13TSB_SynBio: Synthetic biology to improve antibiotic production

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Bacterial infections are a major cause of death world wide and antibiotics provide one key resource for controlling them.Mupirocin is a successful antibiotic used against Gram positive bacteria, particularly MRSA which is associated with bothhospital- and community-acquired infections and is resistant to most currently available antibiotics. It is also a standardtreatment to remove MRSA from the skin and nose of healthcare workers. The market for the antibiotic is growing in Chinaand other parts of the developing world and GSK wish to increase production without expanding production-plant/fermentercapacity or running costs. The Thomas group have carried out extensive research on the mupirocin biosynthetic cluster inthe soil bacterium Pseudomonas fluorescens and have studied how the genes are switched on and controlled. As a result,we have clear strategies for increasing production by manipulating the gene cluster.However, the set of genes coding for the protein factory that makes mupirocin is complex and occupies a segment of about75,000 base pairs of DNA. There are more than 30 genes in the cluster so that it is difficult to manipulate it. Syntheticbiology (building the genes from chemcially made DNA to our own design) should provide a convenient way to do this andthis project gives an opportunity to validate that idea. By rebuilding the genes we can change the code so that it isoptimised for fast and increased protein synthesis and at the same time we can split the DNA into convenient "Biobricks"(the building blocks for Synthetic Biology) which can be assembled in different orders and supplemented with additionalDNA sequences that increase the extent to which they are switched on.Confidence that the current gene cluster is not the only efficient way to configure the genes comes from our discovery thatthe genes that make a related plasmid called thiomarinol are arranged in a different order. On top of that we have foundthat increased production of the activator MupR, in the existing genetic organisation, can increase production up to 20-fold.We will therefore first introduce mutations that produce more MupR and we will then systematically insert DNA thatpromotes expression of these genes in a MupR-dependent way, to increase the productivity of each bacterium. We will usestate of the art techniques to assess the effect of these changes and see how it affects antibiotic production in shake flasksand then on a larger scale in fermenters. If successful, these changes will be incorporated into the design of the newgenes.Another feature of the gene cluster is that the order of genes is not very logical - often genes in a cluster are lined up in theway they work in the biochemical pathway. We will therefore shuffle the mupirocin gene (Biobrick) order to increasepathway efficiency and we will screen derivatives for increased production. We will do this using an enzyme (Int) thatdeliberately shuffles genes in bacteria. We will insert DNA that allows Int to work between Biobricks and then transientlyexpress Int to shuffle the genes to produce many permutations of gene order. This approach has been validated by othersand shown to improve the efficiency of the E. coli tryptophan biosynthetic operon - a well studied model system. Bacteriawill be assessed for increased production in the lab and in fermenters as above.Finally, to explore how the genes can be further improved we will add extra functional units to key modules of the pathwayto increase throughput capacity and to fuse genes to create new multifunctional genes. Gene fusions may increaseefficiency by ensuring that protein partners fold together and subsequently catalyse successive enzymic steps moreefficiently. Examples of both of these sorts of changes are found in other biosynthetic factories.