Deciphering Biosynthetic Enzymes Leading to 4-Chloro-6-Methyl-5,7-Dihydroxyphenylglycine, a Non-Proteinogenic Amino Acid in Totopotensamides.

Totopotensamide A (TPM A, ) is a polyketide-peptide glycoside featuring a nonproteinogenic amino acid 4-chloro-6-methyl-5,7-dihydroxyphenylglycine (ClMeDPG). The biosynthetic gene cluster (BGC) of totopotensamides () was previously activated by manipulating transcription regulators in marine-derived SCSIO 02999. Herein, we report the heterologous expression of the BGC in TK64, and the production improvement of TPM A via in-frame deletion of two negative regulators and . The formation of ClMeDPG was proposed to require six enzymes, including four enzymes TotC1C2C3C4 for 3,5-dihydroxyphenylglycine (DPG) biosynthesis and two modifying enzymes TotH (halogenase) and TotM (methyltransferase). Heterologous expression of the four-gene cassette led to production of 3,5-dihydroxyphenylglyoxylate (DPGX). The aminotransferase TotC4 was biochemically characterized to convert DPGX to -DPG. Inactivation of led to a mutant accumulated a deschloro derivative TPM H1, and the Δi/Δi double mutant afforded two deschloro-desmethyl products TPMs HM1 and HM2. A hydrolysis experiment demonstrated that the DPG moiety in TPM HM2 was -DPG, consistent with that of the TotC4 enzymatic product. These results confirmed that TotH and TotM were responsible for ClMeDPG biosynthesis. Bioinformatics analysis indicated that both TotH and TotM might act on thiolation domain-tethered substrates. This study provided evidence for deciphering enzymes leading to ClMeDPG in TPM A, and unambiguously determined its absolute configuration as .