Cloning, expression in Komagataella phaffii, and biochemical characterization of recombinant sequence variants of Pseudomonas sp. S9 GDSL-esterase

Abstract

Two recombinant Komagataella phaffii (formerly Pichia pastoris) yeast strains for production of two sequential variants of EstS9 esterase from psychrotolerant bacterium Pseudomonas sp. S9, i.e. αEstS9N (a two-domain enzyme consisting of a catalytic domain and an autotransporter domain) and αEstS9Δ (a single-domain esterase) were constructed. However, only one of recombinant K. phaffii strains, namely Komagataella phaffii X-33/pPICZαestS9Δ, allowed to successfully produce and secrete recombinant αEstS9Δ enzyme outside of the host cell. The purified αEstS9Δ esterase was active towards short-chain p-nitrophenyl esters (C2–C8), with optimal activity for the acetate (C2) ester. The single-domain αEstS9Δ esterase exhibits the highest activity at 60oC and pH 9.5. In addition, the enzyme retains 90% of its activity after 3 hour incubation at 70–90oC. What should be also noted is that αEstS9Δ esterase produced in the K. phaffii expression system has a much higher specific activity (0.069 U/mg of protein) than the recombinant EstS9Δ esterase produced in an E. coli expression system (0.0025 U/mg of protein) (Wicka et al., 2016, Acta Biochim Pol 63: 117–125. https://doi.org/10.18388/abp.2015_1074).

A New Expression System Based on Psychrotolerant Debaryomyces macquariensis Yeast and Its Application to the Production of Cold-Active β-d-Galactosidase from Paracoccus sp. 32d

“…Several endogenous and heterologous signal sequences have also been used to secrete proteins in yeast expression hosts [ 6 , 20 , 22 , 23 ]. Due to the fact that the conservation among eukaryotic signal sequences is strong, the pre-pro-sequence of the S. cerevisiae α-mating factor (α-MF) is commonly used for recombinant proteins secretion in non- Saccharomyces yeasts, e.g., K. lactis [ 6 , 22 ], B. adeninivorans [ 55 ], O. polymorpha [ 55 , 64 , 65 ], and K. phaffii [ 6 , 20 , 66 , 67 , 68 ]. Moreover, the signal sequence usually comprises a Kex2 protease recognition site, which is located between the signal peptide and the heterologous protein for proteolytic maturation.…”