Effect of gene dosage and incubation temperature on production of β-mannanase by recombinant Pichia pastoris

High-level expression ofβ-mannanase has been reported in Pichia pastoris under control of the GAP promoter.Two factors that strongly influence protein production and fermentation process development in Pichia pastoris protein expression system are gene dosage and cultivation temperature.The aim of this research was to improve the expression level ofβ-mannanase in Pichia pastoris by proper increasing the gene dosage and decreasing the culture temperature.To this end,a panel of strains harboring different copy numbers ofβ-mannanase gene were obtained by multiple zeocin concentration gradients screening,the influence of gene copy number on the expression ofβ-mannanase in Pichia pastoris X33 was investigated.With the constitutive GAP promoter,the four copies strain exhibited a 4.04-fold higherβ-mannanase yield and a 1.83-fold higher total secretion proteins than the one copy strain,but an increase of the copy number above four resulted in a decrease of expression.Furthermore,the effects of culture temperature were studied in flask.The decreased culture temperature of four copies strain resulted in a 1.8-fold(26℃)and 3.5-fold(22℃)higherβ-mannanase activity compared to that at 30℃.A fed-batch strategy was successfully used for high cell-density fermentation andβ-mannanase activity reached 2124 U/mL after cultivation for 72 h in a 5 L fermenter.

High-level expression of foreign genes via multiple joined operons and a new concept regarding the restricted constant of total amount of plasmid DNA per Escherichia coli cell

OBJECTIVE: To examine the feasibility of linking operons in tandem to enhance expression of heterologous genes in Escherichia coli (E. coli) and clarify the potential control mechanism of the total plasmid DNA amount in each host cell. METHODS: Two series of expression plasmids, CW11 and CW12, containing 1 to 4 and 1 to 3 heterologous gene operon(s) respectively, were constructed. The molecular size of the CW11 series varied from 5.47 kb to 12.26 kb in 2.25 kb increments. The CW12 series varied from 5.40 kb to 9.72 kb in 2.16 kb increments. The expression level of desired protein was assayed by SDS-PAGE and laser density scanning. Plasmid copy number was determined by incorporation with (3)H-thymidine ((3)H-TdR). RESULTS: No influence of the tandem-joined operons on host growth and plasmid stability was observed. Upon induction, the desired protein accumulations in the CW11 series were 44.9% +/- 3.9%, 51.3% +/- 4.1%, 54.8% +/- 3.3% and 58.2% +/- 3.4% of total cell protein. In the CW12 series, the yields were 32.2% +/- 5.0%, 42.8% +/- 4.1% and 46.9% +/- 4.0% of total cell protein. As size increased, the plasmid copy number decreased, but target gene dosage increased significantly (P 0.05) and restricted to some extent. CONCLUSIONS: Increasing the target gene dosage by tandem linking of operons may enhance the expression level of a desired protein. Although the size (kb) and the copy number of each plasmid are negatively interrelated, for certain plasmids in each series, their total DNA amount per cell seems to be a restricted constant for specific E. coli strains under identical incubation condition.

A recent burst of gene duplications in Triticeae

Gene duplication provides raw genetic materials for evolution and potentially novel genes for crop improvement.The two seminal genomic studies of Aegilops tauschii both mentioned the large number of genes independently duplicated in recent years,but the duplication mechanism and the evolutionary significance of these gene duplicates have not yet been investigated.Here,we found that a recent burst of gene duplications(hereafter abbreviated as the RBGD)has probably occurred in all sequenced Triticeae species.Further investigations of the characteristics of the gene duplicates and their flanking sequences suggested that transposable element(TE)activity may have been involved in generating the RBGD.We also characterized the duplication timing,retention pattern,diversification,and expression of the duplicates following the evolution of Triticeae.Multiple subgenome-specific comparisons of the duplicated gene pairs clearly supported extensive differential regulation and related functional diversity among such pairs in the three subgenomes of bread wheat.Moreover,several duplicated genes from the RBGD have evolved into key factors that influence important agronomic traits of wheat.Our results provide insights into a unique source of gene duplicates in Triticeae species,which has increased the gene dosage together with the two polyploidization events in the evolutionary history of wheat.