Improved secretion of Candida antarctica lipase B with its native signal peptide in Pichia pastoris

Vadhana, Ashok Kumar Prasanna; Samuel, P.; Berin, Ronald M; Krishna, Jayachandran; Kamatchi, Kavitha; Meenakshisundaram, S.

doi:10.1016/j.enzmictec.2013.01.001

Cited by 47 publications

(27 citation statements)

References 28 publications

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“…Some examples have demonstrated that protein secretion could be improved through using the strong signal peptides or engineering them. For instance, enhanced secretion of Candida antarctica lipase B with its native signal peptide in Pichia pastoris was achieved [32]. Ng et al improved protein secretion from Lactococcus lactis through engineering the native signal peptide of a major secreted lactococcal protein, Usp45 [33].…”

Section: Discussionmentioning

confidence: 99%

Direct production of itaconic acid from liquefied corn starch by genetically engineered Aspergillus terreus

et al. 2014

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BackgroundItaconic acid is on the DOE (Department of Energy) top 12 list of biotechnologically produced building block chemicals and is produced commercially by Aspergillus terreus. However, the production cost of itaconic acid is too high to be economically competitive with the petrochemical-based products. Itaconic acid is generally produced from raw corn starch, including three steps: enzymatic hydrolysis of corn starch into a glucose-rich syrup by α-amylase and glucoamylase, fermentation, and recovery of itaconic acid. The whole process is very time-consuming and energy-intensive.ResultsIn order to reduce the production cost, saccharification and fermentation were integrated into one step through overexpressing the glucoamylase gene in A. terreus under the control of the native PcitA promoter. The transformant XH61-5 produced higher itaconate titer from liquefied starch than WT. To further increase the titer by enhancing the secretion capacity of overexpressed glucoamylase, a stronger signal peptide was selected based on the major secreted protein ATEG_02176 (an acid phosphatase precursor) by A. terreus under the itaconate production conditions. Under the control of the stronger signal peptide, the transformant XH86-8 showed higher itaconate production level than XH61-5 from liquefied starch. The itaconate titer was further enhanced through a two-step process involving the vegetative and production phase, and the transformant XH86-8 produced comparable itaconate titer from liquefied starch to current one (~80 g/L) from saccharified starch hydrolysates in industry. The effects of the new signal peptide and the two-step process on itaconate production were investigated and discussed.ConclusionsItaconic acid could be efficiently produced from liquefied corn starch by overexpressing the glucoamylase gene in A. terreus, which will be helpful for constructing a highly efficient microbial cell factory for itaconate production and for further lowering the production cost of itaconic acid.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-014-0108-1) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Direct production of itaconic acid from liquefied corn starch by genetically engineered Aspergillus terreus

et al. 2014

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“…For P. pastoris, the appropriate choice of the signal sequence cannot be clearly predicted [27]. The α-mating factor pre-pro sequence is most widely used, but in some cases expressions using different signal sequences, including the native signal sequence, have shown better results [28,29].…”

Section: Fig 1 (A) Expression Of Est1 With E Coli Bl21 (De3)star Pementioning

confidence: 98%

Heterologous production of a feruloyl esterase from Pleurotus sapidus synthesizing feruloyl‐saccharide esters

Kelle

Nieter

Krings

et al. 2015

Biotech and App Biochem

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The feruloyl esterase (FAE) gene EST1 from the basidiomycete Pleurotus sapidus was heterologously expressed in Escherichia coli and Pichia pastoris. Catalytically active recombinant Est1 was secreted using P. pastoris as a host. For expression in P. pastoris, the expression vector pPIC9K was applied. The EST1 gene was cloned with an N-terminal α-mating factor pre-pro sequence and expressed under the control of a methanol inducible alcohol oxidase 1 promotor. Est1 was purified to homogeneity using ion exchange and hydrophobic interaction chromatography. The recombinant Est1 showed optima at pH 5.0 and 50 °C, and released ferulic acid from saccharide esters and from the natural substrate destarched wheat bran. Substrate specificity profile and descriptor-based analysis demonstrated unique properties, showing that Est1 did not fit into the current FAE classification model. Transferuloylation synthesis of feruloyl-saccharide esters was proven for mono- and disaccharides.

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“…coli DH5α was used as the host for vector manipulations. Recombinant pPICZαB vector containing Candida antarctica lipase B (CALB) gene [24] expressed in P . pastoris GS115 with high copy CALB (GSCALB) was used in this study for protein expression analysis.…”

Section: Methodsmentioning

confidence: 99%

Cofactor engineering improved CALB production in Pichia pastoris through heterologous expression of NADH oxidase and adenylate kinase

2017

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The cofactor engineering strategy can relieve the metabolic stress induced by expression of recombinant protein in cellular metabolism related to cofactor and energy reactions. To study the effect of cofactor regeneration on recombinant protein expression, NADH oxidase (noxE) was engineered in P. pastoris expressing lipase B (GSCALB). Expression of noxE in P. pastoris (GSCALBNOX) increased NAD+ levels by 85% with a concomitant reduction in NADH/NAD+ ratio of 67% compared to GSCALB. The change in the redox level positively influenced the methanol uptake rate and made 34% augment in CALB activity. The decline in NADH level (44%) by noxE expression had lowered the adenylate energy charge (AEC) and ATP level in GSCALBNOX. In order to regenerate ATP in GSCALBNOX, adenylate kinase (ADK1) gene from S. cerevisiae S288c was co—expressed. Expression of ADK1 showed a remarkable increase in AEC and co—expression of both the genes synergistically improved CALB activity. This study shows the importance of maintenance of cellular redox homeostasis and adenylate energy charge during recombinant CALB expression in P. pastoris.

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Improved secretion of Candida antarctica lipase B with its native signal peptide in Pichia pastoris

Cited by 47 publications

References 28 publications

Direct production of itaconic acid from liquefied corn starch by genetically engineered Aspergillus terreus

Direct production of itaconic acid from liquefied corn starch by genetically engineered Aspergillus terreus

Heterologous production of a feruloyl esterase from Pleurotus sapidus synthesizing feruloyl‐saccharide esters

Cofactor engineering improved CALB production in Pichia pastoris through heterologous expression of NADH oxidase and adenylate kinase

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