High-level expression of two thermophilic β-mannanases in Yarrowialipolytica

Wang, Yaping; Ben, Rao; Zhang, Ling; Ma, Lixin

doi:10.1016/j.pep.2017.02.008

Cited by 9 publications

(10 citation statements)

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“…The highest yield by shake flask was 1114 U ml −1 with productivity of only 7.7 U ml −1 h −1 after 144 h cultivation in P. pastoris GS115 using pPIC9K vector [ 55 ]. Besides these commonly used strains, some other host strains such as A. niger [ 77 ], A. sojae [ 26 ], Yarrowia lipolytica [ 78 ], Lactobacillus plantarum [ 79 ] and Trichoderma reesei [ 31 ] were also used for β-mannanases production. The extracellular activity of β-mannanase from A. aculeatus MRC11624 was found to be 16,596 nkat ml −1 (about 996 U ml −1 ) after 192 h cultivation in A. niger D15 [ 77 ].…”

Section: Resultsmentioning

confidence: 99%

Characterization and high-efficiency secreted expression in Bacillus subtilis of a thermo-alkaline β-mannanase from an alkaliphilic Bacillus clausii strain S10

Zhou

Xue

2018

Microb Cell Fact

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Backgroundβ-Mannanase catalyzes the cleavage of β-1,4-linked internal linkages of mannan backbone randomly to produce new chain ends. Alkaline and thermostable β-mannanases provide obvious advantages for many applications in biobleaching of pulp and paper, detergent industry, oil grilling operation and enzymatic production of mannooligosaccharides. However, only a few of them are commercially exploited as wild or recombinant enzymes, and none heterologous and secretory expression of alkaline β-mannanase in Bacillus subtilis expression system was reported. Alkaliphilic Bacillus clausii S10 showed high β-mannanase activity at alkaline condition. In this study, this β-mannanase was cloned, purified and characterized. The high-level secretory expression in B. subtilis was also studied.ResultsA thermo-alkaline β-mannanase (BcManA) gene encoding a 317-amino acid protein from alkaliphilic Bacillus clausii strain was cloned and expressed in Escherichia coli. The purified mature BcManA exhibited maximum activity at pH 9.5 and 75 °C with good stability at pH 7.0–11.5 and below 80 °C. BcManA demonstrated high cleavage capability on polysaccharides containing β-1,4-mannosidic linkages, such as konjac glucomannan, locust bean gum, guar gum and sesbania gum. The highest specific activity of 2366.2 U mg−1 was observed on konjac glucomannan with the Km and kcat value of 0.62 g l−1 and 1238.9 s−1, respectively. The hydrolysis products were mainly oligosaccharides with a higher degree of polymerization than biose. BcManA also cleaved manno-oligosaccharides with polymerization degree more than 3 without transglycosylation. Furthermore, six signal peptides and two strong promoters were used for efficiently secreted expression optimization in B. subtilis WB600 and the highest extracellular activity of 2374 U ml−1 with secretory rate of 98.5% was obtained using SPlipA and P43 after 72 h cultivation in 2 × SR medium. By medium optimization using cheap nitrogen and carbon source of peanut meal and glucose, the extracellular activity reached 6041 U ml−1 after 72 h cultivation with 6% inoculum size by shake flask fermentation.ConclusionsThe thermo-alkaline β-mannanase BcManA showed good thermal and pH stability and high catalytic efficiency towards konjac glucomannan and locust bean gum, which distinguished from other reported β-mannanases and was a promising thermo-alkaline β-mannanase for potential industrial application. The extracellular BcManA yield of 6041 U ml−1, which was to date the highest reported yield by flask shake, was obtained in B. subtilis with constitutive expression vector. This is the first report for secretory expression of alkaline β-mannanase in B. subtilis protein expression system, which would significantly cut down the production cost of this enzyme. Also this research would be helpful for secretory expression of other β-mannanases in B. subtilis.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0973-0) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Characterization and high-efficiency secreted expression in Bacillus subtilis of a thermo-alkaline β-mannanase from an alkaliphilic Bacillus clausii strain S10

Zhou

Xue

2018

Microb Cell Fact

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“…The most evident effect of this metabolic load is the reduced growth capacity of host cells. Specific growth rate and biomass yield both suffer from resource diversion [32,33,36,37]. On a molecular level, limitations in specific amino acids and energy provision may result in translational errors, impacting recombinant protein activity, stability, and possible immunogenicity [32].…”

Section: Protein Productionmentioning

confidence: 99%

“…Integration of expression cassette in yeast chromosome shall be favored over episomal plasmids, whose maintenance in the cells requires a substantial amount of energy. When codon bias differs significantly between heterologous gene and recipient cells, the synthesis of a codon-optimized gene variant can be considered [16,17,20,37,43]. Codon usage frequency of the synthetic gene shall reflect that of the native producer to allow fluent elongation on the ribosome and correct protein folding [44].…”

Section: Protein Productionmentioning

confidence: 99%

“…Compared to mono-copy integration, multi-copy strategy resulted in a 3.5-fold and 4.5-fold increased enzyme yield and productivity for manA and manB, respectively. By contrast, biomass yield was reduced by 2.2- and 1.9-fold, respectively [37]. The only report on a positive effect of multi-copy integration on biomass growth is for SUC2 gene encoding invertase in sucrose-based batch, whose double integration resulted in a higher growth rate (0.132 versus 0.096 h −1 ) along with an improved enzyme yield (1991 versus 597 U·L −1 ) and productivity (27.65 versus 8.29 U·L −1 ·h −1 ).…”

Section: Molecular Strategiesmentioning

confidence: 99%

“…The transition from shake-flask to bioreactor cultures offers improved oxygenation conditions and medium homogeneity [16]. For culture scale-up from flask to pilot-scale reactors, a 3- to 5-fold higher enzyme yield, and an even higher productivity, can be expected as compared to shake-flasks [22,37,53,110,111]. For complex non-enzymatic therapeutic proteins, the gain can be even greater, with a 416-fold increase of human interferon concentration for a culture scale-up from a 250 mL shake-flask to a 5 L batch reactor [17].…”

Section: Process Strategiesmentioning

confidence: 99%

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Bioreactor-Scale Strategies for the Production of Recombinant Protein in the Yeast Yarrowia lipolytica

Vandermies

Fickers

2019

Microorganisms

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Recombinant protein production represents a multibillion-dollar market. Therefore, it constitutes an important research field both in academia and industry. The use of yeast as a cell factory presents several advantages such as ease of genetic manipulation, growth at high cell density, and the possibility of post-translational modifications. Yarrowia lipolytica is considered as one of the most attractive hosts due to its ability to metabolize raw substrate, to express genes at a high level, and to secrete protein in large amounts. In recent years, several reviews have been dedicated to genetic tools developed for this purpose. Though the construction of efficient cell factories for recombinant protein synthesis is important, the development of an efficient process for recombinant protein production in a bioreactor constitutes an equally vital aspect. Indeed, a sports car cannot drive fast on a gravel road. The aim of this review is to provide a comprehensive snapshot of process tools to consider for recombinant protein production in bioreactor using Y. lipolytica as a cell factory, in order to facilitate the decision-making for future strain and process engineering.

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Strategies at Bioreactor Scale for the Production of Recombinant Proteins in Yarrowia lipolytica

Vandermies

Fickers

2019

Non-Conventional Yeasts: From Basic Research to Application

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High-level expression of two thermophilic β-mannanases in Yarrowialipolytica

Cited by 9 publications

References 11 publications

Characterization and high-efficiency secreted expression in Bacillus subtilis of a thermo-alkaline β-mannanase from an alkaliphilic Bacillus clausii strain S10

Characterization and high-efficiency secreted expression in Bacillus subtilis of a thermo-alkaline β-mannanase from an alkaliphilic Bacillus clausii strain S10

Bioreactor-Scale Strategies for the Production of Recombinant Protein in the Yeast Yarrowia lipolytica

Strategies at Bioreactor Scale for the Production of Recombinant Proteins in Yarrowia lipolytica

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