Identification of SARP family regulators involved in milbemycin biosynthesis in S. bingchenggensis
Li et al developed a highly efficient transcriptional repression system by CRISPR-ddCpf1 in S. bingchenggensis (2018). For SARP family regulators usually involved in biosynthesis of secondary metabolites, all the 19 SARP candidates of S. bingchenggensis were tested for their effect on milbemycin production by this ddCpf1-based CRISPRi system. The milbemycin A3/A4 production of all mutants were listed in Fig. 2a. Notably, the titer of milbemycin A3/A4 in TMB/pSETddCpf1-milR3 (targeting the sbi_06842,sbi_06842 was designed as milR3 for its effect on the production of milbemycin) was significantly decreased by 87.3% compared to that of TMB/pSETddCpf1.
Phylogenetic relation of these SARPs with several well-studied SARP family regulators showed that these putative SARPs could be divided into three groups (Fig. 2b). MilR3 was close to three well-studied SARP family regulatory proteins: SrrZ from S. rochei, FarR3 from Streptomyces lavendulae and BulZ from Streptomyces tsukubaensis (Kurniawan et al. 2014; Ma et al. 2018; Suzuki et al. 2010). SrrZ and FarR3 have been proved as specific-pathway positive regulators of their adjacent biosynthetic gene clusters. BulZ, encoding gene located in GBL gene cluster, influenced tacrolimus production through regulation of the biosynthesis of GBL by directly binding to the upstream region of bulS2 (GBL synthetase). Interestingly, milR3 is located 7.16 MB from the mil cluster (sbi_00726 - sbi_00790) and adjacent to a putative type II PKS genes (sbi_06843 - sbi_06846 encoding minimal PKS synthetase). MilR3 probably was a unique SARP regulator in S. bingchenggensis.
MilR3 is a pleiotropic regulator in S. bingchenggensis
The milR3 in-frame deletion mutants (designed as △milR3) were constructed on basis of the parental strain S. bingchenggensis TMB with CRISPR-Cpf1 system. The △milR3 mutant was further confirmed by PCR and DNA sequencing (Figure S1). The complemented plasmid pSET152-milR3, in which milR3 was cloned in the integrative plasmid pSET152 and its expression was driven by its native promoter, was constructed and then transferred into the △milR3 mutant and TMB, resulting in the complemented strain CmilR3 and the overexpression strain OmilR3. As controls, the plasmid pSET152 was introduced into △milR3 and TMB generating DmilR3 and TMB/pSET152, respectively. Milbemycin A3/A4 production of strains TMB/pSET152, DmilR3, CmilR3 and OmilR3 were presented in Fig. 3a. Milbemycin A3/A4 production was abolished in DmilR3 and can be restored in CmilR3. These results clearly demonstrated that MilR3 played as an indispensable activator for milbemycin production in S. bingchenggensis TMB. Milbemycin A3/A4 production in OmilR3 was slightly lower than that of TMB/pSET152(Fig. 3a).
Interestingly, a yellow pigment produced in S. bingchenggensis TMB was abolished in DmilR3 strain and restored in CmilR3 strain (Fig. 3b). The yellow pigment production patterns of these four strains were similar both under liquid fermentation and on solid agar plates (Fig. 3c). It seemed that more yellow pigment was produced in the milR3 overexpression strain. For the milR3 was adjacent to a putative type II polyketide gene cluster, we proposed that this gene cluster was responsible for production of the yellow pigment.
Obviously, the SARP family protein MilR3 acted as a pleiotropic regulator to modulate not only milbemycin biosynthesis but also the yellow pigment biosynthesis in S. bingchenggensis TMB.
The type II PKS was responsible for the biosynthesis of yellow pigments
Four genes (sbi_06843, sbi_06844, sbi_06845 and sbi_06846) neighboring to milR3 were predicted to encode putative type II PKS (Fig. 7). The 3297-bp internal fragment in the putative type II PKS encoding genes (from sbi_06843 to sbi_06845) was deleted by CRISPR-Cpf1 system in S. bingchenggensis TMB (Figure S2). This mutant was named as Dsbi_06844., Dsbi_06844 was incapable to produce the yellow pigment on MS plate (Fig. 4a). This situation was in consistent with DmilR3, indicating that the putative type II PKS encoding genes are responsible for the yellow pigment biosynthesis. The milbemycin A3/A4 yield of Dsbi_06844 strainwas nearly same to that in TMB/pKCCpf1 (Fig. 4b). Meanwhile, the transcription of milbemycin biosynthesis and type II PKS cluster were analyzed through qRT-PCR. The transcriptional time course pattern of milA2, milD, milE, milF, milR and milR3 in Dsbi_06844 were similar to that in TMB/pKCCpf1 (Fig. 4c). These results demonstrated that the yellow pigment was uncoupled with milbemycin biosynthesis. And the biosynthesis of yellow pigment was regulated by MilR3.
MilR3 regulates the transcription of targeted genes involved in milbemycin biosynthesis and the yellow pigment biosynthesis
The dynamic transcription of milbemycin and yellow pigment biosynthetic genes of was investigated by quantitative real-time PCR (RT-qPCR) with time course RNA samples of TMB/pSET152, DmilR3, CmilR3 and OmilR3 strain fermentation culture (2 day, 4 day, 6 day and 8 day). The results showed that transcription of milA1, milA2, milA3, milA4, milC, milD, milE, milF andmilR were almost undetectable in DmilR3, but restored in CmilR3 compared with those of TMB (Fig. 5). Transcription of sbi_06844 in DmilR3 was expressed extremely lower than that in TMB/pSET152, and restored in CmilR3. And the time course transcription patterns of tested genes were all similar. These results indicated that MilR3 functions as an indispensable activator for both milbemycin and yellow pigment biosynthetic gene cluster.
However, the transcription of milR2,aprevious reported TetR family activator involved in 5-oxomilbemycin A3/A4 biosynthesis was increased in DmilR3. And its transcription level can restore in the complementary strain CmilR3 (Fig. 5). This indicates that milR3 had negative effect on the transcription of milR2.
The milbemycin biosynthesis was regulated by MilR3-MilR cascade
MilR has been reported as a LAL family (large ATP-binding regulator of the LuxR family) pathway-specific transcriptional activator of milbemycin biosynthesis through specifically binding the promoters of the milA4-milE operon and of milF with its C-terminal HTH domain (Zhang et al. 2016). The transcription of representative genes involved in milbemycin and yellow pigment biosynthesis was investigated by qRT-PCR analysis of MilR overexpression strain OmilR (TMB strain carried an integrative plasmid pSET152 containing milR driven by a strong constitutive kasOp* promoter) and wildtype strain in fermentation culture. The transcriptional level of milR was up-regulated in OmilR at each time points. The transcription of previously reported target genes of MilR also showed same tendency in the OmilR strain. The milbemycin A3/A4 production in OmilR was increased by 29% compared with that of TMB/pSET152, which was consistent with the results of qRT-PCR (Fig. 6a).
Notably, the expression level of milR3 in OmilR was similar to that in TMB/pSET152 (Fig. 6b). It was indicated that transcription of milR3 was not affected by milR. Probably the milbemycin production was regulated through MilR3 to MilR cascade.