Pathway and efflux engineering to utilize endogenous high isoprenoid flux in <i>Azospirillum brasilense</i> Sp7 for sesquiterpene production

ABSTRACT Sesquiterpenes, mainly produced by plants as secondary metabolites, are synthesized from farnesyl pyrophosphate (FPP, an intermediate of the carotenoid biosynthesis pathway) by sesquiterpene synthases (STPSs). The ever-increasing commercial applications have increased the demand for several sesquiterpenes, and the limitations associated with their natural extraction and chemical synthesis have shifted the interest toward heterologous sesquiterpene production using engineered microbes. However, most of the engineered microbes lack the industrial potential yield. Since a high carotenoid yield reflects an equally high flux through FPP, we developed improved carotenoid-producing strains of Azospirillum brasilense Sp7 and explored sesquiterpene production using α-humulene and (+)-valencene as two model compounds. A combination of strain construction and media optimization improved the biomass and carotenoid yield &gt;2 and ≈35-fold, respectively. The developed strains were tolerant to the exogenously added sesquiterpenes and organic solvents used for biphasic cultures. Expression of STPSs in the developed strains produced 3.2 ± 0.9 mg/L valencene and 3.25 ± 0.46 mg/L α-humulene, which was improved &gt;10-fold (up to ≈30 mg/L) by co-expressing the rate-limiting enzymes of the 1-deoxy-D-xylulose 5-phosphate pathway. Furthermore, selection and co-expression of the efflux pumps improved the yield &gt;1.5-fold (up to 50 mg/L). We also present evidence that heterologous sesquiterpenes downregulate the isoprenoid/carotenoid pathway in this bacterium. Altogether, our results indicate that A. brasilense Sp7 has evolved to carry an endogenous high-flux, squalene-mediated, and dispensable carotenoid pathway, and improvement in the carotenoid productivity improves the sesquiterpene yields up to a certain level. However, sesquiterpene-mediated downregulation of the isoprenoid pathway limits the efficient utilization of its high isoprenoid flux for sesquiterpene production. IMPORTANCE To date, several microbes have been engineered, and very few of these are being used for industrial-level sesquiterpene production. However, since most of the engineered microbes lack the industrially potential yield, and since high-yielding microbes are the industrial proprietaries, there is a need for accessible and efficient sesquiterpene-producing microbial hosts, which can be developed by engineering new microbes carrying intrinsic high isoprenoid/carotenoid flux. This study develops Azospirillum brasilense strains carrying endogenous, high flux, and dispensable carotenoid pathway and demonstrates the diversion of this flux toward the heterologous sesquiterpenes as well as the bottlenecks limiting the diversion. Although the used engineering approaches could divert only 10% of the total carotenoid flux, it highlights the bottlenecks, which can be addressed to develop this bacterium as an efficient sesquiterpene-producing microbial host. Moreover, for the first time, this work provides an insight into heterologous sesquiterpene-mediated downregulation of the native isoprenoid pathway in bacteria.