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 ECHHOe, 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.
Highlights
Lupeol is a commercially important pentacyclic triterpene, which exists in a variety of vegetables, fruits and medicinal plants[1,2,3]
The metabolic flux up to oxidosqualene naturally exists in S. cerevisiae, and lupeol biosynthesis was ever successfully engineered in a yeast strain called NK2-LUP, the lupeol amount (8.2 mg l−1) yielded from that yeast strain remains pretty low[17]
With an aim to improve lupeol biosynthesis in microorganisms, this study presented the following efforts: Firstly, the three lupeol pathway genes (SQS, SE and LUP) derived from different organisms were codon-optimized based on E. coli or S. cerevisiae preference and their in vivo performance with regard to their enzymatic products was evaluated; Secondly, by recruiting the better pathway candidate genes, the lupeol pathway was reconstituted, and the utility of this recruited pathway was evaluated under two different yeast strains-WAT1118 and EPY30019
Summary
Lupeol is a commercially important pentacyclic triterpene, which exists in a variety of vegetables, fruits and medicinal plants[1,2,3]. Lupeol naturally occurs at very low levels in plant tissues[14], which has seriously limited its industrial application For these reasons, engineering lupeol production in microbes is an attractive alternative to extraction from plant sources. The metabolic flux up to oxidosqualene naturally exists in S. cerevisiae, and lupeol biosynthesis was ever successfully engineered in a yeast strain called NK2-LUP, the lupeol amount (8.2 mg l−1) yielded from that yeast strain remains pretty low[17]. With an aim to improve lupeol biosynthesis in microorganisms, this study presented the following efforts: Firstly, the three lupeol pathway genes (SQS, SE and LUP) derived from different organisms were codon-optimized based on E. coli or S. cerevisiae preference and their in vivo performance with regard to their enzymatic products was evaluated; Secondly, by recruiting the better pathway candidate genes, the lupeol pathway was reconstituted, and the utility of this recruited pathway was evaluated under two different yeast strains-WAT1118 and EPY30019. A serious bottleneck of engineering lupeol biosynthesis in E. coli was firstly discovered in this study, which would provide a valuable guidance for a prokaryotic production of lupeol in the future work
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