8 General Discussion
8.2.9 Could MmfR, MMFs and MARE operators be used as a multi host efficient novel inducible expression system for GC rich
bacteria? Would this allow the purification of recombinant
proteins?
In terms of developing a novel inducible expression system, the lux vectors have certainly shown promise with high levels of expression produced clearly measureable levels of luminescence, with up to an 18 times increase in luminescence once the MMF inducer has been added (Figure 4.7). The lux genes can easily be replaced with a gene of interest to be over expressed, under the control of MmfR/MMF/MARE operator, which should hypothetically be able to be controlled in the same way as the lux genes were. Two separate GbnB analogues from S. mutans and S. enterica were inserted between the L1 intergenic region and luxCDABE in the L1 vector for use as a novel inducible expression system.
The optimisation of the inducible expression system proved to be challenging however, with little known about how to purify proteins from S. coelicolor compared to the better-known E. coli systems. In particular there were a number of challenges that were encountered when trying to develop a heterologous expression super host meaning that trials were done in S. coelicolor M145 instead, a strain which has limitations when trying to grow it in liquid culture. While optimising this system there appeared to be a good improvement in protein yield of secreted protein of the expected molecular weight when using a selection of protease inhibitors, indicating that protein degradation may have a significant impact on protein yield. There is hope that with even further optimisation and more extensive purification of the
The presence of a band of the right size for the GbnB analogues does indicate that this actinomycete system may allow the purification of recombinant proteins from GC rich bacteria. This however still needs to be confirmed with further tests on the purified proteins to identify them as the desired GbnB-like proteins, as the LC-MS analysis done was largely inconclusive.
The development of an optimised heterologous expression host was met with very limited success. It did however, help to shed some light on the cross-species promiscuity of GBL-like receptors, through the in vivo and in silico work done with S. albus. The lux system was added to S. albus with the potential of it being used as a host for the novel inducible expression system due to its very small, streamlined genome size. It was found however that luminescence for the L3+pCC4 positive control was repressed (Figure 7.1). A BLAST search for MmfR homologues found the TetR family member SSHG_01258, which shared over 40% identity with MmfR (across 86% of its sequence) and was potentially binding to the MARE operator and repressing mmyBp. No significant MmyR homologue could be found however. This indicates an extra hurdle when developing a multi-host efficient inducible expression system. TetR family members share a homologous helix-turn-helix DNA binding region which sometimes may share enough sequence identity to bind to one another’s target DNA sequences. This poses a limitation when transferring the MmfR/MMF/MARE operator expression system between hosts that also contain homologues with high sequence identity to MmfR/MmyR. Even weak binding to the MARE operator by native host receptors could be enough to considerably interfere with an inducible expression system and its regulation. Of course, with the advancing and increasing availability of many bacterial genomes, an examination could be run in potential expression hosts for MmfR homologues. However, the cut off for sequence identity that would result in these analogues binding the methylenomycin cluster MARE operators is as yet unknown.
In summary, the MmfR/MMF/MARE operator system still shows promise as an inducible expression system in streptomycetes. However, much more optimisation is needed before it could be widely used.
8.3
Impact of Data Collected
There is hope that the research presented in this thesis can shed some light on the biosynthesis of some other natural products from strains of Streptomyces that contain homologues of the MmfLHP/MmfR/MmyR system. For example, tyrosine 85 and 144 (believed be used in
also use furan molecules as ligands. Some of these homologues are from otherwise silent and less well understood pathways and so have the potential of revealing otherwise undiscovered antibiotics if manipulated in the correct way.
In particular this research has revealed more about the functioning of TetR family pairs analogous to GBL receptors and pseudoreceptors and how they have distinct roles in regulating secondary metabolism. It is apparent that whereas the GBL-like receptors often have similar and predictable mechanisms of action where they bind a hormonal ligand and then are released from a DNA operator, the paralogous ‘pseudoreceptors’ are much hard to predict the function of. Despite the knockouts of these pseudoreceptors often bringing about the same phenotype of overproduction of the cognate secondary metabolite, the mechanism by which they achieve repression appears to vary hugely. Sometimes the pseudoreceptor will only be released by a different, non-GBL ligand, possibly the cognate natural product. The findings of this thesis on the other hand, indicate that MmyR and MmfR may both bind the MMFs but this will bring about a completely different effect on MmyR, that of increased repression compared to a release of repression that these ligands bring about for MmfR. There are also quite distinct differences in the affinity of MmfR versus MmyR for the DNA operators. All this information is helpful when trying to switch on secondary metabolite production for silent gene clusters, with a broadened selection of possible alternatives to be trialled when trying to manipulate the activity of MmyR analogues.
The research into the DNA binding domains of MmfR, MmyR and their homologues (Table 7.7) revealed a number of common motifs, which could also be of relevance when manipulating TetR family binding to a chosen DNA operator. This does need further investigation but could prove to be very useful when seeking to control other biosynthetic clusters as well as engineering the novel inducible expression system. In particular, the work on MmfR/MmyR may help to better understand the regulation by SAV_2270 and SAV_2268
in S. avermitilis and SHJG_7318 and SHJG_7322 in S. hygroscopicus, of which the natural
product they regulate is unknown. These homologues shared the identical DNA binding motifs to those found in MmfR and MmyR and so possibly will reveal similar DNA binding profiles to those in the methylenomycin regulatory system studied here.