Enhancement of alkaline phytase production in Pichia pastoris: Influence of gene dosage, sequence optimization and expression temperature

Yang, Mimi; Johnson, Steven C.; Murthy, Pushpalatha P. N.

doi:10.1016/j.pep.2012.06.001

Cited by 19 publications

(10 citation statements)

References 49 publications

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“…None of the recombinant colonies of K. lactis YCT390 contained mutations in the αAI - OPT gene sequence, and there were no differences in cell growth among the recombinant K. lactis YCT390/αAI-OPT colonies when compared to the control strain (data not shown). As previously described for the expression of recombinant phytase in P. pastoris [52], it seems that the level of processed αAI produced by K. lactis YCT390 was not related to gene dosage, mutations or toxic effects on the host.…”

Section: Discussionmentioning

confidence: 72%

“…The integration of cassettes into the chromosome of K. lactis regularly produces strains having multiple copies of the vector [33], and it has been reported that gene dosage can affect the yield of some heterologous proteins expressed in yeasts [33, 51, 52]. Here q-PCR data showed that all the K. lactis YCT390/αAI-OPT colonies contained an average of six copies of the gene, indicating that there was no correlation between the gene dosage and the differences in processing of αAI.…”

Section: Discussionmentioning

confidence: 99%

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Heterologous expression of an α-amylase inhibitor from common bean (Phaseolus vulgaris) in Kluyveromyces lactis and Saccharomyces cerevisiae

2017

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BackgroundPhaseolamin or α-amylase inhibitor 1 (αAI) is a glycoprotein from common beans (Phaseolus vulgaris L.) that inhibits some insect and mammalian α-amylases. Several clinical studies support the beneficial use of bean αAI for control of diabetes and obesity. Commercial extracts of P. vulgaris are available but their efficacy is still under question, mainly because some of these extracts contain antinutritional impurities naturally present in bean seeds and also exhibit a lower specific activity αAI. The production of recombinant αAI allows to overcome these disadvantages and provides a platform for the large-scale production of pure and functional αAI protein for biotechnological and pharmaceutical applications.ResultsA synthetic gene encoding αAI from the common bean (Phaseolus vulgaris cv. Pinto) was codon-optimised for expression in yeasts (αAI-OPT) and cloned into the protein expression vectors pKLAC2 and pYES2. The yeasts Kluyveromyces lactis GG799 (and protease deficient derivatives such as YCT390) and Saccharomyces cerevisiae YPH499 were transformed with the optimised genes and transformants were screened for expression by antibody dot blot. Recombinant colonies of K. lactis YCT390 that expressed and secreted functional αAI into the culture supernatants were selected for further analyses. Recombinant αAI from K. lactis YCT390 was purified using anion-exchange and affinity resins leading to the recovery of a functional inhibitor. The identity of the purified αAI was confirmed by mass spectrometry. Recombinant clones of S. cerevisiae YPH499 expressed functional αAI intracellularly, but did not secrete the protein.ConclusionsThis is the first report describing the heterologous expression of the α-amylase inhibitor 1 (αAI) from P. vulgaris in yeasts. We demonstrated that recombinant strains of K. lactis and S. cerevisiae expressed and processed the αAI precursor into mature and active protein and also showed that K. lactis secretes functional αAI.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0719-4) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 72%

Section: Discussionmentioning

confidence: 99%

Heterologous expression of an α-amylase inhibitor from common bean (Phaseolus vulgaris) in Kluyveromyces lactis and Saccharomyces cerevisiae

2017

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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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“…The recombinant P. pastoris capable of resisting higher concentration of Geneticin were inferred to have multiple copies of integrated plasmid and higher expression level of the heterologous protein (26,27). However, the correlation between expression level of the recombinant protein and copy numbers of heterologous gene was more empirical than guaranteed (28), which called for an expression test to determine the actual productivities of the transformants. After screening, only a few transformants could grow in the presence of 2.0 and 4.0 mg/mL Geneticin.…”

Section: Vol XX 2014mentioning

confidence: 99%

Production of LYZL6, a novel human c-type lysozyme, in recombinant Pichia pastoris employing high cell density fed-batch fermentation

Zhou

Tao

et al. 2014

Journal of Bioscience and Bioengineering

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Enhancement of alkaline phytase production in Pichia pastoris: Influence of gene dosage, sequence optimization and expression temperature

Cited by 19 publications

References 49 publications

Heterologous expression of an α-amylase inhibitor from common bean (Phaseolus vulgaris) in Kluyveromyces lactis and Saccharomyces cerevisiae

Heterologous expression of an α-amylase inhibitor from common bean (Phaseolus vulgaris) in Kluyveromyces lactis and Saccharomyces cerevisiae

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

Production of LYZL6, a novel human c-type lysozyme, in recombinant Pichia pastoris employing high cell density fed-batch fermentation

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