CRONOGRAMA DE TESIS

The production of a heterologous expression super host was investigated by adding the

luxCDABE/mmfR system to the genetically streamlined Streptomyces albus host (93) to

establish whether it would be suitable to use for the inducible expression system. This then lead onto an investigation of other alternative streptomycete hosts as well as a literature review of current expression systems.

Following on from this, MmfR/MMF/MARE operator interactions were optimised so they could be adapted for use as a novel inducible expression system. Vectors were designed for the overexpression of a gene of choice to be trialled in this system and a protocol developed for the collection of recombinant proteins from S. coelicolor. An explanation of why a novel inducible expression system is needed can be found in Section 1.5.3.

1.5.2

Choice of Reporter System Used

There are a number of reporter systems available and it was necessary to choose a suitable one for answering the research questions in this project. Unfortunately a number of reporter systems are not suitable for use in GC rich bacteria such as Streptomyces. The lacZ system for example, is a usually easy and sensitive reporter system and is widely used. Unfortunately, streptomycetes have an enzyme that has beta-galactosidase activity and therefore interferes with lacZ expression making it an unsuitable reporter system.(51) Efforts to produce

Streptomyces knockout of this beta-galactosidase enzyme have often been unsuccessful and

results in poorly growing colonies.

A beta-glucorinidase system (94) has been used to study the regulation of virginiamycin biosynthesis by BarA and its cognate ligand VB from S. virginiae (Section 1.2.2) in tobacco plant cells in 2006.(95) This system was then later developed for use in actinomycetes, showing promise as a viable reporter system for these bacteria.(96) This is a colorimetric assay however and therefore limited in the quantitative results that can be obtained. It was therefore decided that it would be best to utilise a reporter system that produces more quantitative results such as fluorescence or bioluminescence.

The gpf system has been trialled in Streptomyces with a degree of success. Streptomycetes will auto-fluoresce in blue light, limiting the range of colours that can be used to study different components of a system but otherwise appears to be fairly successful in these Gram- positive bacteria. The disadvantage of the technique however, is that it tends to photo-bleach rapidly, limiting the genes it can be used to study as well as posing problems if multiple readings need to be taken.(92)

A luxAB system from Vibrio harveyii has shown also a lot of potential for use in

streptomycetes. The downside of this system is that luxAB only produce the enzyme luciferase and therefore there is the need to add a substrate to produce luminescence. This substrate may not pass through all bacterial cell walls with the same efficiency and may disrupt cell growth if overlaid onto a solid culture.(92) For this reason, the full luxCDABE

system, which was optimised for GC high bacteria by Justin Nodwell and his team in Canada, (92) was used to investigate MmfR, MmyR, the MMFs and MARE operators further. LuxA and LuxB together form the heterodimeric luciferase protein whereas LuxC, D and E are the enzymes needed to form tetradecanal, the fatty aldehyde substrate of luciferase.(97) All other biochemicals required by this reaction are found naturally in bacterial cells allowing a self generated bioluminescent response with no need for external manipulations.(98) A diagram of

Figure 1.11. Arrangement of open reading frames in the luxCDABE reporter system and the assigned functions of individual components of this operon

This image was taken directly from the 2009 paper by Lin et al., (99)

1.5.3

Why Do We Need A Novel Inducible Expression System?

Currently, there are a number of commercially available inducible expression systems. Famous examples of these are the vectors regulating gene expression via the lac operon, inducible upon the addition of IPTG. IPTG is a lactose mimic that is not enzymatically broken down like lactose and so remains at a constant level, this will bind to the lac repressor (LacI) and cause its release from DNA thereby allowing the expression of a gene of interest.(100)

Specificity of suitable expression hosts is little understood with each protein requiring a slightly different set of optimal expression conditions.(101) Many current systems are largely based in the Gram-negative Escherichia coli due to its fast growth, ease of culturing and well understood uses as a ‘cell factory’.(102) It is possible to optimise current expression systems to some extent to improve the expression of heterologous genes. For example genomic GC content varies widely across bacteria, ranging from anything between under 20% GC content to over 70% and so codon usage is also a key factor when designing recombinant genes.(103) There are a number of proteins that still cannot be efficiently over produced and purified using existing methods however. This can be due to a variety of complications such as physiological conditions not being suitable for the correct folding of the protein, low expression levels or because the host cannot carry out the required post-translational modifications.(101) Alternative expression systems have been developed to those in E. coli

including systems based in yeast, other bacteria and fungi as well as those for mammalian cells.(104) These hosts will all provide slightly different conditions for protein expression, which may prove optimal for some proteins, but again these systems again cannot express all genes. There is no universal heterologous expression host. There is hope that the

provide an alternative expression system for the overexpression of recombinant genes in the Gram-positive Streptomyces species. This system would hopefully be useful in the production of proteins currently not possible in existing systems.

One benefit of using streptomycetes as an expression host is their high innate protein secretion capacity.(101, 105) This has the advantage of an increased chance of the protein folding properly (106) as well as a reduced requirement for expensive purification techniques. This is therefore something that would be beneficial to include in the expression system being designed in this project.

In recent years there have been a number of systems developed for heterologous expression in streptomycetes that have shown promise. For example, the work by Noda et al. in 2015 showed great success with the production of streptavidin from a streptomycete host.(107) Streptavidin is originally from Streptomyces avidinii so seems logical therefore that it is expressed better in these GC high bacteria as conditions are likely to be closer to the native conditions needed for streptavidin production. The work by Noda et al. resulted in the production of a much more thermostable streptavidin product compared to those produced by

E. coli systems, thereby expanding the potential applications of streptavidin-biotin

interactions. Not all streptomycete expression systems produce a high protein yield however and much optimisation is needed. An example of the type of optimisation done includes the work by Wilkinson et al. who investigated improving expression systems in actinomycetes based on optimising promoters. This lead to 100 times more product than when using than using wild type promoters.(108) Despite these successes however, there a still many instances where a heterologous protein cannot be purified from streptomycetes and so novel inducible expression systems are still very much in demand and it is for this reason that an additional inducible expression system is being developed in this project.

1.6

Outline of Thesis Structure

Chapter two specifies all of the stock solutions and protocols used to obtain results for this thesis as well as specifics on the source of all consumables used. Included in this section are details on primers used as well as lists of vectors and strains created. Further information on how these techniques developed based on experimental findings can also be found throughout the following research chapters.

Chapter three presents the optimisation of the luxCDABE reporter gene system for GC high bacteria for use in studying the interactions between MmfR (and paralogue MmyR) with the

vectors for this method and how these come together to create an arrangement that can be adapted to study different aspects of the regulation of methylenomycin biosynthesis. In addition, results from the investigation into the strength of different promoters in the methylenomycin biosynthetic cluster are reported within this chapter.

Chapter four further expands on this luxCDABE reporter system specifically looking at MmfR as a transcriptional repressor. This chapter is divided into two main sections, the first looking at MmfR/MARE operator interactions and how binding varies at the three operators. The second is an investigation into MmfR/MMF interactions and includes details on all five methylenomycin furan ligands and their binding potentials to MmfR as well as an investigation into the MmfR ligand binding pocket and the production of mutants that were then also tested using the luciferase assay.

Chapter five follows on from the investigation into MmfR, this time looking at its paralogue MmyR. Again both interactions with the MARE operator and the MMF ligands were investigated. Due to the functionality of MmyR being different to MmfR, this chapter then goes in a slightly different direction, investigating other possible ligands for this second repressor rather than studying key residues in ligand binding.

Chapter six further explores the self-regulatory mechanism of MmfR, also using the luciferase assay. Investigations were carried out into the differences in MmfR repression and release when it is under the control of its own promoter. This chapter also briefly examines the potential of MmyR auto-regulation. This chapter is concluded with a proposed mechanism, combining the function of MmfR/MmyR in the regulation on methylenomycin biosynthesis, based on all of the investigative findings up until this point.

Chapter seven is the final investigative chapter and summarises all of the findings of chapter three and four to develop a novel expression system for use in GC rich bacteria, utilising MmfR/MMF/MARE operator interactions to induce transcription. This chapter first looks at the potential of creating an optimised streptomycete expression host followed by details on the creation of vectors for this novel expression system as well as preliminary trials into using it with S. coelicolor as a heterologous expression host.

Chapter eight and nine then discuss and conclude all of the findings from the previous five chapters as well as commenting on the implications of this work in wider research and explaining the possible future work that could be carried out.