Production of biotechnology products using Thermus expression systems

With the aim of use T. thermophilus HB27 as a production host, improved expression vectors and host strains are needed and the functional expression of heterologous genes should be demonstrated. Two new reporter genes whose products can be conveniently and quantitatively detected were developed to study gene expression in T. thermophilus HB27: β-galactosidase and malate dehydrogenase. These are the first reporter genes suitable for studying gene expression at temperatures as high as 85°C. The β-gal and mdh reporter genes were used to study three promoters. The inducible promoter PdnaK (regulating DnaK heat shock inducible protein induced by shifting the incubation temperature from 70 to 85°C), a carbon regulated promoter, Pscs-mdh (regulating expression of succinyl-coA and malate dehydrogenase) and an arginine-inducible promoter Parg (regulating expression of an unknown arginine-inducible protein) were cloned upstream of the β-gal and/or mdh genes in pTEX series expression vector. The PdnaK promoter was shown to undergo 2-fold induction upon heat shock at 85°C. The Pscs-mdh promoter was shown to vary 3-fold depending upon the carbon sources and/or nutrients present. Complex medium yielded low activity while the highest activity observed with Pscs-mdh was minimal medium with malate as the carbon source. The Parg promoter was maximally induced by the presence of 10mM arginine but was repressed at higher arginine concentrations.; In order to use Thermus as an in vivo protein engineering host, two mesophilic enzymes, dibenzothiophene monooxygenase (DszC) responsible for the first step in the biodesulfurization pathway which catalyzes the oxidation of dibenzothiophene (DBT) to dibenzothiophene sulfone (DBTSO₂) and p-nitrobenzyl esterase (PNBE) catalyzing hydrolysis of several β-lactam antibiotic p-nitrobenzyl (PNB) esters to the corresponding free acid and PNB alcohol were studied. The dszC and pnbA genes from Rhodococcus erythropolis and Bacillus subtilus respectively were cloned into Thermus sp. expression vector (pGTI-TEX-1) resulting in pGTI-TEX-1 dszC and pGTI-TEX-1 pnbA, respectively and introduced into Thermus thermophilus HB27. Both growing cells and crude cell extracts of T. thermophilus HB27/pGTI-TEX-1 dszC were capable of metabolizing DBT to DBTSO₂ at 55°C and thermostable PNBE was stably produced from T. thermophilus HB27/pGTI-TEX-1 pnbA host at 65°C. These results indicate that Thermus sp. can be used as production hosts in industrial biotechnology processes, especially processes that can benefit from operating at thermophilic temperatures.