Cell surface display of cold-active esterase EstPc with the use of a new autotransporter from Psychrobacter cryohalolentis K5T

Abstract

We have cloned the gene coding for AT877-a new predicted member of the autotransporter protein family with an esterase passenger domain from permafrost bacterium Psychrobacter cryohalolentis K5(T). Expression of AT877 gene in Escherichia coli resulted in accumulation of the recombinant autotransporter in the outer membrane fraction and at the surface of the induced cells. AT877 displayed maximum hydrolytic activity toward medium-chain p-nitrophenyl esters (C8-C10) at 50 °C and was resistant to the presence of several metal ions, organic solvents and detergents. Previously, we have described a cold-active esterase EstPc from the same bacterium which possesses high activity at low temperatures and relatively high thermal stability. To construct a cell surface display system for EstPc, the hybrid autotransporter gene coding for EstPc with the α-helical linker and the translocator domain from AT877 was constructed and expressed in E. coli. According to the results of the cell fractionation studies and esterase activity measurements, the EstPc passenger was successfully displayed at the surface of the induced cells. It demonstrated a temperature optimum at 15-25 °C and a substrate preference toward p-nitrophenyl butyrate (C4). Obtained results provide a new example of the biotechnologically relevant enzyme from the permafrost microbial community with potential applications for the conversion of short- and medium-chain ester substrates and a basis for the construction of a new cell surface display platform.

Earth's perennially frozen environments as a model of cryogenic planet ecosystems

“…Both esterase and lipase, produced by a psychrotolerant Psychrobacter cryohalolentis K5 T isolated from Kolyma cryopegs, were shown to be active and stable over a wide temperature range from 0 to 30‐35°С. For the esterase EstPc, the level of relative activity, 85‐90%, remained essentially the same in the range between 30°С and 0°С …”

“…For the esterase EstPc, the level of relative activity, 85-90%, remained essentially the same in the range between 30°С and 0°С. [134][135][136] In the last 15 years permafrost microbiology has adopted modern molecular techniques, such as metagenomics, PCR amplification and phylogenetic identification of bacterial, archaeal and eukaryotic microorganisms. Long-term preservation of bacterial, archaeal and fungal DNA has been observed in relatively young sediments of Late Pleistocene and Holocene permafrost in the Canadian Arctic 55 and Tibet, 54,137,138 as well as in older Late Pliocene and Early Pliocene sediments in Siberia 119,126,139 and Antarctica.…”

Functional Autodisplay of Phenolic Acid Decarboxylase using a GDSL Autotransporter on Escherichia coli for Efficient Catalysis of 4-Hydroxycinnamic Acids to Vinylphenol Derivatives

“…SP GDSL and the passenger domain were replaced with a PelB signal peptide sequence and BLPAD protein in the BLPAD chimera with a PelB signal peptide ( Figure 1B), or, alternatively, only the passenger domain was replaced with BLPAD protein in the BLPAD chimera with a GDSL autotransporter signal peptide ( Figure 1C). In this GDSL autotransporter-based cell surface display system, the translated fusion protein will be transported into the periplasmic space via Sec machinery guiding the signal peptide [27]. After the signal peptide is cleaved, the fusion protein can be translocated across the outer membrane with the help of the β-barrel domain.…”

“…GDSL autotransporters have proved to be excellent vehicles for the E. coli surface display of various enzymes, particularly for members of the lipase and esterase families [27,[29][30][31]. However, not all proteins can successfully be autodisplayed on the cell surface in active forms due to limitations of the passage domains, such as molecular weight and number of disulfide bonds [32,33].…”

“…Membrane fraction isolation was performed as previously described [27]. After induction, the expressed cells were harvested by centrifugation (6000× g, 30 min, 4 • C).…”