2.3 CIRCADO DE VETA ANGOSTA
3.2.1 Geología regional
7.1
Aims at Strategy of Investigation
7.1.1
Existing Heterologous Expression Systems
There are a number of well-known and validated E. coli expression systems that are utilised for controlling the production of a huge variety of proteins.(102) E. coli is not always suitable for every protein of interest however, with a number of polypeptides proving to be insoluble or difficult to purify in these Gram-negative bacteria. It can be hard to predict the conditions a specific protein needs for the synthesis of an active product, with problems encountered in proteins folding into the correct conformation, poor expression levels as well as an inability to carry out the necessary post-translational modifications. It is unsurprising therefore that the same system cannot be used to achieve the successful purification of soluble proteins in all cases. Expression systems have been developed for a number of different types of organism, increasing the variety of conditions present in hosts and broadening the number of recombinant genes that can be expressed but again this does not cover all cases. The Gram- positive Streptomyces have shown promise as heterologous expression hosts, with the possibly of improved expression of genes from other GC high or Gram-positive bacteria.(139, 140) There is hope that the MmfR/MMF/MARE operator system, analogous to LacI/IPTG/lac operator, could possibly be used to provide an alternative inducible expression system for the overexpression of recombinant genes.
7.1.2
MmfR/MMF/MARE Operator as an Inducible Expression
System in Gram-Positive Bacteria
In the previous four chapters, the MmfR/MMF/MARE operator system from the methylenomycin cluster of the SCP1 plasmid of S. coelicolor has been investigated. It has shown promise as an inducible expression system in terms of promoter strength as well as removal of MmfR repression by the furans ligands. Using these findings, research has been carried out in two main areas, firstly an investigation into the choice of a Streptomyces host suitable for an inducible expression system. Secondly, preliminary work was undertaken to start to develop an inducible expression system that can be used to control the production of recombinant proteins that are otherwise difficult to obtain e.g. because they are toxic to the host.
To turn the modified lux system into a novel inducible expression system that can be adapted to study and produce recombinant proteins of interest, using a strain with a reduced genome
well as metabolically streamlining the host to conserve resources for the over production of the protein of interest. Up until now, all of the investigations into MmfR/MMF/ operator using the luciferase assay were done using S. coelicolor M145. This is a genetically reduced derivative of the wild type and model organism Streptomyces coelicolor A3(2). The M145 variant was developed via the removal of the SCP1 and SCP2 plasmids. Conveniently, all of the methylenomycin cluster, including all biosynthetic, regulatory and resistance genes are found on the SCP1 plasmid. This made M145 a suitable host strain for the luciferase reporter gene assay with no background interactions from the methylenomycin cluster being present in this strain. In particular, the absence of the native mmfR, mmyR and mmfLHP were particularly beneficial. Components of the methylenomycin cluster were added as and when needed. For the development of the novel inducible expression system, a further investigation was carried out into whether an even more streamlined host could be achieved, trialling
Streptomyces albus as a potential superior expression host.
7.2
Streptomyces albus as a Potential Host
7.2.1
Introduction to S. albus
S. albus has one of the smallest known genomes of any in the streptomycete genus at only 6.8
Mb.(27, 93) This strain provides a very interesting case study when looking at phylogenic relationships and the evolution of genetic elements due to the natural removal of any apparently unneeded genetic material from the genome. S. albus has recently started to be widely studied with the potential of it being used as a premium host for heterologous expression of natural products.(93, 141) In this report, genomic, transcriptomic and in vivo
analyses have been carried out on S. albus strain J1074 to better understand how it can be used as a super host and whether there will be any background interactions with MmfR/MMF/MARE operator from native gene expression.
7.2.2
Luminescence Assay in S. albus
To check S. albus for suitability as an expression host, the previously used luciferase reporter gene assay was transferred over to this strain. For this strain to be a suitable host for the MmfR/MMF/MARE operator inducible expression system, results collected for the lux strains created would need to be akin to those collected for S. coelicolor M145. Comparable results would indicate that the MmfR/MMF/MARE operator system works in S. albus as well as S.
coelicolor without any background interactions from existing S. albus networks.
The L3 vector (containing mmyBp) used in the earlier luciferase assay as well as mmfR and
Streptomycetes
L3+mmyR strains.1 These were then analysed via the measurement of luminescence produced using the Photek CCD camera in the same way that the S. coelicolor M145 strain was in the previous four chapters. Measurements were again taken at 21, 24, 27, 48 and 72 hours growth and the luminescence compared to a negative control strain with no luxCDABE insert and a positive control strain with no repressor (L3+pCC4). The findings of this investigation are shown in Figure 7.1 and Figure 7.2. Figure 7.1 shows the luminescence produced at the five time points over 72 hours for all samples and Figure 7.2 shows a bar chart that compares luminescence at just the 48 hour time point. A t-test analysis was then run with data from Figure 7.2, the results of which can be found in Table 7.1.
Figure 7.1. A comparison of luminescence produced by the luxCDABE operon in S. albus, as regulated by the presence and absence MmfR, MmyR and MMF4 over 72 hours
Average light production is calculated as a relative ratio of luminescence produced by the S. albus negative control with no insert (giving this sample a value of 1). Strains used: S. albus
– wild type negative control strain, S. albus L3+pCC4 - luxCDABE under the control of
mmyBp and pCC4, S. albus L3+mmfR – luxCDABE under the control of mmyBp and
mmfR under the control of ermEp* (pKMS01), S. albus L3+mmyR – luxCDABE under the control of mmyBp and mmyR under the control of ermEp* (pKMS03)
1 This nomenclature is the same as that which was used for the equivalent investigation in
0 10 20 30 40 50 60 70 80 0 20 40 60 80 100 120 140 Time (hours)
Ratio of luminescence produced
Luminescence produced by different S. albus strains
S. albus no insert S. albus L3:pCC4 S. albus L3+mmfR S. albus L3+mmyR S. albus L3+pCC4 MMF4 S. albus L3+mmfR MMF4 S. albus L3+mmyR MMF4
Figure 7.2. Bar chart comparing luminescence produced by the luxCDABE operonin S. albus, as regulated by the presence and absence MmfR, MmyR and MMF4 at 48 hours
Average light production is calculated as a relative ratio of luminescence produced by the S. albus negative control (giving this sample a value of 1). Strains used: same as Figure 7.1
Table 7.1. A t-test analysis of the luciferase assay results collected from S. albus data at 48 hours
Average light production is calculated as a relative ratio of luminescence produced by S. albus with no insert (giving this sample a value of 1). The p-value was also calculated based on S. albus with no insert. Strains used: same as Figure 7.1
Strain p-value difference? Significant production at 48 Average light hr (R.R)
S. albus L3+pCC4 5.06E-01 FALSE 0.82
S. albus L3+mmfR 8.27E-01 FALSE 0.95
S. albus L3+mmyR 8.64E-12 TRUE 77.07
S. albus L3+pCC4 MMF4 9.87E-01 FALSE 1.01
S. albus L3+mmfR MMF4 2.27E-02 TRUE 2.25
S. albus L3+mmyR MMF4 1.32E-13 TRUE 97.72
It was found that the MmfR/MMF/MARE operator inducible expression system in S. albus
was not comparable to that in S. coelicolor M145 indicating that S. albus is not a suitable heterologous expression host for this particular expression system. As can be seen in both Figure 7.1 and Figure 7.2, the S. albus L3+pCC4 positive control and L3+mmfR both appear to have almost entirely repressed levels of luminescence. The L3+mmyR strain on the other hand produces high levels of luminescence both in the presence of the MMFs and without. The findings of the t-test in Table 7.1 show that there is a significant increase in luminescence produced by the L3+mmfR strain in the presence of MMF4 but this is only minimal with
S. albus
no insert L3+pCC4S. albus L3+S. albusmmfR L3+S. albusmmyR L3+pCC4 S. albus MMF4 S. albus L3+mmfR MMF4 S. albus L3+mmyR MMF4 0 50 100 150 200
Streptomycetes
This is still nowhere near close to the levels of luminescence produced by S. albus L3+mmyR
or the levels of induction seen for the equivalent system in S. coelicolor.
A possible interpretation as to why the L3+pCC4 and L3+mmfR strains produce no luminescence is that a protein from the S. albus genome could be causing repression at the MARE operator sequence. The L3+mmyR strain still produces high levels of luminescence and the conclusion inferred from this is that MmyR can bind genetic elements in the S. albus
genome and thereby repress the expression of this native TetR that might otherwise bind the MARE operator.
Where it is possible that a native protein from S. albus is binding to the MARE operator sequence and preventing the expression of the luciferase genes, it appears unlikely that this protein also has the correct binding pocket for the MMFs and for this reason the addition of 100 µM MMF4 causes little or no induction of lux expression. This potential native S. albus
TetR, homologous to MmfR/MmyR, is discussed further in the following paragraphs.
It is also necessary to consider that, despite the plasmid inserts in S. albus being checked by PCR and the ex-conjugants gaining the selective apramycin and hygromycin resistance from the vectors inserted, it is still entirely possible that the inserted genes are not being expressed properly in S. albus. As mentioned previously, S. albus is known to have a streamlined genome due to genetic reshufflings and deletions of ‘unnecessary’ genes, this is something which may have occurred to the L3, pKMS01 and pKMS03 inserts after they had been screened by PCR. In hindsight it may have been helpful to run extra screenings of the ex- conjugants during and after the luciferase assays to check for maintenance of the insert. Until this has been investigated further therefore, the analyses just discussed should be studied with caution.