Keywords: natural products,Streptomyces, antibiotics, regulation, biosynthesis, formicamycins, fasamycins, two-component system

Formicamycin biosynthesis requires 24 genes expressed on nine transcripts

We previously showed that Cas9-mediated deletion of 46 kbp of DNA encompassing thefor BGC inS. formicae abolished formicamycin biosynthesis. Production was restored in theS. formicae Δfor strain by introducing the phage-derived artificial chromosome (ePAC) pESAC13-215-G, which carries the entirefor BGC plus 40–80 kbp of DNA on each side, thus proving this genomic region encodes biosynthesis of these molecules (Qin et al., 2017). Chromatin immunoprecipitation (ChIP) sequencing and cappable RNA sequencing cc (see below) suggested 24 genes form this cluster, and we confirmed this by deleting genes on each side of this 24-gene cluster contained on pESAC13-215-G and demonstrating their ability to induce formicamycin biosynthesis in theS. formicae Δfor strain. To elucidate the transcriptional organization of thefor BGC we mapped the transcription start sites (TSSs) using cappable RNA sequencing (accession number E-MTAB-7975) and identified 10 TSSs, nine of which are in intergenic regions. We thus conclude that the 24 genes required for formicamycin biosynthesis and export are expressed as nine transcripts comprisingforN,forMLK,forJ,forHI,forGF,forTSRABCD,forUVWXY,forZ, andforAA. The other TSS is located within theforV coding region and likely maintains the expression levels offorWXY. TheforX andforY gene products are required for the unique ring conversion that changes a fasamycin precursor into a formicamycin (Qin et al., 2020) (Figure 1).

Formicamycin biosynthesis is repressed by the MarR-family regulator ForJ and activated by the TCS ForGF

Thefor BGC encodes three CSRs that were predicted to regulate the expression of the transcripts required for formicamycin biosynthesis and export. There are two putative MarR-family transcriptional regulators encoded byforJ andforZ and a TCS encoded byforGF. To determine the roles of these CSRs, we made CRISPR/Cas9-mediated deletions in each of their coding genes and measured formicamycin biosynthesis in the resulting mutants. Formicamycin and fasamycin biosynthesis were abolished in the ΔforGF mutant, whereas ectopic expression of an additional copy offorGF from the native promoter led to an almost doubling (1.9-fold increase) in formicamycin biosynthesis when grown in solid culture. The loss of production caused by deletion offorGF was rescued by ectopic expression offorGF. In contrast, formicamycin production increased 5-fold in the ΔforJ mutant compared with the wild-type, with the levels of fasamycin precursors increasing more than 28-fold. This suggests that the conversion of a fasamycin to a formicamycin is the limiting step in this biosynthetic pathway (the absolute titers of fasamycins are generally lower than those of the formicamycins). Overall, the combined production of fasamycin and formicamycin metabolites increased 6.7-fold in the ΔforJ mutant grown in solid culture. Introduction offorJ cloned downstream of either the putativeforJ promoter or the constitutiveermE∗ promoter failed to complement theΔforJ mutant for reasons we cannot explain, but overexpression offorJ under control of theermE∗ promoter in the wild-type strain significantly reduced fasamycin and formicamycin biosynthesis. Deletion offorZ reduced formicamycin biosynthesis to approximately 60% of the wild-type levels (~70% of total metabolites). Taken together, these results show that formicamycin biosynthesis is activated by ForGF and repressed by ForJ, while ForZ may be involved in activation of formicamycin biosynthesis but is not absolutely required and most likely regulates the divergent transporter geneforAA. We then deleted bothforJ andforGF in combination and this resulted in a mutant that over-produced formicamycins and accumulated fasamycins, even though loss offorGF was expected to result in biosynthesis being abolished. Furthermore, as noted above, the ectopic expression of a second copy offorJ under a native promoter is enough to almost abolish biosynthesis in the wild-type strain, even in the presence of the activating TCS. These combined data suggest thatforJ sits at the top of thefor BGC regulatory network as the effects of manipulating the other CSRs are overcome by its activity (Figure 2,Table 1).

To determine how these CSRs control the expression of thefor biosynthetic genes, we generated 3x-FLAG-tagged fusion constructs to complement the deletion mutants and used ChIP sequencing to identify where these CSRs bind across theS. formicae chromosome (accession number E-MTAB-8006). The results show that ForF binds to a single site in thefor BGC at the promoter region between theforGF operon and the divergentforHI operon, and therefore likely autoregulates and controls expression offorHI. ForZ binds to a single site between the divergentforZ andforAA genes and likely acts as a typical MarR-family regulator by controlling expression of itself and the MFS transporter geneforAA. ForJ, the apparent master CSR, binds to multiple locations across the BGC. There are binding sites between the divergentforN andforMLK operons, in the intergenic regions between the divergentforHI andforGF operons, and between theforTSRABCDE andforUVWXY operons that encode the majority of the core biosynthetic machinery. These data are consistent with the observation that ForJ represses the biosynthesis of fasamycins and formicamycins by repressing the transcription of most of the biosynthetic genes. ForJ also binds to its own coding region, presumably to autorepress via a roadblock mechanism, as well as binding within the coding region offorE at the end of the longforTSRABCDE transcript. We predict that its likely function here is to act as a roadblock to prevent the RNA polymerase transcribing theforTSRABCDE operon from running into RNA polymerase transcribingforGF since this transcript is required for activation of thefor BGC (Figure 3). Only three significant enrichments occurred outside thefor BGC: ForF binds upstream ofKY5_0375, which encodes a putative NLP/P60 family protein, while ForJ binds upstream of bothKY5_3182, which encodes a putative MoxR-type ATPase, andKY5_5812, which encodes a hypothetical protein. The significance of these binding events is not known, and they were not considered further in this study.

To determine the roles of ForGF, ForJ, and ForZ in regulatingfor BGC expression, we compared the mRNA levels of allfor transcripts in the ΔforJ, ΔforGF, and ΔforZ mutants with those in the wild-type strain, with the exception offorN as no suitable primers for qRT-PCR could be found within this relatively short transcript (Figure 4). The results show that levels of all transcripts encoding core biosynthetic machinery were higher in the ΔforJ mutant compared with the wild-type strain, consistent with the hypothesis that binding of ForJ represses the expression of these transcripts. Since theforJ transcript is missing from the ΔforJ mutant, we made a transcriptional fusion between theforJ promoter andgusA (encoding β-glucuronidase [GUS]) and found that GUS activity was 2-fold higher in ΔforJ relative to the wild-type, suggesting ForJ is autorepressed (Table S1). In contrast, levels of the core biosynthetic transcripts were greatly reduced in the ΔforGF mutant compared with the wild-type strain, which is consistent with the fact that this mutant does not make fasamycins or formicamycins. Activity levels of theforG andforH promoters was also reduced in the ΔforGF mutant, suggesting ForGF auto-activates. Interestingly, levels of theforJ transcript were slightly increased in theforGF mutant, suggesting levels of repression fromforJ are higher in the absence of activation byforGF. In the ΔforZ mutant, levels of theforAA transcript were decreased more than 3-fold, suggesting that ForZ is required to activate the production of this putative transporter. Levels of some of the transcripts encoding biosynthetic machinery were also reduced in theforZ mutant compared with the wild-type strain, suggesting thatforZ may play an indirect role in activating formicamycin biosynthesis (Figure 4). Interestingly, the activity of theforZ promoter was increased in the ΔforZ mutant, suggesting that ForZ may be an example of a dual activator-repressor MarR regulator that activates expression of the divergent transporter while repressing its own transcription. Together these results show that ForJ represses the majority of the core biosynthetic genes by binding to multiple regions across thefor BGC. We also propose that ForGF is required to activate the divergentforGF andforHI promoters. TheforHI genes encode subunits of the acetyl-CoA carboxylase, which converts acetyl-CoA into malonyl-CoA, the substrate of thefor PKS, and are essential for the initiation of formicamycin biosynthesis. We hypothesize that expression of the remaining biosynthetic genes is repressed by ForJ in order to ensure biosynthesis only begins when sufficient levels of malonyl-CoA for formicamycin biosynthesis have been achieved. The results also indicate that ForZ auto-represses while activating the transporter geneforAA to ensure that compounds do not accumulate intracellularly once biosynthesis has been initiated.

BGC de-repression results in the production of additional formicamycins and induces biosynthesis in liquid culture

Wild-typeS. formicae does not produce fasamycins or formicamycins during liquid culture and production levels on solid agar are low. This limits the scope for further investigation of the antibiotic potential of these compounds since they cannot easily be purified on a large scale. As noted, the ForGF TCS is essential for biosynthesis in the wild-type, while deletion offorJ leads to 5-fold higher production of formicamycins (6.7-fold increase in combined fasamycin and formicamycins) on solid medium. We therefore expressed a second copy of theforGF operon in theΔforJ strain and found the resulting strain makes 10-fold higher levels of formicamycins on solid culture compared with the wild-type (Figure 2,Table 1). Further analysis of theΔforJ andΔforJ +forFG strains gave the surprising result that deletion offorJ induced production of the formicamycins in liquid medium. The ΔforJ mutant produced 1.5-fold more formicamycins when grown in liquid culture compared with on solid culture, although with significantly reduced levels of accumulated fasamycins; this equates to approximately the same overall production level of both sets of metabolites combined, and to an overall 7.1-fold increase in total productivity versus the wild-type strain grown on solid culture. The ΔforJ +forGF mutant was even more impressive, producing 1.6-fold the total levels of metabolites compared with the ΔforJ mutant in liquid culture and 11.8-fold more total metabolites (9.3-fold more formicamycins) than the wild-type strain grown in solid culture. By enabling production of the formicamycins during liquid culture, these mutants will facilitate fermenter-scale production to accelerate their further investigation. Deletion offorJ also induced the production of two additional formicamycin congeners (formicamycins S and R), each carrying five chlorine atoms (Figure 5) (Devine et al., 2020). Previously we had observed a maximum of four chlorination events for any congener. Both molecules exhibited potent antibacterial activity against both methicillin-sensitiveS. aureus (MSSA) and MRSA (Table 2).

Combining mutations in regulatory and biosynthetic genes results in the accumulation of additional fasamycin congeners

Our observations suggested that combining de-repression (deletion offorJ) with deletion of key biosynthetic genes would lead to strains that accumulate elevated levels of pathway intermediates and, potentially, more congeners. We previously showed that the halogenase ForV performs a gatekeeper function, with chlorination of fasamycin intermediates controlling the ability of downstream enzymes to utilize these molecules as substrates and their conversion into the formicamycin skeleton (Qin et al., 2017). We thus constructed anS. formicae ΔforJΔforV strain and found that it accumulates fasamycin C exclusively at a titer similar to that of the combined fasamycins and formicamycins produced by the ΔforJ strain in solid culture; this corresponds to a titer 90-fold higher than the total fasamycins produced by the wild-type strain (Table 1,Figure 2). This provides a powerful route for selectively producing non-halogenated fasamycin congeners, and the lack of formicamycin biosynthesis by this strain is consistent with the proposed gatekeeper function of ForV.

In a similar vein, we next made a strain lackingforJ andforX, which encodes the flavin-dependent monooxygenase ForX, the enzyme responsible for the first ring-expansion step involved in converting fasamycin precursors into formicamycins (Qin et al., 2020). The resultingΔforJΔforX strain did not make formicamycins but instead accumulated chlorinated fasamycin congeners to approximately 120 times the level of the wild-type strain (8.9-fold increase in total metabolites) when grown in solid culture (Table 1,Figure 2). Moreover, the congener profile of this strain changed considerably and six additional fasamycin congeners (L-Q) were isolated (Figure 5) (Devine et al., 2020). These include molecules carrying up to four chlorine atoms, whereas a maximum of two had previously been observed for fasamycins isolated from the wild-type strain. These congeners all displayed potent antibacterial activity against both MSSA and MRSA (Table 2).

The structures of these fasamycin and formicamycin congeners were assigned using high-resolution liquid chromatography-mass spectrometry and 2D NMR based on our published data (Qin et al., 2017). The substituent variations in chlorination andO-methylations were determined from the 2D heteronuclear single quantum coherence spectroscopy (HSQC) and¹H-¹H correlation spectroscopy (NOESY) data. Supplementary chemistry data are available here:https://doi.org/10.6084/m9.figshare.13222070.v1.

Key resources table

Resource availability

Experimental model and subject details

For details of strains used and generated in this study seeTable S3. Generally,Streptomyces strains were grown at 30°C and other organisms at 37°C, with shaking at 200 rpm for liquid cultures, unless otherwise stated (Table S3).Streptomyces spores were harvested from confluent lawns streaked from single colonies using a sterile cotton bud and stored in 1.5 ml 20% glycerol at -80°C. Glycerol stocks were made by resuspending overnight culture in 50:50 LB and glycerol (final concentration 20%). All plasmids and ePACs used in this study are described inTable S4 and all primers inTable S5. Standard DNA sequencing was conducted by Eurofins Genomics using the Mix2Seq kit (Ebersberg, Germany).

Method details

Chemicals and reagents were laboratory standard grade or above and purchased from Sigma Aldrich (UK) or Thermo Fisher Scientific (UK) unless otherwise stated. All media and solutions were made using deionised water (dH₂O) except where stated otherwise (Table S2).

Quantification and statistical analysis

In general, data were analysed in Excel and figures plotted in R using ggplot2. Details of replicates and data analysis for each experiment can be found in the figure legends.

Author contributions

R.D., H.M., Z.Q., C.A., K.N., B.W., and M.I.H. designed the research. R.D., H.M., K.N., B.W., and M.I.H. wrote the paper and all authors commented. R.D., H.M., K.N., and C.A. performed the molecular genetics experiments, H.M. and Z.Q. performed the chemical experiments. G.C. conducted the bioinformatics analysis.

Declaration of interests

The authors declare no competing interests.

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Supplementary Materials

Data Availability Statement

Sequencing data are available at EMBL-EBI (accession numbers E-MTAB-7975 and E-MTAB-8006). Other data and code are available on request via the lead contact.