Total enzymatic synthesis of cis-α-irone from a simple carbon source

Abstract

Metabolic engineering has become an attractive method for the efficient production of natural products. However, one important pre-requisite is to establish the biosynthetic pathways. Many commercially interesting molecules cannot be biosynthesized as their native biochemical pathways are not fully elucidated. Cis-α-irone, a top-end perfumery molecule, is an example. Retrobiosynthetic pathway design by employing promiscuous enzymes provides an alternative solution to this challenge. In this work, we design a synthetic pathway to produce cis-α-irone with a promiscuous methyltransferase (pMT). Using structure-guided enzyme engineering strategies, we improve pMT activity and specificity towards cis-α-irone by >10,000-fold and >1000-fold, respectively. By incorporating the optimized methyltransferase into our engineered microbial cells, ~86 mg l−1 cis-α-irone is produced from glucose in a 5 l bioreactor. Our work illustrates that integrated retrobiosynthetic pathway design and enzyme engineering can offer opportunities to expand the scope of natural molecules that can be biosynthesized.

High-Yield Biosynthesis of trans-Nerolidol from Sugar and Glycerol

“…The transcriptional efficiencies of TM1, TM2, and TM3 promoters are about 92, 37, and 16% of that of the wildtype T7 promoter, respectively . T7 and TM1/2/3 promoters have been successfully applied in modular metabolic engineering and MHP methods to produce various high-value metabolites by systematically balancing the biosynthetic pathway. ,,, Among the nine constructs we tested, the two strains #211 and #321 produced the highest titers of nerolidol (Figure A). Details of the nine strains are shown in Tables S1 and S2.…”

Mutagenesis of Dimer Interfacial Residues Improves the Activity and Specificity of Methyltransferase for cis-α-Irone Biosynthesis

“…Similar to our previous study, BL21 cells transformed with the pMT mutant were directly grown in 10 mL of autoinduction media at 20 °C, 300 rpm. Then, the cells were collected and resuspended in 1 mL of PBS.…”

“…Three random colonies were inoculated into 1 mL of LB media with antibiotics. Cells at 0.1 OD from the overnight culture were then inoculated into 1 mL of chemically defined autoinduction media in a 20 mL HS–SPME vial . The culture was cultivated at 28 °C for 72 h and was extracted with 1 mL of ethyl acetate for GCMS analysis.…”

“…The three-dimensional structures from our previous study were used to generate models for pMT7, pMT7_Q60V, pMT12, and pMT12_Q60V. One dimeric unit was used, which was complex with 1RS for cis -α-irone and SAH.…”

“…This reaction mechanism has been harnessed to introduce structural diversity to natural products and expand the biosynthetic capacity. Many methyltransferases have been engineered to accept non-native substrates, − produce alternative products, , and introduce a different donor group. , Recently, we have tailored a promiscuous methyltransferase (pMT) to convert psi-ionone to irone, a premium flavor and fragrance molecule . By mutating the active site residues, we generated the pMT7 mutant, containing Y200F, S182E, L273V, L180A, A202L, and Y65F mutations, which was the most specific toward cis -α-irone production from psi-ionone.…”