on of C09 strain overexpressing distinct biosynthetic genes encoding 2-HIS and HID and relevant genetic characteristics in the resultant strains. For the supply of chosen plant genes: Mt, Medicago truncatula; Tp, Trifolium pretense. See Fig. 1 legend relating to abbreviations of other plant species. Cells were grown inside a defined minimal medium with 30 g L-1 glucose as the sole carbon source, and cultures had been sampled just after 72 h of development for metabolite detection. All data represent the imply of n = three biologically independent samples and error bars show typical deviation. The source data underlying figures (b-d) are supplied in a Supply Information file.CCCCThe entry point enzyme inside the isoflavonoid biosynthetic pathway is 2-hydroxyisoflavanone synthase (2-HIS), which belongs towards the cytochrome P450 family and catalyzes the intramolecular aryl migration from the B-ring yielding the intermediate 2-hydroxyisoflavanones25. Subsequently, dehydration on the resultant intermediate products, catalyzed by 2-hydroxyisoflavanone dehydratase (HID), gives rise to corresponding isoflavones30 (Fig. 2a). The 2-HIS and HID-coding genes had been primarily identified in legumes that have been confirmedto create isoflavonoids25. To recognize effective biosynthetic enzymes for DEIN formation, a group of leguminous 2-HIS and HID homologs had been screened. Particularly, 5 2-HIS-coding genes, such as Pl2-HIS, Gm2-HIS1, Mt2-HIS1 (Medicago truncatula), Tp2-HIS (Trifolium pretense), and Ge2-HIS (Glycyrrhiza echinata), and 3 HID-coding genes, such as PlHID, GmHID, and GeHID, have been combined and overexpressed in strain C09 (Fig. 2d). While most engineered strains generated detectable amounts of DEIN, strain C28, harboring the gene mixture 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 6 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. 3 Tailoring the redox partner of Ge2-HIS for effective DEIN production. a Schematic illustration from the biosynthetic pathways major towards the production of DEIN and associated byproducts. P450 enzymes are indicated in magenta. Moreover, a common catalytic mechanism of your membrane-bound plant P450 is shown in the inset. See Fig. 1 and its legend concerning abbreviations of metabolites and gene information. b mGluR8 custom synthesis Diverse redox partners (RPs) like 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 αvβ8 Formulation RhFRED with ferredoxin (Fdx) from spinach. See Fig. 1 and its legend with regards to abbreviations of metabolites and also other gene specifics. c Impact of distinctive RPs on the production of DEIN. Cells wer
