Combination of Genetic Manipulation Improved <i>Saccharomycopsis fibuligera</i> α-Amylase Secretion by <i>Pichia pastoris</i>

Abstract

This study assessed the combinations of genetic manipulation; signal peptide modification, gene dosage increment and co-expression of folding component, to increase Saccharomycopsis fibuligera R64 α-amylase (Sfamy) secretion in Pichia pastoris. Sfamy native signal peptide was replaced with modified signal peptide which contained 15 amino acid of mouse salivary α-amylase signal peptide fused to the pro-region of the signal peptide of Saccharomyces cerevisiae α-mating factor (α-MF). Increase in gene dosage was identified by screening for P. pastoris harboring multicopies of the Sfamy gene. Whereas, co-expression of folding component was done by addition of Protein Disulfide Isomerase (PDI). Expression plasmids harboring Sfamy containing modified signal sequence (pPICZA-MS-Sfamy) was used to transform P. pastoris GS115, and gene dosage increment was screened using zeocin. Effect of PDI co-expression on secretion levels of Sfamy was assessed by constructing the pPIC3.5K-Pdi1 plasmid and introducing into P. pastoris harboring multicopies of MS-Sfamy for expression of Sfamy. Signal peptide modification consequently increased Sfamy secretion by P. pastoris by 3.3-fold compared to native signal peptide. Gene dosage increment had improved Sfamy secretion by 11-fold in P. pastoris [MS-Sfamy] resistant to 2000 μg/mL zeocin, compared to P. pastoris harboring one copy of WT-Sfamy. Hence, PDI co-expression increased the secretion of Sfamy by 2-fold as compared without PDI co-expression. In summary, the combination of genetic manipulation successfully increased Sfamy secretion by 20-fold compared to P. pastoris harboring one copy of WT-Sfamy.

Development of synthetic biology tools to engineer Pichia pastoris as a chassis for the production of natural products

“…In addition to single-copy integration, multi-copy integration is generally demanded for high-level expression of the target proteins. The pPIC series of vectors are commonly used integrative vectors in P. pastoris [ [23] , [24] , [25] , [26] ], which enable the screening of the multi-copy integration strains under high concentration of antibiotics, a mechanism known as post-transformation amplification [ [27] , [28] , [29] , [30] ]. In addition to the formation of tandem repeats via post-transformation amplification, multi-copy strains can be constructed by integrating into the repetitive sequences of the P. pastoris genome, such as the ribosomal DNA (rDNA) sequences [ 13 ].…”

Expression of human serum albumin in Pichia pastoris protease-deficient host and conjugation with gadolinium-diethylenetriamine pentaacetate for application as a contrast agent

“…Confirmation of rHSA sequence by using the dideoxy Sanger method using universal primers for pD912 shows 100% homology to HSA sequence (Supplement material 2). The expression of HSA by P. pastoris was carried out extracellularly by using methanol as an inducer (Gaffar et al, 2019).…”

“…Pichia pastoris demonstrates a remarkably attractive host for HSA production due to several benefits of this expression system. Pichia pastoris is a widely used host for recombinant protein expression due to the fact that it is easy to manipulate, its genetic structure is stable, it has a high cell density when growing in an inexpensive basal salt medium, it has strong inducible alcohol oxidase 1 (AOX1) promoter, it has the ability to perform posttranslational modification, and it has a high capacity of secretory expression and low endogenous protein secretion (Ahmad et al, 2014;Baghban et al, 2018;Gaffar et al, 2019). Recently, Zhu et al (2018) have reported the production of rHSA in P. pastoris GS115 hosts in a shake flask and fermenter culture yielded 1.6 and 8.86 g/l, respectively.…”