Sesquiterpenes from Streptomyces qinglanensis and Their Cytotoxic Activity.

Nine sesquiterpenes, including eight pentalenenes (1-8) and one bolinane derivative (9), were isolated from the culture broth of a marine-derived actinobacterium Streptomyces qinglanensis 213DD-006. Among them, 1, 4, 7, and 9 were new compounds. Their planar structures were determined by spectroscopic methods (HRMS, 1D, and 2D NMR), and the absolute configuration was established by biosynthesis consideration and electronic-circular-dichroism (ECD) calculations. All the isolated compounds were screened for their cytotoxicity against six solid and seven blood cancer cell lines. Compounds 4-6 and 8 showed a moderate activity against all of the tested solid cell lines, with GI50 values ranging from 1.97 to 3.46 µM.

A Single Active-Site Mutagenesis Confers Enhanced Activity and/or Changed Product Distribution to a Pentalenene Synthase from Streptomyces sp. PSKA01.

Pentalenene is a ternary cyclic sesquiterpene formed via the ionization and cyclization of farnesyl pyrophosphate (FPP), which is catalyzed by pentalenene synthase (PentS). To better understand the cyclization reactions, it is necessary to identify more key sites and elucidate their roles in terms of catalytic activity and product specificity control. Previous studies primarily relied on the crystal structure of PentS to analyze and verify critical active sites in the active cavity, while this study started with the function of PentS and screened a novel key site through random mutagenesis. In this study, we constructed a pentalenene synthetic pathway in E. coli BL21(DE3) and generated PentS variants with random mutations to construct a mutant library. A mutant, PentS-13, with a varied product diversity, was obtained through shake-flask fermentation and product identification. After sequencing and the functional verification of the mutation sites, it was found that T182A, located in the G2 helix, was responsible for the phenotype of PentS-13. The site-saturation mutagenesis of T182 demonstrated that mutations at this site not only affected the solubility and activity of the enzyme but also affected the specificity of the product. The other products were generated through different routes and via different carbocation intermediates, indicating that the 182 active site is crucial for PentS to stabilize and guide the regioselectivity of carbocations. Molecular docking and molecular dynamics simulations suggested that these mutations may induce changes in the shape and volume of the active cavity and disturb hydrophobic/polar interactions that were sufficient to reposition reactive intermediates for alternative reaction pathways. This article provides rational explanations for these findings, which may generally allow for the protein engineering of other terpene synthases to improve their catalytic efficiency or modify their specificities.

A dynamic and multilocus metabolic regulation strategy using quorum-sensing-controlled bacterial small RNA.

Metabolic regulation strategies have been developed to redirect metabolic fluxes to production pathways. However, it is difficult to screen out target genes that, when repressed, improve yield without affecting cell growth. Here, we report a strategy using a quorum-sensing system to control small RNA transcription, allowing cell-density-dependent repression of target genes. This strategy is shown with convenient operation, dynamic repression, and availability for simultaneous regulation of multiple genes. The parameters Ai, Am, and RA (3-oxohexanoyl-homoserine lactone [AHL] concentrations at which half of the maximum repression and the maximum repression were reached and value of the maximum repression when AHL was added manually, respectively) are defined and introduced to characterize repression curves, and the variant LuxRI58N is identified as the most suitable tuning factor for shake flask culture. Moreover, it is shown that dynamic overexpression of the Hfq chaperone is the key to combinatorial repression without disruptions on cell growth. To show a broad applicability, the production titers of pinene, pentalenene, and psilocybin are improved by 365.3%, 79.5%, and 302.9%, respectively, by applying combinatorial dynamic repression.

Renewable production of high density jet fuel precursor sesquiterpenes from Escherichia coli.

BACKGROUND: Aviation fuels are an important target of biofuels research due to their high market demand and competitive price. Isoprenoids have been demonstrated as good feedstocks for advanced renewable jet fuels with high energy density, high heat of combustion, and excellent cold-weather performance. In particular, sesquiterpene compounds (C15), such as farnesene and bisabolene, have been identified as promising jet fuel candidates. RESULTS: In this study, we explored three sesquiterpenes-epi-isozizaene, pentalenene and α-isocomene-as novel jet fuel precursors. We performed a computational analysis to calculate the energy of combustion of these sesquiterpenes and found that their specific energies are comparable to commercial jet fuel A-1. Through heterologous MVA pathway expression and promoter engineering, we produced 727.9 mg/L epi-isozizaene, 780.3 mg/L pentalenene and 77.5 mg/L α-isocomene in Escherichia coli and 344 mg/L pentalenene in Saccharomyces cerevisiae. We also introduced a dynamic autoinduction system using previously identified FPP-responsive promoters for inducer-free production and managed to achieve comparable amounts of each compound. CONCLUSION: We produced tricyclic sesquiterpenes epi-isozizaene, pentalenene and α-isocomene, promising jet fuel feedstocks at high production titers, providing novel, sustainable alternatives to petroleum-based jet fuels.