rocess improvement; and (5) Sigma 1 Receptor Biological Activity fine-tuning of gene expression inside the competing metabolic pathways. The systematic engineering enabled the production of 85.4 mg L-1 DEIN from glucose in shake flask cultivations. Ultimately, in the course of application phase III, we demonstrated the efficient conversion of DEIN to bioactive glycosylated isoflavonoids by introducing plant glycosyltransferases. Supplementary Fig. 2 delivers an overview of all strains constructed in the different phases of your development method. Results Phase I–Establishing the biosynthesis of scaffold isoflavone DEIN. In plants, the general phenylpropanoid pathway makes use of the aromatic amino acid (AAA) L-phenylalanine as a precursor for the biosynthesis of isoflavonoids too as other flavonoids24. The initial steps engage phenylalanine ammonia lyase (PAL), cinnamic acid 4-hydroxylase (C4H), and 4-coumarate-coenzyme A ligase (4CL), resulting within the conversion of L-phenylalanine to p-coumaroyl thioester. Subsequently, the chalcone precursors, naringenin chalcone (NCO) and deoxychalcone isoliquiritigenin (ISOLIG), are synthesized in the condensation of p-coumaroyl CoA and three molecules of malonyl-CoA by chalcone synthase (CHS) alone or with all the co-action of NADPH-dependent chalcone reductase (CHR), respectively25. Chalcone isomerase (CHI) is responsible for the additional isomerization of chalcone to flavanone26. When naringenin (NAG) acts as the shared structural core in isoflavone GEIN and flavonoids pathways, the flavanone liquiritigenin (LIG) is used for the biosynthesis of isoflavone DEIN. The efficient generation of LIG represents hence the first step towards developing a yeast platform for generating DEIN. To facilitate the screening of biosynthetic enzymes for LIG production, we used a yeast platform strain (QL11) which has previously been reported to generate a moderate degree of p-coumaric acid (p-HCA) (exceeding 300 mg L-1) from glucose devoid of notable development deficit27. The plant candidate genes have been chosen in accordance with their supply and enzymatic specificity/ activity. We initially evaluated the combinations of candidate CHS, CHR, and CHI homologs, alongside the well-characterized At4CL1 from Arabidopsis thaliana, for the biosynthesis of LIG (Fig. 2a). Especially, three CHS-coding genes, which includes leguminous GmCHS8 (Glycine max) and PlCHS (Pueraria lobate) as well as non-leguminous RsCHS (Rhododendron simsii), had been chosen (Supplementary Fig. 3a). CHR activity has been largely demonstrated in leguminous species28; hence GmCHR5, PlCHR, and MsCHR (Medicago sativa) were screened (Supplementary Fig. 3a). Plant CHIs may be categorized into distinct isoform groups based on their evolutionary path and enzymatic profiles. Whereas kind I CHIs, frequent to all vascular plants, convert only NCO to NAG, legume-specific type II CHIs are capable of yielding each NAG and LIG26. Correspondingly, type II CHI-coding genes PlCHI1 and GmCHI1B2 have been evaluated, collectively using a kind I CHI-coding gene PsCHI1 (Paeonia suffruticosa) being employed as a control for enzymatic activity. All biosynthetic genes have been chromosomally integrated and transcriptionally controlled by sturdy constitutive MMP-13 Species promoters. Cooverexpression of At4CL1, GmCHR5, GmCHS8, and GmCHI1B2 resulted inside the ideal LIG production at a level of 9.8 mg L-1 (strainNATURE COMMUNICATIONS | (2021)12:6085 | | nature/naturecommunicationsNATURE COMMUNICATIONS | IIGlu