MARCO TEÓRICO

To our current knowledge, the putative GBL-dependent signalling systems have not been investigated and the exploitation of regulation systems is expected to unlock the production of novel natural compounds.

In 7 of the GBL-dependent gene clusters, the MEME enrichment analysis successfully identified ARE sequences upstream of putative transcriptional activators (highlighted in green in Table 4-4). The specific overexpression of pathway-specific transcriptional activator is expected to increase expression of neighbouring cryptic biosynthetic gene clusters. Furthermore, in the partially characterised GBL-dependent regulatory systems of SCB/ScbR/ScbR2 in S. coelicolor and SVB/JadR3/JadR2 in S. venezuelae, the deletion of specific transcriptional repressors have resulted in the production of compounds from cryptic biosynthetic

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gene clusters; for instance, the deletion of scbR2 and jadR2 "pseudo" GBL receptors in S. coelicolor and S. venezuelae unlocked the production of coelimycin and jadomycin respectively. 121,148 _{To identify specific TetR repressor to inactivate, a} phylogenetic analysis of all of the TetR repressors in the AHFCA/GBL dependent signalling systems were carried out (Figure 4-5).

The phylogenetic tree reveals 4 main clades of receptors (Figure 4-5). Other “smaller” clades were also observed in the phylogenetic tree (Figure 4-5) and were primary found divergently adjacent to biosynthetic genes; the exact biological function or the cognate ligand for these TetR repressors are not known.

The “cognate AHFCA binding receptor” clade consists of the analogous MmfR repressors and have been experimentally confirmed to bind to AHFCA signalling molecules (Malet, N. Zhou, S., Styles, K. and Fullwood, A. Personal communication). In the ““pseudo” AHFCA binding receptor” clade, which includes the paralogous MmyR repressors, in silico analysis and early investigations have shown the MmyR repressors do not bind to the same AHFCA signalling molecules (Styles, K. Personal communication).

The deletion of "psuedo" AHFCA binding receptors in S. coelicolor and S. venezuelae

resulted in the overproduction of methylenomycin and gaburedins respectively and hence, the deletion of MmyR orthologues is also expected to result in a similar phenotype in other cryptic biosynthetic gene clusters (Figure 4-5). 57,62

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Figure 4-5 – Phylogenetic analysis reveals the TetR repressors are clustering into

four clades. TetR repressor protein sequences were aligned using MUSCLE and phylogenetic tree was generated using maximum likelihood method. Bootstrap consensus tree was inferred from 1000 iterations.

“pseudo” AHFCA binding receptor Cognate AHFCA binding receptor “pseudo” GBL binding receptor Cognate GBL binding receptor SVEN 4187 M878 26365 IG96 RS0135515 HMPREF1211 06615 SAV 2268 SHJG 7322 KCH 13290 SCP1.246 KCH 13230 SCP1.242c M878 26345 SAV 2270 HMPREF1211 06613 SHJG 7318 IG96 RS0135495 SVEN 4182 M271 07490 SCO6265 SFUL 272 SSEG 02734 SVEN 5968 BN159 2280 DC74 7322 BN159 4439 SCAB 12091 M878 11425 ArpA SHJG 8651 Sfla 6322 DC74 7608 SHJG 4003 SCAB 12101 SSEG 02731 SFUL 269 SCO6286 M878 11395 SVEN 5972 BN159 2278 SHJG 8654 Sfla 6319 HMPREF1211 06623 SFUL 260 SFUL 258 SFUL 262 SVEN 6001 TetR 95 93 93 98 75 74 70 66 64 57 43 40 42 35 62 89 44 34 29 28 28 28 25 64 23 23 24 62 79 40 45 36 22 19 24 18 70 67 21 17 32 15 55

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Intriguingly, the M271_07490 repressor from S. rapamycinicus also falls into the “cognate AHFCA binding receptor” clade (Figure 4-5); this implies that the M271_07490 repressor share similar properties with MmfR and may interact with AHFCA-like signalling molecules. To determine whether M271_07490 could bind to AHFCA-like signalling molecules, the key amino acid residues required for binding with the cognate ligand were compared with analogous MmfR repressors. Co-crystallisation of MmfR with MMF2 has revealed Y85 and Y144 are directly involved in hydrogen bonding with the cognate ligand (Figure 4-6) and these amino acid residues are not present in the analogous MmyR repressors (Dean., R. et al. Manuscript in preparation).

All of the MmfR analogues contain the conserved Y85 and Y144 residue, except for SHJG_7318, which has a phenylalanine residue instead of tyrosine at position 144. The M271_07490 repressor contains phenylalanine in both of the key amino acid positions and suggests M271_07490 may also bind to AHFCA-like signalling molecules. Of particular interest is that the cryptic biosynthetic gene cluster in

S. rapamycinicus does not contain analogous mmfH or mmfP genes; therefore, the signalling system in S. rapamycinicus is proposed to be dependent on a novel class of signalling molecule. Alternatively, the cryptic biosynthetic gene cluster could cross-talk with other Streptomyces bacteria that produce AHFCA signalling molecules to coordinate the biosynthesis of specialised metabolites.

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Figure 4-6 – Clustal Omega alignment of the cognate AHFCA binding receptor clade.

Residues proposed to be involved in hydrogen bond with the cognate AHFCA ligand are highlighted in pale red.

There are also two separate clades for the GBL binding receptors (Figure 4-5). Based on the TetR repressors in jadomycin and coelimycin biosynthetic gene clusters, the ““real” GBL binding receptor” were reported to bind to GBL signalling molecules, whilst the ““pseudo” GBL binding receptor” do not bind to the same GBL molecules, but to structurally diverse metabolites, such as actinorhodin, undecylprodigiosin and jadomycin. 98,99,117,139 _{In addition, the deletion of sco6286, which encodes for the} ScbR2 “pseudo” binding receptor, resulted in the production of coelimycin in

S. coelicolor. 115,121 _{Therefore, the deletion of these specific “pseudo” GBL receptors} in putative GBL-dependent regulatory systems is expected to unlock the production of compounds from cryptic biosynthetic gene clusters.

Some of the putative GBL-dependent signalling systems contain a varied number of TetR repressor genes; for example, the putative GBL-dependent signalling systems in S. albulus, S. davawensis and S. rapamycinicus appears to be under the control of a single TetR repressor whilst S. fulvissimus contains five TetR repressors. From the predictions of ARE sequences, the transcriptional regulation of these cryptic biosynthetic gene clusters could function differently and novel mechanisms could have evolved. M271_07490 VVEEHFATWPPLIERF-20-AFRDDIVVRAGARLWAERTLIEAPMPPPFVGWID KCH_13230 VVEALYATWPRTLEAV-20-AFLNDPIMQAGTRLQNERDVIDVELPLPYVDWTT SCP1.242c VVEEHYARWPAAMEEI-20-AFRDDPVMQAGARLQSERAFIDAELPLPYVDWTH M878_26345 VVEAHYSRWPELLEGI-20-AFATDIVVQAGARLQLERSLIDAQLPQPYVGWTQ SAV_2270 VLEEFYRRMQEAVNGA-21-AFHEDVFIHAGARLQIERPYIKAELPVPYVGTLK SHJG_7318 VTTEFYKRLPAISDTV-20-ALRDEPMMKAGARLQIERGLIDADLPIPFQDYTE HMPREF1211_06613 VAEEFYHVLGVIGQEV-20-AFRDDTMVQAGARLQIERSLIGAEMPMPYLGYTE IG96_RS0135495 VIMEHYARWEPLVSEV-20-AFRDDVMVQAGARLQIERSLIKADLPVPYVGWQE SVEN_4182 VVQEHYARWPEILKGA-20-AFARDIVVQAGARLQIERALIDAELPEPYVGWED * : . *: : .::**:** ** * . :* *:

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4.4. Conclusions

The GBL-dependent signalling systems are widely distributed in Streptomyces

bacteria; in fact, the genome analysis of 20 sequenced Streptomyces bacteria identified 36 cryptic biosynthetic gene clusters proposed to be under the control of putative GBL-dependent signalling systems. To test the effectiveness of the MEME enrichment analysis at discovering TetR binding sites, the bioinformatic analysis was performed on 36 gene clusters.

From the 36 gene clusters, the MEME enrichment analysis was successful at discovering the ARE sequences for 18 biosynthetic gene clusters. The ARE sequences were identified in the promoters of putative transcriptional activators and TetR repressors; the deletion of specific “pseudo” ArpA-like transcriptional repressors or overexpression of pathway-specific transcriptional activators is expected to unlock the production of novel compounds from cryptic biosynthetic gene clusters.

The SCB/ScbR/ScbR2 system, which is involved in regulating coelimycin biosynthesis, has been extensively studied by global transcriptomic analysis and ChIP-seq. MEME enrichment analysis was unable to identify all of the experimentally confirmed binding sites and is speculated to be due to the strict nature of the searching algorithm.