Improved Production of Recombinant Carboxylesterase FumDM by Co-Expressing Molecular Chaperones in Pichia pastoris

Abstract

Fumonisins (FBs) are mycotoxins that threaten public health and food safety worldwide. Enzymatic degradation of Fumonisin B1 (FB₁) through decarboxylation has attracted much attention, whereas application of FB₁ carboxylesterase in detoxification requires more effective expression of the recombinant carboxylesterase. In this study, the carboxylesterase FumDM from Sphingopyxis sp. ASAG22 was codon-optimized and co-expressed with five different molecular chaperones (PDI, CPR5, ERO1, HAC1, and Bip) in order to improve the expression level of FumDM in Pichia pastoris (also known as Komagataella phaffii) GS115. The co-expression of different chaperones caused varying degrees of improvement in FumDM activity for FB₁. The enzyme activities of recombinant strains over-expressing PDI and CPR5 reached the highest levels of 259.47 U/mL and 161.34 U/mL, 635% and 357% higher than the original enzyme activity, respectively. Transcriptomic analysis of the two recombinant strains in comparison with the control strain showed that the correct folding of proteins assisted by molecular chaperones played a key role in the improvement of FumDM expression and its enzyme activity. This study demonstrated that co-expression of carboxylesterase FumDM and folding chaperones was an efficient strategy and therefore might inspire new perspectives on the improvement of carboxylesterase for detoxification of FB₁.

Keywords: carboxylesterase; chaperones; detoxification; fumonisins.

Figure 1

Construction and expression of recombinant strains GS115-FumDO and GS115-FumDM. (A) Electrophoresis pattern of PCR products for FumDO and FumDM. Lane M, DL 2000 DNA Marker (2000, 1000, 750, 500, 250, and 100 bp); Lanes 1–2, PCR results for the purpose of FumDO and FumDM, respectively. (B) Enzyme activity of recombinant strains GS115-FumDO/GS115-FumDM and western blot analysis (Lane M, molecular mass marker. Lane 1, the recombinant strains GS115-FumDO. Lane 2, the recombinant strains GS115-FumDM).

Figure 2

Construction and expression of recombinant strains co-expressing of molecular chaperones (A) Electrophoresis pattern of PCR products for five molecular chaperone genes. Lane M, Trans 5k DNA Marker (5000, 3000, 2000, 1500, 1000, 800, 500, and 300 bp); Lanes 1–5, PCR results for the purpose of PDI, CPR5, ERO1, Bip and HAC1, respectively. (B) Enzyme activity of recombinant strains co-expressing of molecular chaperones and western blot analysis (Lane M, molecular mass marker. Lane 1, the FumDM without molecular chaperone co-expression. Lanes 2–6, the FumDM with PDI, CPR5, ERO1, HAC1, and Bip co-expression, respectively).

Figure 3

Gene expression in different groups. (A) Principal component analysis of samples. The color and shape of the scatter plot represented the grouping of samples. CK represented the FumDM sample without molecular chaperone co-expression. CP and PD were used to denote the FumDM co-expression with CPR5 and PDI, respectively. (B) Correlation analysis of comparison groups. CK-1, CK-2, and CK-3 were parallel samples, and the rest were numbered similarly. The color indicated the level of correlation for each sample, from low (blue) to high (red).

Figure 4

Volcano plots of DEGs in different groups. (A) The DEGs in the PD group. (B) The DEGs in the CP group.

Figure 5

KEGG analysis of the top 20 differentially regulated pathways. (A) Differentially regulated pathways in the PD group. (B) Differentially regulated pathways in the CP group.

Figure 6

Relative expression of DEGs in the two groups was verified by qRT-PCR. The horizontal coordinate was the gene name, while the vertical coordinate was the difference in ploidy of the experimental group’s genes compared with those of the control group. A difference multiplier greater than 1 indicated upregulation, and vice versa for downregulation. The expression of DEGs in PD and CP groups was illustrated by the red and blue bars. Each column represents the mean of three replicates, and the results are presented as the mean ± standard error.