Engineering and optimization of the 2-phenylethylglucosinolate production inNicotiana benthamianaby combining biosynthetic genes fromBarbarea vulgarisandArabidopsis thaliana

Abstract

Among the glucosinolate (GLS) defense compounds characteristic of the Brassicales order, several have been shown to promote human health. This includes 2phenylethylglucosinolate (2PE) derived from homophenylalanine (HPhe). In this study, we used transient expression in Nicotiana benthamiana to validate and characterize previously predicted key genes in the 2PE biosynthetic pathway from Barbarea vulgaris and 2 demonstrate the feasibility of engineering 2PE production. We used genes from B. vulgaris and Arabidopsis thaliana, in which the biosynthesis of GLSs is predominantly derived from HPhe and dihomomethionine, respectively. The resulting GLS profiles partially mirrored GLS profiles in the gene donor plant, but in both cases the profiles in N. benthamiana were wider than in the native plants. We found that BvBCAT4 is a more efficient entry enzyme for biosynthesis of both HPhe and dihomomethionine and that MAM1 enzymes determine the chain-elongated profile. Co-expression of the chain elongation pathway and CYP79F6 from B. vulgaris with the remaining aliphatic GLS core pathway genes from A. thaliana, demonstrated the feasibility of engineering production of 2PE in N. benthamiana. Noticeably, the HPhe-converting enzyme BvCYP79F6 in the core GLS pathway belongs to the CYP79F subfamily, a family believed to have substrate specificity towards chain-elongated methionine derivatives. Replacing the B. vulgaris chain elongation pathway with a chimeric pathway consisting of BvBCAT4, BvMAM1, AtIPMI and AtIPMDH1 resulted in an additional 2-fold increase in 2PE production, demonstrating that chimeric pathway with genes from different species can increase flux and boost production in an engineered pathway. Our study provides a novel approach to produce the important HPhe and 2PE in a heterologous host. Chimeric engineering of a complex biosynthetic pathway enabled detailed understanding of catalytic properties of individual enzymes -a prerequisite for understanding biochemical evolution -and with biotechnological and plant breeding potentials of new-tonature gene combinations.

Systematic engineering of plant cytochrome P450 system identifies a comprehensive strategy for expression of highly functional P450 enzymes inEscherichia coli

“…To validate transmembrane domain truncation as a strategy that could be applied to a broad range of P450s, we chose four additional CYP79 enzymes with diverse N-terminal sequences, distinct substrate preferences and different plant origins. CYP79A1 from Sorghum bicolor converts tyrosine into its corresponding oxime (32), CYP79B2 from A. thaliana accepts tryptophan as the sole substrate (33), CYP79D2 from Manihot esculenta acts on both valine and isoleucine (34), and CYP79F6 from Barbarea vulgaris accepts only non-canonical, carbon chain-elongated amino acids (here homophenylalanine) (35). We cloned the four CYP79s, CYP83 and ATR1 into operons where both CYP79 and CYP83 were either native full-length or with the transmembrane domain truncated.…”

Production of benzylglucosinolate by engineering and optimizing the biosynthetic pathway inSaccharomyces cerevisiae

“…This has primed a desire to engineer the production of health-promoting GLSs to enable a stable and rich source as dietary supplements for the benefit of human health. Biotechnological approaches have been used to engineer and optimize the production of the desirable GLSs (Petersen et al, 2019(Petersen et al, , 2018Wang et al, 2020).…”