Construction of an Artificial Biosynthetic Pathway for the Styrylpyrone Compound 11-Methoxy-Bisnoryangonin Produced in Engineered Escherichia coli

Abstract

A cDNA clone (named pnpks), which shows high homology to the known chalcone synthase (CHS)-like type III PKS, was obtained from the leaves of Piper nigrum. The PnPKS protein with ferulic acid catalyzed lactonization instead of chalcone or stilbene formation. The new product was characterized as a styrylpyrone, 11-methoxy-bisnoryangonin, which is the lactonization compound of a linear triketide formed as the reaction product of PnPKS protein with ferulic acid. These results show that pnpks encodes a styrylpyrone synthase (SPS)-like PKS that catalyzes two-chain elongation with feruloyl CoA-linked starter substrates. Although these styrylpyrone compounds are promising for use in human healthcare, they are mainly obtained by extraction from raw plant or mushroom sources. For de novo synthesis of 11-methoxy-bisnoryangonin in the heterologous host Escherichia coli from a simple sugar as a starter, the artificial biosynthetic pathway contained five genes: optal, sam5, com, and 4cl2nt, along with the pnpks gene. The engineered L-tyrosine overproducing E. coli ∆COS1 strain, in which five biosynthetic genes were cloned into two vectors, pET-opT5M and pET22-4P, was cultured for 24 h in a minimal glucose medium containing ampicillin and kanamycin. As a result, 11-methoxy-bisnoryangonin production of up to 52.8 mg/L was achieved, which is approximately 8.5-fold higher than that in the parental E. coli strain harboring a plasmid for 11-methoxy-bisnoryangonin biosynthesis. As a potential styrylpyrone compound, 11-methoxy-bisnoryangonin, was successfully produced in E. coli from a simple glucose medium, and its production titer was also increased using engineered strains. This study provides a useful reference for establishing the biological manufacture of styrylpyrone compounds.

Construction of an Artificial Biosynthetic Pathway for Zingerone Production in Escherichia coli Using Benzalacetone Synthase from Piper methysticum

“…23 The ferulic acid biosynthetic gene cluster has already been used to successfully produce some ferulic acid-derived compounds in E. coli, particularly in L-tyrosine-overproducing E. coli ΔCOS1. [23][24][25]32 In addition, we used an engineered E. coli strain (ΔCOS4), generated by deleting bioC on the ΔCOS1 strain. 22 BioC is believed to be an O-methyltransferase that methylates the free carboxyl of either malonyl-CoA or malonylacyl carrier protein.…”

“…coli, particularly in l -tyrosine-overproducing E. coli ΔCOS1. − , In addition, we used an engineered E. coli strain (ΔCOS4), generated by deleting bioC on the ΔCOS1 strain .…”

“…Quantification data were generated from independent experiments performed in triplicate. For LC/MS analysis, the samples were dissolved in methanol and analyzed by electrospray ionization (ESI) on an UltiMate U3000-LTQ XL linear ion trap mass spectrometer system (Thermo Fisher Scientific), under conditions reported by us previously . Two microliters of the sample was injected into an HSS T3 C18 column (150 × 2.1 mm; 2.5 μm particle size) from Waters; the mobile phase used for gradient elution consisted of CH 3 CN (0.1% formic acid) as system A and H 2 O (0.1% formic acid) as system B.…”

De novo biosynthesis of diverse plant-derived styrylpyrones in Saccharomyces cerevisiae

“…Our heterologous fermentation results indicate that the low efficiencies of PmSPS1 and PmKOMT1 are the major bottleneck hindering efficient styrylpyrone production. Discovery and functional expression of enzymes from other plants might solve this problem: Equisetum arvense styrylpyrone synthase 3 ( Beckert et al, 1997 ), Humulus lupulus chalcone synthase ( Okada et al, 2004 ) Neonothopanus nambi hispidin synthase ( Kotlobay et al, 2018 ), and Piper nigrum polyketide synthase ( Heo et al, 2021 ) that have been reported to catalyze the formation of styrylpyrone scaffolds are potential candidates for future investigations; A. zerumbet that exhibits high DDK productivity might provide novel styrylpyrone synthases and O -methyltransferases. In addition, the development of computational protein design has opened up the opportunity of designing novel enzymes from a known template ( Planas-Iglesias et al, 2021 ).…”

“…By exploiting de novo transcriptome assembly, phylogenomics, and expression analysis, a recent work has identified the genes encoding SPS and downstream tailoring enzymes (methyltransferases catalyzing methylation at multiple sites, including the C4 site that distinguishes plant and fungal styrylpyrones, and oxidoreductases catalyzing the reduction of the 5,6-olefin and the formation of the C11-C12 methylenedioxy bridge) from the kava plant for the first time ( Pluskal et al, 2019 ). An SPS-like enzyme, PnPKS, has also been reported from the black pepper ( Piper nigrum ) transcriptome, catalyzing feruloyl-CoA into 11-methoxy-bisnoryangonin ( Heo et al, 2021 ). Although the discovery of the core styrylpyrone pathway genes in plants has laid the foundation for the engineered biosynthesis of plant styrylpyrones heterologously, microbial production of plant-specific styrylpyrone has not been achieved: while yangonin and methysticin can be synthesized in the model plant Nicotiana benthamiana via transient gene co-expression ( Pluskal et al, 2019 ), microbial production of plant-specific styrylpyrones has never been reported.…”