Codon optimization of Bacillus licheniformis β-1,3-1,4-glucanase gene and its expression in Pichia pastoris

Teng, Da; Fan, Ying; Yang, Yalin; Tian, Zhigang; Luo, Jianxun; Wang, Jianhua

doi:10.1007/s00253-006-0765-z

Cited by 73 publications

(47 citation statements)

References 42 publications

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“…To obtain high-purity and high-yield expression of secreted CysC protein, we chose the pPIC9K expression vector and the P. pastoris GS115 strain as the host to express CysC, since a number of studies have concluded that many proteins produced in P. pastoris are similar in structure and bioactivity to the native proteins produced in mammalian cells (Ouchkourov et al, 2002;Hu et al, 2006;Teng et al, 2007;Fu et al, 2011;Huang et al, 2012;Yang et al, 2012). Moreover, the pPIC9K vector has an α-factor secretion signal sequence, which has been used with great success.…”

Section: Discussionmentioning

confidence: 99%

Effect of codon optimization on expression levels of human cystatin C in Pichia pastoris

Li¹,

Li²,

Xu³

et al. 2014

Genet. Mol. Res.

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ABSTRACT. Human cystatin C (CysC) is a cysteine proteinase inhibitor with many potential applications. To facilitate further studies of the functions and applications of CysC, we improved the heterologous expression of CysC using a basic codon optimization method. In this study, we cloned the high-GC content wild-type sequence of the CysC gene and also designed a slightly AT-biased sequence, with codons optimized for expression in the Pichia pastoris GS115 strain. Our results showed that the optimized coding sequence of human CysC increased the expression and secretion of the CysC protein by approximately 3-to 5-fold (90-96 mg CysC/L) in yeast, compared with the expression levels of the native CysC gene (17.9-18.4 mg CysC/L). We designed, Codon optimization increases CysC expression in P. pastoris constructed, and applied an optimized version of the CysC gene for the Pichia expression system. Our results demonstrate that the optimized coding sequence provides a higher yield of secreted CysC than that produced using the wild-type gene. Our data also serve as a practical example demonstrating a rational design strategy for the heterologous expression of secreted proteins.

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Section: Discussionmentioning

confidence: 99%

Effect of codon optimization on expression levels of human cystatin C in Pichia pastoris

Li¹,

Li²,

Xu³

et al. 2014

Genet. Mol. Res.

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“…To date, many studies have been carried out to improve its activity and performance by DNA recombinant techniques (Chen et al, 2001;Teng et al, 2007;Li et al, 2009). However, little attention has been paid to the practicability and safety of the application of β-1,3-1,4-glucanase in the food industry.…”

Section: Discussionmentioning

confidence: 99%

“…Traditionally, β-1,3-1,4-glucanase is made by separation and purification from natural sources, such as Penicillium emersonii, Aspergillus niger, Bacillus subtilis, and Trichoderma reesei (Harman and Kubicek, 1998).microorganisms, including Lactococcus lactis , Escherichia coli (Teng et al, 2006;Qiao et al, 2009), Pichia pastoris (Teng et al, 2007), and Saccharomyces cerevisiae (Hinchliffe and Box, 1984;van Rensburg et al, 1997;Zhang Q. et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

A food-grade industrial arming yeast expressing β-1,3-1,4-glucanase with enhanced thermal stability

Guo

Zhang

et al. 2010

J. Zhejiang Univ. Sci. B

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Abstract:The aim of this work was to construct a novel food-grade industrial arming yeast displaying β-1,3-1,4-glucanase and to evaluate the thermal stability of the glucanase for practical application. For this purpose, a bi-directional vector containing galactokinase (GAL1) and phosphoglycerate kinase 1 (PGK1) promoters in different orientations was constructed. The β-1,3-1,4-glucanase gene from Bacillus subtilis was fused to α-agglutinin and expressed under the control of the GAL1 promoter. α-galactosidase induced by the constitutive PGK1 promoter was used as a food-grade selection marker. The feasibility of the α-galactosidase marker was confirmed by the growth of transformants harboring the constructed vector on a medium containing melibiose as a sole carbon source, and by the clear halo around the transformants in Congo-red plates owing to the expression of β-1,3-1,4-glucanase. The analysis of β-1,3-1,4-glucanase activity in cell pellets and in the supernatant of the recombinant yeast strain revealed that β-1,3-1,4-glucanase was successfully displayed on the cell surface of the yeast. The displayed β-1,3-1,4-glucanase activity in the recombinant yeast cells increased immediately after the addition of galactose and reached 45.1 U/ml after 32-h induction. The thermal stability of β-1,3-1,4-glucanase displayed in the recombinant yeast cells was enhanced compared with the free enzyme. These results suggest that the constructed food-grade yeast has the potential to improve the brewing properties of beer.

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“…The yield of the recombinant β-glucanase was higher than those by previous studies. A β-1,3-1,4-glucanase from Bacillus licheniformis was expressed with a yield of 256.7 U/mL in P. pastoris (Teng et al 2007) and a truncated β-1,3-1,4-glucanase from Fibrobacter succinogenes was produced with a yield of 1,940 U/mL in P. pastoris (Wen et al 2005). The characterization of the recombinant β-1,3-1,4-glucanase was studied in a detail.…”

Section: Characterization Of the Recombinant β-13-14-glucanasementioning

confidence: 99%

Codon optimization, expression and characterization of Bacillus subtilis MA139 β-1,3-1,4-glucanase in Pichia pastoris

et al. 2010

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β-1,3-1,4-Glucanase has been broadly used in feed and brewing industries. According to the codon bias of Pichia pastoris, the Bacillus subtilis MA139 β-1,3-1,4-glucanase gene was de novo synthesized and expressed in P. pastoris X-33 strain under the control of the alcohol oxidase 1 promoter. In a 10-L fermentor, the β-1,3-1,4-glucanase was overexpressed with a yield of 15,000 U/mL by methanol induction for 96 h. The recombinant β-1,3-1,4-glucanase exhibited optimal activity at 40• C and pH 6.4. The activity of the recombinant β-1,3-1,4-glucanase was not significantly affected by various metal ions and chemical reagents. To our knowledge, the expression of this β-1,3-1,4-glucanase from Bacillus sp. in P. pastoris is in relatively high level compared to previous reports. These biochemical characteristics suggest that the recombinant β-1,3-1,4-glucanase has a prospective application in feed and brewing industries.

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Codon optimization of Bacillus licheniformis β-1,3-1,4-glucanase gene and its expression in Pichia pastoris

Cited by 73 publications

References 42 publications

Effect of codon optimization on expression levels of human cystatin C in Pichia pastoris

Effect of codon optimization on expression levels of human cystatin C in Pichia pastoris

A food-grade industrial arming yeast expressing β-1,3-1,4-glucanase with enhanced thermal stability

Codon optimization, expression and characterization of Bacillus subtilis MA139 β-1,3-1,4-glucanase in Pichia pastoris

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