Production of the bioactive plant‐derived triterpenoid morolic acid in engineered Saccharomyces cerevisiae

Abstract

Morolic acid is a plant‐derived triterpenoid that possesses pharmacological properties such as cytotoxicity, as well as anti‐HIV, anti‐HSV, anti‐inflammatory, and antidiabetic effects. The significant therapeutic properties of morolic acid are desirable in the context of pharmacological and drug development research, but the low accessibility of morolic acid from natural resources limits its applications. In the present study, we developed a microbial system for the production of morolic acid. Using a combinatorial biosynthesis approach, a novel production pathway was constructed in Saccharomycescerevisiae by coexpressing BfOSC2 (germanicol synthase) from Bauhinia forficata and CYP716A49 (triterpene C‐28 oxidase) from Beta vulgaris. Moreover, we reconstructed the cellular galactose regulatory network by introducing a chimeric transcriptional activator (fusion of Gal4dbd.ER.VP16) to overdrive the genes under the control of the galactose promoter. We also overexpressed truncated HMG1, encoding feedback‐inhibition‐resistant form of 3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase 1 and sterol‐regulating transcription factor upc2‐1, to increase the isoprenoid precursors in the mevalonate pathway. Using this yeast system, we achieved morolic acid production up to 20.7 ± 1.8 mg/L in batch culture. To our knowledge, this is the highest morolic acid titer reported from a heterologous host, indicating a promising approach for obtaining rare natural triterpenoids.

Comparative Analysis of NADPH-Cytochrome P450 Reductases From Legumes for Heterologous Production of Triterpenoids in Transgenic Saccharomyces cerevisiae

“…PSIII is an engineered yeast strain that has been optimized for triterpenoid biosynthesis by means of some modifications in the upstream mevalonate pathway. This strain is modified from the PSII strain constructed in previous research, which can produce a 100 × times higher triterpene backbone as a substrate than its parental strain ( Srisawat et al, 2020 ). In contrast to the INV Sc 1 results, in the PSIII strain, almost all the triterpenoid sapogenin production profiles had a lower ratio of β-amyrin, and the trends of CYP-CPR preferences observed in the INV Sc 1 strain seem to be less obvious in this strain ( Figure 9 ).…”

“…Two different yeast strains were used in this research, INV Sc1 (MATa his3D1 leu2 trp1-289 ura3-52; Thermo Fisher Scientific) and PSIII strain. PSIII strain was generated by transforming pESC-TRP[P GAL 10 / t HMG1-T2A-upc2-1 ][P GAL 1 /Lj bAS ]) as described in Supplementary Methods 2 into PSI strain (BY4742/P erg 7 :P MET 3 - ERG7 / trp1 :P ACT 1 - Gal4dbd-ER-VP16 ) constructed in a previous study ( Srisawat et al, 2020 ). INV Sc 1 strain carrying pYES3-ADH-aAS, pYES3-ADH-OSC1, or pYES3-ADH-LUS ( Fukushima et al, 2011 ) and PSIII strain were then co-transformed with pAG415-CPR, pYES-DEST52-CYP, and pESC-HIS-CYP consecutively using Frozen-EX Yeast Transformation II™ (Zymo Research, Irvine, CA, United States).…”

The Aspartic Protease Yps3p and Cell Wall Glucanase Scw10p Are Novel Determinants That Enhance the Secretion of the Antitumor Triterpenoid GA-HLDOA in Saccharomyces cerevisiae

“…Owing to its genetic tractability and the inherent existence of multiple metabolites, Saccharomyces cerevisiae is the predominant yeast species used in the synthetic bioproduction of triterpenoids. 8 The metabolites contained in S. cerevisiae (e.g., squalene and lanosterol) serve as precursors of triterpenoids. Ganoderic acids (GAs), a group of lanostane-type triterpenoids, are the main bioactive secondary metabolites of the traditional Chinese medicinal mushroom Ganoderma lucidum.…”

“…Owing to its genetic tractability and the inherent existence of multiple metabolites, Saccharomyces cerevisiae is the predominant yeast species used in the synthetic bioproduction of triterpenoids . The metabolites contained in S. cerevisiae (e.g., squalene and lanosterol) serve as precursors of triterpenoids.…”

Metabolic Engineering of Escherichia coli for Production of α-Santalene, a Precursor of Sandalwood Oil

“…For example, the accumulation of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) induced growth retardation in E. coli expressing a yeast MVA pathway, which could be alleviated by modulation of HMG-CoA reductase or substitution with other homologs. , A similar strategy was also conducted to eliminate the accumulation of IPP, DMAPP, and/or FPP in the engineering process for terpenoid production. , The key concern of this multivariate-modular approach would be to differentiate expression levels on pathway modules and screen the best combinatory for the desired production of interest . Rational optimization of ribosome binding sites (RBSs) offers a very effective way to tune multiple genes of the MVA pathway to improve isoprene production .…”