Thermococcus kodakarensis as a Host for Gene Expression and Protein Secretion

Abstract

Taking advantage of the gene manipulation system developed in Thermococcus kodakarensis, here, we developed a system for gene expression and efficient protein secretion using this hyperthermophilic archaeon as a host cell. DNA fragments encoding the C-terminal domain of chitinase (ChiA⌬4), which exhibits endochitinase activity, and the putative signal sequence of a subtilisin-like protease (TK1675) were fused and positioned under the control of the strong constitutive promoter of the cell surface glycoprotein gene. This gene cassette was introduced into T. kodakarensis, and secretion of the ChiA⌬4 protein was examined. ChiA⌬4 was found exclusively in the culture supernatant and was not detected in the soluble and membrane fractions of the cell extract. The signal peptide was specifically cleaved at the C-terminal peptide bond following the Ala-Ser-Ala sequence. Efficient secretion of the orotidine-5-monophosphate decarboxylase protein was also achieved with the same strategy. We next individually overexpressed two genes (TK1675 and TK1689) encoding proteases with putative signal sequences. By comparing protein degradation activities in the host cells and transformants in both solid and liquid media, as well as measuring peptidase activity using synthetic peptide substrates, we observed dramatic increases in protein degradation activity in the two transformants. This study displays an initial demonstration of cell engineering in hyperthermophiles.

Engineering of the Hyperthermophilic Archaeon Thermococcus kodakarensis for Chitin-Dependent Hydrogen Production

“…2). TK0895 encodes a cell surface glycoprotein (Csg) that is strongly expressed in T. kodakarensis, and its promoter has previously been used to overexpress ChiAΔ4 (the C-terminal domain of T. kodakarensis ChiA) (19), endogenous pantoate kinase (27), and ␣-1,4-glucan phosphorylase from Sulfolobus solfataricus (28) in T. kodakarensis. The csg promoter was placed upstream of chiA (TK1765) in plasmid pUD3 (27) harboring pyrF used for transformant selection (see Fig.…”

“…The entire genome sequence of T. kodakarensis KOD1 has been determined (13), and gene manipulation systems based on homologous recombination and shuttle vectors have been developed (14)(15)(16)(17)(18). These have frequently been used to study gene function in vivo as well as to engineer T. kodakarensis for biotechnological purposes (10,19).…”

Polysaccharide-Degrading Thermophiles Generated by Heterologous Gene Expression in Geobacillus kaustophilus HTA426

“…In S. acidocaldarius, a heterologous gene from S. solfataricus is expressed from the mal promoter with a yield of 1 mg of purified protein per liter (32). Although the mass yields of recombinant proteins are not specified, thermophile genes are also functionally expressed from the csg and gdh promoters in Thermococcus kodakarensis (33)(34)(35)(36), the nar and slp promoters in the Gram-negative bacterium Thermus thermophilus (37)(38)(39)(40)(41)(42), and the ldh and pfl promoters in the Gram-positive bacterium G. thermoglucosidasius (5). The pheA promoter from G. thermoglucosidasius acts as an efficient promoter in E. coli (43), implying its functionality in Geobacillus spp.…”

“…Examples include protein degradation by T. kodakarensis cells genetically modified to overproduce thermostable proteases (35) and ethanol production by G. thermoglucosidasius cells that overproduce thermostable pyruvate dehydrogenase (5). The expression of heterologous genes in G. kaustophilus generated notable thermophiles capable of degrading plant biomass at high temperatures, e.g., strain MK204 to degrade cellulose paper (Fig.…”

On archaeal homologs of the human RNase P proteins Pop5 and Rpp30 in the hyperthermophilic archaeon Thermococcus kodakarensis

“…Thermococcus kodakarensis KOD1, a sulfurreducing hyperthermophilic archaeon belonging to Euryarchaeota, 4) has attracted a great deal of interest because it is one of the few archaea, in which genetic manipulations have been established. 5,6) Recently, we have described the T. kodakarensis mutant TkoΔP3, in which genetic manipulation of RNase P RNA (TkopRNA) resulted in impaired tRNA precursor processing. 7) It is thus anticipated that whole-transcriptome analysis of T. kodakarensis and TkoΔP3 will provide valuable information about the functional roles of the archaeal pre-tRNA processing ribozyme, RNase P. In this context, the correspondence of T. kodakarensis RNase P components with their counterparts of the well-studied archaeon Pyrococcus horikoshii should provide a better understanding of the structure-function relationships of archaeal RNase P. In this study, we describe biochemical and structural information about TkoPop5 and TkoRpp30, homologs of PhoPop5 and PhoRpp30 in P. horikoshii, respectively.…”