Improving lupeol production in yeast by recruiting pathway genes from different organisms

Abstract

Lupeol is a pentacyclic triterpene that shows a variety of pharmacological properties. Compared to engineering the production of sesquiterpenes and diterpenes, it is much more challenging to engineer the biosynthesis of triterpenes in microbial platforms. This study showed our efforts on engineering the triterpene pathway in Escherichia coli and Saccharomyces cerevisiae cells by recruiting the codon-optimized three lupeol pathway genes from different organisms. By comparing their activities with their respective counterparts, the squalene synthase from Thermosynechococcus elongates (tSQS), the squalene epoxidase from Rattus norvegicus (rSE) and the lupeol synthase from Olea europaea (OeLUP) were introduced into E . coli BL21(DE3), a break-through from zero was observed for lupeol biosynthesis in a prokaryotic host. We also assessed the lupeol pathway under two different yeast backgrounds-WAT11 and EPY300, and have found that the engineered strains based on EPY300, named E CHHOe , processed the best lupeol-producing ability with the maximum lupeol titer being 200.1 mg l −1 at 30 °C in a 72 h-flask culture, which so far was the highest amount of lupeol obtained by a microbial system and provides a basis for further industrial application of lupeol in the future.

Extension of cell membrane boosting squalene production in the engineered Escherichia coli

“…It suggests that gene expressions from the engineered squalene synthesis module and the MVA pathway do not impair cell growth of E. coli host, and their expression level is crucial to the improved squalene production. A peak titer of 272 mg/L was observed at culture of 48 hr, which was comparable to the achieved titer using squalene synthases of Homo sapiens and Thermosynechococcus elongatus (Katabami et al, 2015; Qiao et al, 2019). It suggested that these employed squalene synthases probably possess a similar capability to synthesize squalene.…”

“…(Figure 1a) As squalene synthase catalyzes this abrupt transition from water‐soluble FPP to highly insoluble squalene, it generally anchors to the membrane by C‐terminal tail and exposes its catalytic domain to the cytosol (D. Zhang, Jennings, Robinson, & Poulter, 1993). Membrane‐anchored squalene synthases were thus truncated to improve solubilization for squalene production (Katabami et al, 2015; Qiao et al, 2019). A truncated S. cerevisiae squalene synthase gene erg 9 ( erg 9 TC ) was designed (Figure S1) and tandemly coupled to E. coli FPP synthase gene ispA to construct the squalene synthesis module IspA‐Erg9 TC (pTispAErg9 TC ).…”

“…The eukaryotic squalene synthases are anchored to the membrane by a short C-terminal transmembrane helix. The use of truncated Erg9 (Erg9 TC ) titrated squalene of 272 mg/L, an equivalent catalytic efficiency to the engineering of H. sapiens and T. elongatus squalene synthases in E. coli (Katabami et al, 2015;Qiao et al, 2019). It was to note that expression and activity of Erg9 TC was not quantified in recombinant strain bearing pTispAErg9 TC and pSNA.…”

“…and Thermosynechococcus elongatus (Katabami et al, 2015;Qiao et al, 2019). It suggested that these employed squalene synthases probably possess a similar capability to synthesize squalene.…”

Biocatalysis in the Chemistry of Lupane Triterpenoids

“…Introducing Lotus japonicus oxidosqualene cyclase and Medicago truncatula cytochrome P450 along with its native reductase enabled the production of betulin ( 8 ) and its precursor lupeol ( 9 ). In 2019, Qiao et al [ 118 ] presented biosynthetic production of lupeol ( 9 ) in Escherichia coli and Saccharomyces cerevisiae cells by recruiting three optimized lupeol pathway genes from different organisms. The authors introduced squalene synthase from Thermosynechococcus elongates , squalene epoxidase from Rattus norvegicus and lupeol synthase from Olea europaea into E. coli BL21(DE3).…”