Concluding Remarks

Polyketide synthases (PKSs) are enzymes that synthesize a biologically important family of complex chemicals called polyketides, which include antibiotics, anticancer, and immunosuppressant drugs. Due to their highly complex architectures, polyketides are difficult to synthesize via traditional organic chemistry means. Through bioengineering, scientists are able to genetically modify PKSs to selectively produce polyketides of interest such as adipic acid, methyl ketones, and chiral building blocks. However, commonly used heterologous hosts for PKS production, such as Escherichia coli and Streptomyces, often produce misfolded, truncated, and non-functional PKSs or they can be challenging to genetically manipulate and grow slowly. In response to this need, we seek to develop Mycobacterium smegmatis as a new PKS host due to its more easily manipulated genome, availability of genetic tools, relatively fast growth rate, and ability to naturally produce precursors for biosynthesis. To assay for phosphopantetheinylation, an essential post-translational modification of PKSs made by phosphopantetheinyl transferase (PPTase), we introduced the gene bpsA, which encodes a blue-pigment indigoidine synthetase, into wild type M. smegmatis. We found that wild type M. smegmatis produced indigoidine, which indicates the presence of an active PPTase. Furthermore, we genomically integrated a second copy of a PPTase under the control of a constitutive promoter to improve the ability of the host to phosphopanteheinylate heterologous PKSs and integrated T7 RNA polymerase to make the host compatible with IPTG inducible T7 promoter expression systems. Additionally, we developed an IPTG inducible T7 promoter expression plasmid, containing a his-tagged RFP under an inducible T7 promoter for protein purification. Flow cytometry was used to study the inducibility of the system by measuring fluorescence levels of induced and non-induced M. smegmatis containing the T7 expression plasmid, further providing evidence that these genetic tools will be a useful platform for purifying PKSs or for fermentative production of polyketides.