Development of simple random mutagenesis protocol for the protein expression system in Pichia pastoris

Abstract

BackgroundRandom mutagenesis is a powerful technique to obtain mutant proteins with different properties from the wild-type molecule. Error-prone PCR is often employed for random mutagenesis in bacterial protein expression systems, but has rarely been used in the methylotrophic yeast Pichia pastoris system, despite its significant advantages, mainly because large (μg-level) amounts of plasmids are required for transformation.ResultsWe developed a quick and easy technique for random mutagenesis in P. pastoris by sequential Phi29 DNA polymerase-based amplification methods, error-prone rolling circle amplification (RCA) and multiple displacement amplification (MDA). The methodology was validated by applying it for random mutation of the gene encoding cellulase from the basidiomycete Phanerochaete chrysosporium (PcCel6A), a key enzyme in degradation of cellulosic biomass. In the error-prone RCA step, the concentrations of manganese ion (Mn2+) and cellulase gene-containing plasmid were varied, and the products obtained under each condition were subjected to the second MDA step in the absence of Mn2+. The maximum error rate was 2.6 mutations/kb, as evaluated from the results of large-scale sequencing. Several μg of MDA products was transformed by electroporation into Pichia cells, and the activities of extracellularly expressed PcCel6A mutants towards crystalline and amorphous celluloses were compared with those of wild-type enzyme to identify key amino acid residues affecting degradation of crystalline cellulose.ConclusionsWe present a rapid and convenient random mutagenesis method that does not require laborious steps such as ligation, cloning, and synthesis of specific primers. This method was successfully applied to the protein expression system in P. pastoris.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0613-z) contains supplementary material, which is available to authorized users.

Crystal structure of a family 6 cellobiohydrolase from the basidiomycetePhanerochaete chrysosporium

“…Mutagenesis work performed on PcCel6A revealed several properties of noncatalytic residues of this enzyme (see Heinzelman et al, 2009;Ito et al, 2013;Tachioka et al, 2016). In our recent work, the substitution mutation W267C was found to be a critical mutation for the degradation of crystalline cellulose III I but not of amorphous phosphoric acid-swollen cellulose (Tachioka et al, 2016), which was a similar finding to the work on TrCel6A (Koivula et al, 1998).…”

“…In our recent work, the substitution mutation W267C was found to be a critical mutation for the degradation of crystalline cellulose III I but not of amorphous phosphoric acid-swollen cellulose (Tachioka et al, 2016), which was a similar finding to the work on TrCel6A (Koivula et al, 1998). Trp267 is located at the entrance to the catalytic site at subsite +4 and probably plays a specialized role in the recognition and recruitment of a single cellulose chain from the crystalline surface (Fig.…”

“…In a previous study, we cloned and recombinantly expressed PcCel6A and found that the hydrolysis rate of PcCel6A was greatly accelerated by a polymorphic change of crystalline cellulose (Igarashi et al, 2012). PcCel6A has been the target of protein engineering aimed at improving its thermostability (Heinzelman et al, 2009;Ito et al, 2013), and we have also recently applied random mutagenesis to this enzyme (Tachioka et al, 2016). Here, we report the structure of the CD of PcCel6A in its apo form and in complex with the ligand cellobiose.…”

Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins

“…Furthermore, P. pastoris is a suitable microorganism in the secretory production of recombinant proteins directly into the supernatant of the culture medium. In the P. pastoris expression system due to its limited production of endogenous secretory proteins, the purification of recombinant protein is easy (Tachioka et al, 2016).…”

Fermentation, Purification, and Tumor Inhibition of a Disulfide-Stabilized Diabody Against Fibroblast Growth Factor-2

“…Currently, several expression systems are used to produce recombinant proteins such as bacteria, yeasts and mammals ( 22 ). Compare to bacteria expression systems, yeast expression systems like Pichia pastoris are preferred tools for high expression of properly folded secretory proteins and limited production of endogenous secretory proteins ( 23 , 24 ). Moreover, they are easy to handle, less costly, and well suited for fermentation processes ( 25 ).…”