Engineering a carbohydrate-binding module to increase the expression level of glucoamylase in Pichia pastoris

Abstract

Background: Glucoamylase is an important industrial enzyme for the saccharification of starch during sugar production, but the production cost of glucoamylase is a major limiting factor for the growth of the starch-based sugar market. Therefore, seeking strategies for high-level expression of glucoamylase in heterologous hosts are considered as the main way to reduce the enzyme cost.

Results: ReGa15A from Rasamsonia emersonii and TlGa15B-GA2 from Talaromyces leycettanus have similar properties. However, the secretion level of ReGa15A was significantly higher than TlGa15B-GA2 in Pichia pastoris. To explore the underlying mechanisms affecting the differential expression levels of glucoamylase in P. pastoris, the amino acid sequences and three-dimensional structures of them were compared and analyzed. First, the CBM region was identified by fragment replacement as the key region affecting the expression levels of ReGa15A and TlGa15B-GA2. Then, through the substitution and site-directed mutation of the motifs in the CBM region, three mutants with significantly increased expression levels were obtained. The eight-point mutant TlGA-M4 (S589D/Q599A/G600Y/V603Q/T607I/V608L/N609D/R613Q), the three-point mutant TlGA-M6 (Q599A/G600Y/V603Q) and the five-point mutant TlGA-M7 (S589D/T607I/V608L/N609D/R613Q) have the same specific activity with the wild-type, and the enzyme activity and secretion level have increased by 4-5 times, respectively. At the same time, the expression levels were 5.8-, 2.0- and 2.4-fold higher than that of wild type, respectively. Meanwhile, the expression of genes related to the unfolded protein responses (UPR) in the endoplasmic reticulum (ER) did not differ significantly between the mutants and wild type. In addition, the most highly expressed mutant, TlGA-M7 exhibits rapidly and effectively hydrolyze raw corn starch.

Conclusions: Our results constitute the first demonstration of improved expression and secretion of a glucoamylase in P. pastoris by introducing mutations within the non-catalytic CBM. This provides a novel and effective strategy for improving the expression of recombinant proteins in heterologous host expression systems.

Keywords: Carbohydrate-binding module; Glucoamylase; Pichia pastoris; Protein expression level; Site-directed mutagenesis.

Fig. 1

Enzymatic properties of ReGa15A and TlGa15B-GA2. a Effect of pH on enzyme activity. b Effect of temperature on enzyme activity. c pH stability. d Effect of temperature (70℃) on the stability. Each value in the panel represents the means ± SD (n = 3)

Fig. 2

SDS-PAGE analysis of the recombinant ReGa15A and TlGa15B-GA2. Lane 1, 2, the culture supernatant of transformants ReGa15A and TlGa15B-GA2; lane 3, 4, the purified ReGa15A and TlGa15B-GA2

Fig. 3

Schematic diagram of the construction of the chimeric mutant. The CD sequences of TlGa15B-GA2 and ReGa15A are marked in yellow, and the CBM region is marked in green and blue, respectively. A CBM substitution. B Segment replacement. C Construction of combinatorial mutants

Fig. 4

SDS-PAGE analysis of culture supernatants among ReGa15A, TlGa15B-GA2 and mutants. A Secretion analysis of CBM substitution mutants between ReGa15A and TlGa15B-GA2. B Secretion analysis of segment replacement mutants on TlGa15B-GA2. C Secretion analysis of combinatorial mutants on TlGa15B-GA2

Fig. 5

Effect of pH and temperature on the activity and stability of purified recombinant TlGa15B-GA2 and mutants. a: Effect of temperature on the activity. b: Effect of temperature (70℃) on the stability. c: Effect of pH on the activity. d: Effect of pH on the stability

Fig. 6

Use qRT-PCR to determine the relative expression levels of WT and mutants and the relative expression of genes related to quality control of UPR and ER. CNE1: calnexin (ER chaperone); ERO1: Pdi oxidase; HAC1: UPR activated transcription factor; KAR2: ER chaperone; PDI1: protein disulfide isomerase. The experiment is performed in at least triplicate, and the error bars represent the standard deviation. The 2.^−ΔΔCT method was used to determine the relative expression, and the expression level of ARG4 in the strain was used as a reference. **: P value < 0.01 (t test)

Fig. 7

Scanning electron micrograph images of raw corn starch after incubation with TlGA-M7 at 50 °C, for A 0, B 6, C 12, and D 24 h