palmer, Miller et al 2020, and terpenoids (Jin, Zhang et al 2019 in Yarrowia lipolytica. However, substantial portion of acetyl-CoA and malonyl-CoA is channeled into lipid synthesis in the engineered strains.…”

Optimizing mevalonate pathway for squalene production inYarrowia lipolytica

“…Yarrowia lipolytica is an industrial oleaginous yeast that has been extensively engineered to synthesize lipophilic compounds, including lipids (Qiao, Wasylenko, Zhou, Xu, & Stephanopoulos, 2017), oleochemicals (P. Xu, Qiao, Ahn, & Stephanopoulos, 2016), carotenoids (Gao et al, 2017;Macarena Larroude et al, 2018), terpenoids (Jin, Zhang, Song, & Cao, 2019) and aromatic polyketides (Lv, Marsafari, Koffas, Zhou, & Xu, 2019) et al The lipogeneity of this yeast makes it a superior host to produce chemicals that are derived from acetyl-CoA, malonyl-CoA, HMG-CoA and NADPHs. The compartmentalization of oil droplets into lipid bodies provides a hydrophobic environment to sequestrate many lipid-related compounds and mitigate the toxicity issues associated with lipophilic membrane damages.…”

“…A large collection of customized genetic toolboxes, including YaliBricks gene assembly (Wong, Engel, Jin, Holdridge, & Xu, 2017), CRISPR-Cas9 (Bae, Park, Kim, & Hahn, 2020;Macarena Larroude, Trabelsi, Nicaud, & Rossignol, 2020) or CRISPR-Cpf1 (Yang, Edwards, & Xu, 2020) genome editing, Cre-LoxP-based iterative chromosomal integrations (Lv, Edwards, Zhou, & Xu, 2019), transposon-based mutagenesis (Wagner, Williams, & Alper, 2018) and Golden-gate cloning (Celińska et al, 2017;Egermeier, Sauer, & Marx, 2019;M. Larroude et al, 2019), enabled us to rapidly modify its genome and evaluate many metabolic events to explore the catalytic diversity of this yeast beyond its regular portfolio of fatty acids, fatty alcohols, biofuels et al Recent metabolic engineering effort in this yeast has allowed us to access more specialized secondary metabolites with pharmaceutical values, including sesquiterpenes (Marsafari & Xu, 2020), triterpenoids (Jin et al, 2019) and flavonoids (Lv, Marsafari, et al, 2019;Palmer, Miller, Nguyen, & Alper, 2020) et al Isoprenoids are a large group of natural products with diverse biological functions. An estimation of more than 70,000 isoprenoids, ranging from monoterpenes, sesquiterpenes, diterpenes and triterpenes have been discovered from nature (Moser & Pichler, 2019).…”