Summary Triterpenoids are widely distributed among plants of the legume family. However, most studies have focused on triterpenoids and their biosynthetic enzymes in model legumes. We evaluated the triterpenoid aglycones profile of the medicinal legume tree Bauhinia forficata by gas chromatography–mass spectrometry. Through transcriptome analyses, homology‐based cloning, and heterologous expression, we discovered four oxidosqualene cyclases (OSCs) which are responsible for the diversity of triterpenols in B. forficata. We also investigated the effects of the unique motif TLCYCR on α‐amyrin synthase activity. B. forficata highly accumulated α‐amyrin. We discovered an OSC with a preponderant α‐amyrin‐producing activity, which accounted for at least 95% of the total triterpenols. We also discovered three other functional OSCs (BfOSC1, BfOSC2, and BfOSC4) that produce β‐amyrin, germanicol, and cycloartenol. Furthermore, by replacing the unique motif TLCYCR from BfOSC3 with the MWCYCR motif, we altered the function of BfOSC3 such that it no longer produced α‐amyrin. Our results provide new insights into OSC cyclization, which is responsible for the diversity of triterpenoid metabolites in B. forficata, a non‐model legume plant.

Triterpenoids are plant specialized metabolites with various pharmacological activities. They are widely distributed in higher plants, such as legumes. Because of their low accumulation in plants, there is a need for improving triterpenoid production. Cytochrome P450 monooxygenases (CYPs) play critical roles in the structural diversification of triterpenoids. To perform site-specific oxidations, CYPs require the electrons that are transferred by NADPH-cytochrome P450 reductase (CPR). Plants possess two main CPR classes, class I and class II. CPR classes I and II have been reported to be responsible for primary and specialized (secondary) metabolism, respectively. In this study, we first analyzed the CPR expression level of three legumes species, Medicago truncatula, Lotus japonicus, and Glycyrrhiza uralensis, showing that the expression level of CPR class I was lower and more stable, while that of CPR class II was higher in almost all the samples. We then co-expressed different combinations of CYP716As and CYP72As with different CPR classes from these three legumes in transgenic yeast. We found that CYP716As worked better with CPR-I from the same species, while CYP72As worked better with any CPR-IIs. Using engineered yeast strains, CYP88D6 paired with class II GuCPR produced the highest level of 11-oxo-β-amyrin, the important precursor of high-value metabolites glycyrrhizin. This study provides insight into co-expressing genes from legumes for heterologous production of triterpenoids in yeast.

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.

The use of autotrophic microorganisms to fabricate biochemical products has attracted much attention in both academia and industry. Unlike heterotrophic microorganisms that require carbohydrates and amino acids for growth, autotrophic microorganisms have evolved to utilize either light (photoautotrophs) or chemical compounds (chemolithotrophs) to fix carbon dioxide (CO2) and drive metabolic processes. Several biotechnological approaches, including synthetic biology and metabolic engineering, have been proposed to harness autotrophic microorganisms as a sustainable/green production platform for commercially essential products such as biofuels, commodity chemicals, and biopolymers. Here, we review the recent advances in natural autotrophic microorganisms (photoautotrophic and chemoautotrophic), focusing on the biopolymer production. We present current state-of-the-art technologies to engineer autotrophic microbial cell factories for efficient biopolymer production.

Improving polyhydroxyalkanoate (PHA, a biodegradable plastic) production under photoautotrophic cultivation is challenging for sustainable bioproduction. In this study, we demonstrated the use of engineered nanogel particles to enhance PHA accumulation in the marine photosynthetic bacterium Rhodovulum sulfidophilum under photoautotrophic culture. We screened the effect of 13 engineered nanogel particles on the cell growth and PHA accumulation of R. sulfidophilum. The addition of anionic nanogel particles significantly enhanced PHA accumulation in R. sulfidophilum up to 157-fold compared to that without nanogel particles. By performing 13 C tracer experiments and gas chromatography−mass spectrometry analysis, we confirmed that HCO 3 − was assimilated throughout the central carbon metabolism and that the accumulated PHA was indeed incorporated from HCO 3 − . Our results indicate successful PHA production with the supplementation of engineered nanogel particles under photoautotrophic cultivation in R. sulfidophilum. Furthermore, the strategy of using engineered nanoparticles demonstrated in this study may be applicable to other microbial cell factories to produce other commodity metabolites.