Abstract
BackgroundMicrobial production of monoterpenes provides a promising substitute for traditional chemical-based methods, but their production is lagging compared with sesquiterpenes. Geraniol, a valuable monoterpene alcohol, is widely used in cosmetic, perfume, pharmaceutical and it is also a potential gasoline alternative. Previously, we constructed a geraniol production strain by engineering the mevalonate pathway together with the expression of a high-activity geraniol synthase.ResultsIn this study, we further improved the geraniol production through reducing the endogenous metabolism of geraniol and controlling the precursor geranyl diphosphate flux distribution. The deletion of OYE2 (encoding an NADPH oxidoreductase) or ATF1 (encoding an alcohol acetyltransferase) both involving endogenous conversion of geraniol to other terpenoids, improved geraniol production by 1.7-fold or 1.6-fold in batch fermentation, respectively. In addition, we found that direct down-regulation of ERG20 expression, the branch point regulating geranyl diphosphate flux, does not improve geraniol production. Therefore, we explored dynamic control of ERG20 expression to redistribute the precursor geranyl diphosphate flux and achieved a 3.4-fold increase in geraniol production after optimizing carbon source feeding. Furthermore, the combination of dynamic control of ERG20 expression and OYE2 deletion in LEU2 prototrophic strain increased geraniol production up to 1.69 g/L with pure ethanol feeding in fed-batch fermentation, which is the highest reported production in engineered yeast.ConclusionAn efficient geraniol production platform was established by reducing the endogenous metabolism of geraniol and by controlling the flux distribution of the precursor geranyl diphosphate. The present work also provides a production basis to synthesis geraniol-derived chemicals, such as monoterpene indole alkaloids.
Highlights
Microbial production of monoterpenes provides a promising substitute for traditional chemical-based methods, but their production is lagging compared with sesquiterpenes
During S. cerevisiae fermentation, Oye2p is the main enzyme involved in the conversion of geraniol to citronellol, and Atf1p is the main contributor to synthesis of terpenyl acetates from geraniol [27]
Geraniol was produced at 285.9 mg/L by YZG14 with OYE2 deletion, which was improved by 1.7-fold compared with the control strain (YZG13-GE1), and 259.8 mg/L was produced by YZG15 with ATF1 deletion, which was improved by 1.6-fold compared with the control strain (YZG13-GE1) (Fig. 2a)
Summary
Microbial production of monoterpenes provides a promising substitute for traditional chemical-based methods, but their production is lagging compared with sesquiterpenes. We constructed a geraniol production strain by engineering the mevalonate pathway together with the expression of a high-activity geraniol synthase. The universal precursors of isoprenoids, isopentenyl diphosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), are derived from both pathways. Engineering microbial organisms for monoterpene synthesis provides a potential effective route for their production. Given its low yield in aromatic plants, geraniol has been successfully synthesized in Escherichia coli and Saccharomyces cerevisiae through metabolic engineering strategies [7,8,9,10,11]. In S. cerevisiae, the ERG20 gene of the endogenous MVA pathway encodes a farnesyl pyrophosphate synthase (FPPS) that, despite having both GPP and FPP synthase activity, only releases a very low amount of GPP from its catalytic site [20]. Ignea et al demonstrated that the double mutant Erg20p (F96W-N127W) had a strong dominant negative ability to decrease the FPPS function of Erg20p, increasing
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