Multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris

Abstract

We have found a direct relationship between protein production in Pichia pastoris and the number of introduced synthetic genes of miniproinsulin (MPI), fused to the Saccharomyces cerevisiae pre-pro alpha factor used as secretion signal, and inserted between the alcohol oxidase 1 (AOX1) promoter and terminator sequences. Two consecutive approaches were followed to increase the number of integrated cassettes: the head-to-tail expression cassette multimerization procedure and re-transformation with a dominant selection marker. This increased expression from 19 to 250 mg l(-1) when about 11 copies have been integrated. Further, the correct position of one of the disulphide bridges of the purified molecule was verified by digestion with Glu-C endoprotease, followed by mass spectrometry of the isolated fragments.

Cloning and Expression of the Endo-1,3(4)-β-glucanase Gene fromPaecilomycessp. FLH30 and Characterization of the Recombinant Enzyme

“…The presence of multiple integrated copies of a desired expression cassette has been reported to be an important factor in increasing foreign protein production in P. pastoris. 31,32) In the present study, highlevel extracellular production (61,754 U mL À1 ) of PsBg16A was achieved and the highest concentration of secreted proteins (8.4 g L À1 culture) was reached after incubation of transformed P. pastoris cells with methanol for 4 d.…”

Heterologous expression of cellulase genes in natural Saccharomyces cerevisiae strains

“…Exoglucanases (cellobiohydrolases [CBHs]) progressively act on the reducing and non-reducing ends of cellulose chains to release short-chained cello-oligosaccharides that are further hydrolysed through endo-exo synergism with EGs (65). The exoglucanases disrupt the crystalline structure of cellulose and are, therefore, pivotal to the biomass conversion process (2).…”

“…Various hypotheses have been proposed to explain the mechanism of synergistic interactions, however research has shown that the extent of cellulase synergism is influenced by enzyme concentration and the nature of the lignocellulosic substrate (65), therefore synergism is dependent on the ratio of individual enzymes, the substrate saturation and the properties of the substrate (99). It has been demonstrated that the synergistic cooperation not only substantially enhances the hydrolysis content, but also dramatically reduces the required cellulase dosage (in some cases 7-fold) needed to achieve reasonable cellulose hydrolysis yield (65). In order to find a solution to the optimal ratios of different types of cellulases, Yamada and co-workers (103) developed a cocktail of cellulase gene cassettes which were introduced into the S. cerevisiae chromosome simultaneously in one step using a single selection marker and the genes T. reesei cel7B and cel7A, and Aspergillus aculeatus cel3A.…”

“…Results have suggested that higher number of target genes are effective for improving cellulolytic activity, however, it is not proportionate (65). In order to evaluate gene copy number differences between reference and superior natural transformants, episomal plasmids and δ-integration vectors were employed to generate transformants with various gene copy numbers.…”

Optimising expression of the recombinant fusion protein biopesticide ω-hexatoxin-Hv1a/GNA inPichia pastoris:sequence modifications and a simple method for the generation of multi-copy strains

“…al., 2001, Mansur et. al., 2005; with recombinant protein production increasing with increasing copy number to an optimum, after which further increasing the copy number results in decreased expression ( [13] Zhu et.…”