An in-cluster Sfp-type phosphopantetheinyl transferase instead of the holo-ACP synthase activates the granaticin biosynthesis under natural physiological conditions

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 Δgra-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 Δgra-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 Δgra-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.


Supplementary
Δgra-orf32 in-frame deletion of gra-orf32 This study Δgra-orf32::gra-orf32 the complementary strain of the Δgra-orf32 mutant This study Δgra-orf32Δpig-pks in-frame deletion of the minimal PKS genes (ks, clf and acp) of the predicted spore pigment in the genetic background of gra-orf32 deletion This study Δgra-orf32Δkina-likepks in-frame deletion of the minimal PKS genes (ks, clf and acp) of the predicted kinamycin-like cluster in the genetic background of gra-orf32 deletion This study Δgra-orf32Δkina-like-pksΔpig-pks in-frame deletion of the minimal PKS genes (ks, clf and acp) of the predicted spore pigment using Δgra-orf32Δkina-like-pks as a parent strain Apr r , temperature sensitive vector used for construction of the disruption plasmid of the gra-orf32 gene (Bierman et al., 1992) pYH7 Apr r , vector for construction of the disruption plasmids of the predicted kinamycin-like and spore-pigment minimal PKS genes (Sun et al., 2006) pSETAT-KasOp* Amp r , Thio r , pSET152-based and derived from the plasmid pSET-KasOp*, containing the promoter KasOp* (Pan et al., 2017) pKC-Δgra-orf32 Apr r , in-frame deletion plasmid used for construction of the Δgra-orf32 mutant  The EFI-EST and EFI-GNT tools (Zallot et al., 2019) were used to do this analysis with Gra-ORF32 used as query protein. E-value and neighbourhood size were set as 5 and l5, respectively. The retrieved clusters were mannually checked. The graphic was edited by Inkscape editor for display.

Supplementary
Supplementary Figure 10. PCR verification of complementation of the gra-orf32 gene and overexpression of other endogenous PPTase genes in the mutant strain Δgra-orf32. Lane 1, DNA marker DL2000; Lane 2, strain with introduction of the blank plasmid pSETAT-KasOp* as a control; Lanes 3 to 5, three separate clones with the desired PPTase genes under the strong promoter KasOp* as indicated on top of each electrophoregram. Vpsetat-F/Vpsetat-F were used as the primer pair.
Supplementary Figure 11. The predicted biosynthetic gene clusters of secondary metabolites in the genome of Streptomyces vietnamensis GIMV4.0001 by antiSMASH (Blin et al., 2021). Three out of 30 clusters contain type II polyketide synthases. Region 1.11 is the granaticin cluster. The most similar known clusters of regions 1.18 and 1.26 are the clusters of kinamycin and spore pigment with 51% and 83% of genes showing significant similarity, respectively.

Supplementary Figure 15. PCR verification of introduction of other exogenous
PPTase genes into the mutant strain Δgra-orf32. Lane 1, DNA marker DL2000; Lane 2, strain with of the blank plasmid pSETAT-KasOp* as a control; Lanes 3 to 5, three separate clones with the desired PPTase genes under the strong promoter KasOp* as indicated on top of each electrophoregram. Vpsetat-F/Vpsetat-F were used as the primer pair.
Supplementary Figure 16. Sequence alignment showing the Arg-and Pro-rich N terminuses of the in-cluster Sfp-type PPTases of type II PKSs. Arginine and proline are colored in sky blue. The Arg-and Pro-rich N terminuses are highlighted in red square box.
Supplementary Figure 17. Phylogenetic tree of PPTases generated by MEGA 11 (Tamura et al., 2021). PPTases from S. vietnamensis and other known PPTase were highlighted in red and pink, respectively.