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Bacterial aromatic polyketides are mainly biosynthesized by type II polyketide synthases (PKSs). The PKSs cannot be functional unless their acyl carrier proteins (ACPs) are phosphopantetheinylated by phosphopantetheinyl transferases (PPTases). Gra-ORF32 was identified as an in-cluster PPTase dedicated for granaticin biosynthesis in Streptomyces vietnamensis and the Arg- and Pro-rich N terminus was found to be crucial for catalytic activity. Overexpression of the encoding genes of the holo -ACP synthases of fatty acid synthases (FAS ACPSs) of both E. coli and S. vietnamensis could efficiently activate the production of granaticins in the Δ gra-orf32 mutant, suggesting the ACP of granaticin (graACP) is an efficient substrate for FAS ACPSs. However, Gra-ORF32, the cognate PPTase of the graACP, could not compensate the conditional deficiency of ACPS in E. coli HT253, indicating that it has evolved to be functionally segregated from fatty acid biosynthesis. Nine out of eleven endogenous and all the tested exogenous non-cognate PPTases could activate the production of granaticins to varied extents when overexpressed in the Δ gra-orf32 mutant, indicating that ACPs of type II PKSs could also be widely recognized as effective substrates by the Sfp-type PPTases. The exogenous PPTases of type II PKSs activated the production of granaticins with much higher efficiency, suggesting that the phylogenetically distant in-cluster PPTases of type II PKSs could share substrate preferences for the ACPs of type II PKSs. A significantly elevated production of granaticins was observed when the mutant Δ gra-orf32 was cultivated on ISP2 plates, which was a consequence of crosstalk between the granaticin pathway and a kinamycin-like pathway as revealed by transcriptome analysis and pathway inactivations. Although the host FAS ACPS could efficiently activate the production of granaticins when overexpressed, only Gra-ORF32 activated the efficient production of granaticins under natural physiological conditions, indicating that the activity of the host FAS ACPS was strictly regulated, possibly by binding the FAS holo -ACP product with high affinity. Our findings would contribute to a more comprehensive understanding of how the ACPs of type II PKSs are activated and facilitate the future functional reconstitutions of type II PKSs in E. coli ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Deng, Chik, Li and Zhu.)

Granaticins are benzoisochromanequinone polyketides with remarkable antibacterial and anticancer activities. Three sulfur-containing granaticin congeners, mycothiogranaticins A ( 1 ), B ( 2 ) and granaticin MA ( 3 ) were discovered from a granaticin-producing strain of Streptomyces vietnamensis GIMV4.0001. Two of them were structurally determined with mycothiol or N-acetylcysteine moieties and found to be bio-actively reluctant. Disruption of the mshA gene ( SVTN_RS20640 ) that encodes the D-inositol-3-phosphate glycosyltransferase crucial for mycothiol biosynthesis, fully abolished the production of mycothiogranaticins. The result substantiated that the newly discovered mycothiogranaticins are consequences of the combination of the granaticin and mycothiol biosynthetic pathways. The overall granaticin production of the Δ mshA mutant strain was unexpectedly decreased by at least more than 50%, while similar production level of granaticins to that of the wild type strain was observed in an mycothiol-S transferase gene ( SVTN_RS22215 ) disruptant Δ mst . These results indicated that the mycothiol deficiency was responsible for the decreased production of granaticins. Mycothiol may positively regulate the biosynthesis of granaticin possibly by maintaining the cellular redox balance. To the best of our knowledge, this is the first report that mycothiol can not only be a direct building block of polyketides but also play a regulatory role in the polyketide biosynthesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Deng, Li, Luo, Zheng, Chen, Zhang, Zhang, Zhou and Zhu.)