Improving the autotransporter‐based surface display of enzymes in Pseudomonas putida KT2440

Abstract

Summary Pseudomonas putida can be used as a host for the autotransporter‐mediated surface display of enzymes (autodisplay), resulting in whole‐cell biocatalysts with recombinant functionalities on their cell envelope. The efficiency of autotransporter‐mediated secretion depends on the N‐terminal signal peptide as well as on the C‐terminal translocator domain of autotransporter fusion proteins. We set out to optimize autodisplay for P. putida as the host bacterium by comparing different signal peptides and translocator domains for the surface display of an esterase. The translocator domain did not have a considerable effect on the activity of the whole‐cell catalysts. In contrast, by using the signal peptide of the P. putida outer membrane protein OprF, the activity was more than 12‐fold enhanced to 638 mU ml−1 OD −1 compared with the signal peptide of V. cholerae CtxB (52 mU ml−1 OD −1). This positive effect was confirmed with a β‐glucosidase as a second example enzyme. Here, cells expressing the protein with N‐terminal OprF signal peptide showed more than fourfold higher β‐glucosidase activity (181 mU ml−1 OD −1) than with the CtxB signal peptide (42 mU ml−1 OD −1). SDS‐PAGE and flow cytometry analyses indicated that the increased activities correlated with an increased amount of recombinant protein in the outer membrane and a higher number of enzymes detectable on the cell surface.

Highly Crystalline Post‐Consumer PET Waste Hydrolysis by Surface Displayed PETase Using a Bacterial Whole‐Cell Biocatalyst

“…Both antibodies were unable to pass the bacterial outer membrane. Consequently, when monitoring the cells using flow cytometry, the observed increased fluorescence intensity verified the presence of PETase fused to the EhaA autotransporter on the cell surface (Figure S1B) [36] . A suspension of cells displaying PETase with an optical density at 578 nm (OD 578 nm ) of 6 showed an activity of 0.875 g*l −1 *h −1 on the soluble substrate BHET, in comparison to 0.015 g*l −1 *h −1 for the free enzyme at 100 nM (Figure S1C).…”

“…The PETase gene sequence from I. sakaiensis with terminal XhoI and KpnI restriction sites was codon optimized for E. coli (Sequence S1), synthesized (Geneart, Regensburg, Germany) and inserted into the maximized autotransporter expression (MATE) vector pMATE‐EstA [36] via restriction and ligation. The resulting plasmid pDG01 was used for the expression of the PETase‐EhaA fusion protein for surface display.…”

Towards Synthetic PETtrophy: EngineeringPseudomonas putidafor concurrent polyethylene terephthalate (PET) monomer metabolism and PET hydrolase expression

“…First, we used a construct based on the E. coli autotransporter EhaA. 49 This construct consists of the P. putida OprF signal peptide (37 residues), the cargo domain ( i.e ., the PET hydrolase to be displayed), a G 4 SGGS(G 4 S) 3 linker, 50 and the C-terminal EhaA autotransporter domain (488 residues). Second, we employed a construct based on the commonly used ice nucleation protein display anchors, comprising the N-terminal 203 residues fused to the C-terminal 97 residues of the ice nucleation protein inaV from Pseudomonas syringae .…”

When metabolic prowess is too much of a good thing: how carbon catabolite repression and metabolic versatility impede production of esterified α,ω-diols in Pseudomonas putida KT2440

“…In P. putida KT2440, several esterase genes have been reported, such as EstZ and EstA [ 35 ]. However, it is unclear how many esterases were active in the above cases.…”