ABSTRACT
Plant styrylpyrones exerting well-established neuroprotective properties have attracted increasing attention in recent years. The ability to synthesize each individual styrylpyrone in engineered microorganisms is important to understanding the biological activity of medicinal plants and the complex mixtures they produce. Microbial biomanufacturing of diverse plant-derived styrylpyrones also provides a sustainable and efficient approach for the production of valuable plant styrylpyrones as daily supplements or potential drugs complementary to the prevalent agriculture-based approach. In this study, we firstly demonstrated the heterogenous biosynthesis of two 7,8-saturated styrylpyrones (7,8-dihydro-5,6-dehydrokavain (DDK) and 7,8-dihydroyangonin (DHY)) and two 7,8-unsaturated styrylpyrones (desmethoxyyangonin (DMY) and yangonin (Y)), in Saccharomyces cerevisiae. Although plant styrylpyrone biosynthetic pathways have not been fully elucidated, we functionally reconstructed the recently discovered kava styrylpyrone biosynthetic pathway that has high substrate promiscuity in yeast, and combined it with upstream hydroxycinnamic acid biosynthetic pathways to produce diverse plant-derived styrylpyrones without the native plant enzymes. We optimized the de novo pathways by engineering yeast endogenous aromatic amino acid metabolism and endogenous double bond reductases and by CRISPR-mediated δ-integration to overexpress the rate-limiting pathway genes. These combinatorial engineering efforts led to the first three yeast strains that can produce diverse plant-derived styrylpyrones de novo, with the titers of DDK, DMY and Y at 4.40 µM, 1.28 µM and 0.10 µM, respectively. This work has laid the foundation for larger-scale styrylpyrone biomanufacturing and the complete biosynthesis of more complicated plant styrylpyrones.
ABSTRACT
The samurai wasp, Trissolcus japonicus (Ashmead), has been proposed as a biocontrol agent against brown marmorated stink bugs (BMSB), due to its ability to parasitize and kill BMSB eggs. However, the wasps' small size makes it challenging for those untrained in morphological identification to determine the wasps' species. To circumvent this problem, a molecular method was created to identify T. japonicus. The method uses species-specific primers, designed in this study, which target the variable region of the mitochondrial Cytochrome Oxidase 1 (CO1) locus. After confirming successful DNA extraction from samples, the PCR amplification using our primers produced 227-bp PCR products for all T. japonicus specimens and no amplification in other microhymenoptera candidates. Additionally, DNA from BMSB-parasitized eggs gave positive PCR amplification, while the control BMSB samples showed no amplification. This indicates that PCR with our primers specifically and sensitively differentiates T. japonicus specimens from other similar wasp species and discriminates between T. japonicus-parasitized and non-parasitized BMSB eggs. Finally, an in silico analysis of CO1 sequences demonstrated that our primers match the sequences of four different haplotypes of T. japonicus, indicating that our diagnostic method could potentially be applied to analyze T. japonicus populations throughout North America, Europe, and parts of Asia.
