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.
Highlights
Random mutagenesis is a powerful technique to obtain mutant proteins with different properties from the wild-type molecule
We have developed a novel, rapid random mutagenesis strategy that enables the P. pastoris expression system to be used for directed evolution of eukaryotic proteins, despite its low transformation efficiency
This method is composed of sequential plasmid amplification by means of error-prone rolling circle amplification (RCA) and multiple displacement amplification (MDA) of the error-prone RCA products, followed by protein expression in P. pastoris (Fig. 1)
Summary
Random mutagenesis is a powerful technique to obtain mutant proteins with different properties from the wild-type molecule. P. pastoris is able to secrete heterologous proteins directly into the culture medium, which enables easy screening of mutant libraries and simplifies downstream purification Despite these advantages, P. pastoris has rarely been used in random mutagenesis [2], mainly because the transformation efficiency is usually several orders of magnitude lower than that for E. coli and other yeasts [6]. Microgram amounts of plasmids are required for integration into the P. pastoris genome via homologous recombination, and expression plasmids are often designed as shuttle vectors to enable rapid amplification in bacterial cloning hosts [7] This technique is feasible for low-throughput analyses, but is too time-consuming for high-throughput experiments because of the need for thousands of bacterial transformations and plasmid isolations. There are only a few established methods of random mutagenesis in this host [8,9,10,11]
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