on of C09 strain overexpressing different biosynthetic genes encoding 2-HIS and HID and relevant genetic qualities on the resultant strains. For the supply of selected plant genes: Mt, Medicago truncatula; Tp, Trifolium pretense. See Fig. 1 legend Plasmodium Compound relating to TXB2 Purity & Documentation abbreviations of other plant species. Cells had been grown within a defined minimal medium with 30 g L-1 glucose because the sole carbon source, and cultures have been sampled right after 72 h of development for metabolite detection. All information represent the mean of n = three biologically independent samples and error bars show typical deviation. The source information underlying figures (b-d) are offered in a Source Information file.CCCCThe entry point enzyme in the isoflavonoid biosynthetic pathway is 2-hydroxyisoflavanone synthase (2-HIS), which belongs for the cytochrome P450 loved ones and catalyzes the intramolecular aryl migration of the B-ring yielding the intermediate 2-hydroxyisoflavanones25. Subsequently, dehydration on the resultant intermediate items, catalyzed by 2-hydroxyisoflavanone dehydratase (HID), gives rise to corresponding isoflavones30 (Fig. 2a). The 2-HIS and HID-coding genes had been mainly identified in legumes which have been confirmedto make isoflavonoids25. To identify effective biosynthetic enzymes for DEIN formation, a group of leguminous 2-HIS and HID homologs were screened. Especially, five 2-HIS-coding genes, including Pl2-HIS, Gm2-HIS1, Mt2-HIS1 (Medicago truncatula), Tp2-HIS (Trifolium pretense), and Ge2-HIS (Glycyrrhiza echinata), and 3 HID-coding genes, including PlHID, GmHID, and GeHID, had been combined and overexpressed in strain C09 (Fig. 2d). Although most engineered strains generated detectable amounts of DEIN, strain C28, harboring the gene combination ofNATURE COMMUNICATIONS | (2021)12:6085 | doi.org/10.1038/s41467-021-26361-1 | nature/naturecommunicationsCNATURE COMMUNICATIONS | doi.org/10.1038/s41467-021-26361-ARTICLEbNADP+ NADPHa2eHOLIGOOHRPs Ge2-HISHOOO OHPEP L-Phe E4POH OHCPRssurrogate RPsOOHTriHIF FAD/FMN FMN Fe2SHO OGmHIDFAD/FMNBM3R2eNADP+GmCPRRH, ORhFREDOCANADPH, O2 NADP+, H2ODEIN FAD/FMN Fe2S2 FMNOHAtC4H AtATR2 CYBO OHNADPH FAD/FMNHemeROH, H2O ERCrCPRRhF-fdxCPRPHOp-HCAAt4CLPlant P450 reaction scheme ROH+H2O RH+O2+2e-+2H+c15 Titer (mg L-1) 12 9 six 3X Malonyl-CoAGmCHS8 GmCHS8 GmCHRp-Coumaroyl-CoAOH HO OHO O OHISOLIG By-productsGmCHI1BOGe2-HISOGmHIDOHDEIN0 2nd Ge2-HIS Redox partnerLIGNADPH, O2 NADP+, H2OTriHIFED R hFPRPR3RCBMmrCCGR37 CRCCCCFig. three Tailoring the redox partner of Ge2-HIS for effective DEIN production. a Schematic illustration from the biosynthetic pathways top for the production of DEIN and connected byproducts. P450 enzymes are indicated in magenta. Furthermore, a common catalytic mechanism of the membrane-bound plant P450 is shown within the inset. See Fig. 1 and its legend relating to abbreviations of metabolites and gene information. b Diverse redox partners (RPs) including CPR and surrogate redox partners from self-sufficient P450s were tested to enhance the catalytic activity of P450 Ge2-HIS. GmCPR1, cytochrome P450 reductase from G. max; BM3R, the eukaryotic-like reductase domain of P450BM3 from Bacillus megaterium; RhFRED, the FMN/Fe2S2-containing reductase domain of P450RhF from Rhodococcus sp. strain NCIMB 9784; RhF-fdx, a hybrid reductase by substituting Fe2S2 domain of RhFRED with ferredoxin (Fdx) from spinach. See Fig. 1 and its legend concerning abbreviations of metabolites as well as other gene facts. c Impact of different RPs around the production of DEIN. Cells wer
