Overexpression of an optimized Aspergillus sulphureus β-mannanase gene in Pichia pastoris

Abstractβ-Mannanase (EC 3.2.1.78) is a key enzyme to hydrolyze the β-mannosidic linkages in mannan and heteromannan. The expression of a wild type β-mannanase (manWT) of Aspergillus sulphureus in Pichia pastoris is not high enough for its application in feed supplement. To earn a high expression level, the manWT gene was firstly optimized to manM according to the code bias of P. pastoris, which was then inserted into pPICzαA and transformed into P. pastoris strain X-33. In the induction by methanol, β-mannanase was expressed in high level with 32% increase in comparison with the manWT gene expressed in P. pastoris in shaken flask. In a 10-L fermenter, the manM was expressed in 9-fold higher level than that in shaken flask, which yielded the enzyme activity of 1100 U/mL. This is the first study on codon bias effect on the β-mannanase gene expression level, which helps to achieve high β-mannanase yield and enzymatic activity in P. pastoris.

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High-Level Secretory Expression and Purification of Recombinant Human Interleukin 1 Beta in Pichia pastoris

Protein and Peptide Letters ◽

10.2174/0929866523666160530184936 ◽

2016 ◽

Vol 23 (8) ◽

pp. 763-769 ◽

Cited By ~ 2

Author(s):

Pengfei Li ◽

Ganggang Yang ◽

Xiaofang Geng ◽

Jinbao Shi ◽

Bin Li ◽

...

Keyword(s):

Pichia Pastoris ◽

Interleukin 1 ◽

Interleukin 1 Beta ◽

Expression And Purification ◽

Secretory Expression ◽

High Level

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Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01948-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01948-20

Author(s):

Dalin Rifat ◽

Si-Yang Li ◽

Thomas Ioerger ◽

Keshav Shah ◽

Jean-Philippe Lanoix ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Clinical Evidence ◽

Clinical Samples ◽

Loss Of Function ◽

Wild Type ◽

Content Type ◽

Solid Media ◽

High Level ◽

Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

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Allelic Exchange and Mutant Selection Demonstrate that Common Clinical embCAB Gene Mutations Only Modestly Increase Resistance to Ethambutol in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01288-09 ◽

2009 ◽

Vol 54 (1) ◽

pp. 103-108 ◽

Cited By ~ 38

Author(s):

Hassan Safi ◽

Robert D. Fleischmann ◽

Scott N. Peterson ◽

Marcus B. Jones ◽

Behnam Jarrahi ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

In Vitro Selection ◽

Gene Mutations ◽

Wild Type ◽

Mutant Selection ◽

Preferential Growth ◽

Allelic Exchange ◽

High Level ◽

Isogenic Mutants

ABSTRACT Mutations within codon 306 of the Mycobacterium tuberculosis embB gene modestly increase ethambutol (EMB) MICs. To identify other causes of EMB resistance and to identify causes of high-level resistance, we generated EMB-resistant M. tuberculosis isolates in vitro and performed allelic exchange studies of embB codon 406 (embB406) and embB497 mutations. In vitro selection produced mutations already identified clinically in embB306, embB397, embB497, embB1024, and embC13, which result in EMB MICs of 8 or 14 μg/ml, 5 μg/ml, 12 μg/ml, 3 μg/ml, and 4 μg/ml, respectively, and mutations at embB320, embB324, and embB445, which have not been identified in clinical M. tuberculosis isolates and which result in EMB MICs of 8 μg/ml, 8 μg/ml, and 2 to 8 μg/ml, respectively. To definitively identify the effect of the common clinical embB497 and embB406 mutations on EMB susceptibility, we created a series of isogenic mutants, exchanging the wild-type embB497 CAG codon in EMB-susceptible M. tuberculosis strain 210 for the embB497 CGG codon and the wild-type embB406 GGC codon for either the embB406 GCC, embB406 TGC, embB406 TCC, or embB406 GAC codon. These new mutants showed 6-fold and 3- to 3.5-fold increases in the EMB MICs, respectively. In contrast to the embB306 mutants, the isogenic embB497 and embB406 mutants did not have preferential growth in the presence of isoniazid or rifampin (rifampicin) at their MICs. These results demonstrate that individual embCAB mutations confer low to moderate increases in EMB MICs. Discrepancies between the EMB MICs of laboratory mutants and clinical M. tuberculosis strains with identical mutations suggest that clinical EMB resistance is multigenic and that high-level EMB resistance requires mutations in currently unknown loci.

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High-level expression of a thermostable endo-β-1,3-glucanase from Trichoderma harzianum in Pichia pastoris KM71 and its use in the enzymatic hydrolysis of multifunctional oligoglucosides

Process Biochemistry ◽

10.1016/j.procbio.2021.05.010 ◽

2021 ◽

Author(s):

Min-Jie Gao ◽

Jia-Jun Yan ◽

Yue Zhao ◽

Li Zhu ◽

Guo-Shuai Yang ◽

...

Keyword(s):

Enzymatic Hydrolysis ◽

Pichia Pastoris ◽

Trichoderma Harzianum ◽

High Level Expression ◽

High Level ◽

Hydrolysis Of

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Transcriptome Analysis Provides Insights into the Mechanism of Astaxanthin Enrichment in a Mutant of the Ridgetail White Prawn Exopalaemon carinicauda

Genes ◽

10.3390/genes12050618 ◽

2021 ◽

Vol 12 (5) ◽

pp. 618

Author(s):

Yue Jin ◽

Shihao Li ◽

Yang Yu ◽

Chengsong Zhang ◽

Xiaojun Zhang ◽

...

Keyword(s):

Transcriptome Analysis ◽

Underlying Mechanism ◽

Biological Processes ◽

Wild Type ◽

Expression Levels ◽

In The Wild ◽

Cuticle Proteins ◽

Apolipoprotein D ◽

High Level ◽

Lower Expression

A mutant of the ridgetail white prawn, which exhibited rare orange-red body color with a higher level of free astaxanthin (ASTX) concentration than that in the wild-type prawn, was obtained in our lab. In order to understand the underlying mechanism for the existence of a high level of free astaxanthin, transcriptome analysis was performed to identify the differentially expressed genes (DEGs) between the mutant and wild-type prawns. A total of 78,224 unigenes were obtained, and 1863 were identified as DEGs, in which 902 unigenes showed higher expression levels, while 961 unigenes presented lower expression levels in the mutant in comparison with the wild-type prawns. Based on Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes analysis, as well as further investigation of annotated DEGs, we found that the biological processes related to astaxanthin binding, transport, and metabolism presented significant differences between the mutant and the wild-type prawns. Some genes related to these processes, including crustacyanin, apolipoprotein D (ApoD), cathepsin, and cuticle proteins, were identified as DEGs between the two types of prawns. These data may provide important information for us to understand the molecular mechanism of the existence of a high level of free astaxanthin in the prawn.

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Disulfide bond engineering of AppA phytase for increased thermostability requires co-expression of protein disulfide isomerase in Pichia pastoris

Biotechnology for Biofuels ◽

10.1186/s13068-021-01936-8 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Laura Navone ◽

Thomas Vogl ◽

Pawarisa Luangthongkam ◽

Jo-Anne Blinco ◽

Carlos H. Luna-Flores ◽

...

Keyword(s):

Pichia Pastoris ◽

Phytic Acid ◽

Disulfide Bond ◽

Wild Type ◽

Wild Type Enzyme ◽

E Coli ◽

Biochemical Characterisation ◽

Disulfide Bond Isomerase ◽

Microbial Systems ◽

Protein Disulfide

Abstract Background Phytases are widely used commercially as dietary supplements for swine and poultry to increase the digestibility of phytic acid. Enzyme development has focused on increasing thermostability to withstand the high temperatures during industrial steam pelleting. Increasing thermostability often reduces activity at gut temperatures and there remains a demand for improved phyases for a growing market. Results In this work, we present a thermostable variant of the E. coli AppA phytase, ApV1, that contains an extra non-consecutive disulfide bond. Detailed biochemical characterisation of ApV1 showed similar activity to the wild type, with no statistical differences in kcat and KM for phytic acid or in the pH and temperature activity optima. Yet, it retained approximately 50% activity after incubations for 20 min at 65, 75 and 85 °C compared to almost full inactivation of the wild-type enzyme. Production of ApV1 in Pichia pastoris (Komagataella phaffi) was much lower than the wild-type enzyme due to the presence of the extra non-consecutive disulfide bond. Production bottlenecks were explored using bidirectional promoters for co-expression of folding chaperones. Co-expression of protein disulfide bond isomerase (Pdi) increased production of ApV1 by ~ 12-fold compared to expression without this folding catalyst and restored yields to similar levels seen with the wild-type enzyme. Conclusions Overall, the results show that protein engineering for enhanced enzymatic properties like thermostability may result in folding complexity and decreased production in microbial systems. Hence parallel development of improved production strains is imperative to achieve the desirable levels of recombinant protein for industrial processes.

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Expression of novel acidic lipase from Micrococcus luteus in Pichia pastoris and its application in transesterification

Journal of Genetic Engineering and Biotechnology ◽

10.1186/s43141-021-00155-w ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Selfela Restu Adina ◽

Antonius Suwanto ◽

Anja Meryandini ◽

Esti Puspitasari

Keyword(s):

Fatty Acid ◽

Pichia Pastoris ◽

Specific Activity ◽

Signal Sequence ◽

Micrococcus Luteus ◽

Acid Methyl Ester ◽

Industrial Applications ◽

Expression Level ◽

Lipase Gene ◽

Acidic Lipase

Abstract Background Lipases are promising biocatalysts for industrial applications and attract attention to be explored. A novel acidic lipase has been isolated from the lipolytic bacteria Micrococcus luteus EMP48-D (LipEMP48-D) screened from tempeh. The lipase gene had previously been overexpressed in Escherichia coli BL21, but the expression level obtained was relatively low. Here, to improve the expression level, the lipase gene was cloned to Pichia pastoris. We eliminated the native signal sequence of M. luteus and replaced it with α-mating factor (α-MF) signal sequence. We also optimized and synthesized the lipase gene based on codon preference in P. pastoris. Results LipEMP48-D lipase was expressed as an extracellular protein. Codon optimization has been conducted for 20 codons, with the codon adaption index reaching 0.995. The highest extracellular lipase activity obtained reached 145.4 ± 4.8 U/mg under AOX1 promoter in P. pastoris KM71 strain, which was 9.7-fold higher than the previous activity in E. coli. LipEMP48-D showed the highest specific activity at pH 5.0 and stable within the pH range 3.0–5.0 at 40 °C. LipEMP48-D also has the capability of hydrolyzing various long-chain triglycerides, particularly olive oil (100%) followed by sunflower oil (88.5%). LipEMP48-D exhibited high tolerance for various polar organic solvents with low log P, such as isopropanol (115.7%) and butanol (114.6%). The metal ions (Na+, K+, Ca2+, Mg2+, Mn+) decreased enzyme activity up to 43.1%, while Fe2+ increased relative activity of enzymes up to 200%. The conversion of free fatty acid (FFA) into fatty acid methyl ester (FAME) was low around 2.95%. Conclusions This study was the first to report overexpression of Micrococcus lipase in yeast. The extracellular expression of this acidic lipase could be potential for biocatalyst in industrial fields, especially organic synthesis, food industry, and production of biodiesel.

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Overexpression of Mucin 1 Suppresses the Therapeutical Efficacy of Disulfiram against Canine Mammary Tumor

Animals ◽

10.3390/ani11010037 ◽

2020 ◽

Vol 11 (1) ◽

pp. 37

Author(s):

Ying Zhao ◽

Zixiang Lin ◽

Zhaoyan Lin ◽

Chaoyu Zhou ◽

Gang Liu ◽

...

Keyword(s):

Mammary Tumor ◽

Mammary Tumors ◽

Expression Level ◽

Canine Mammary Tumor ◽

Wild Type ◽

Mucin 1 ◽

Mammary Tumor Cell ◽

Mammary Tumor Cell Line ◽

Canine Mammary Tumors ◽

Wild Type Control

Mucin 1 (MUC1), a transmembrane protein, is closely associated with the malignancy and metastasis of canine mammary tumors; however, the role of overexpressed MUC1 in the development of cancer cells and response to drug treatment remains unclear. To address this question, we developed a new canine mammary tumor cell line, CIPp-MUC1, with an elevated expression level of MUC1. In vitro studies showed that CIPp-MUC1 cells are superior in proliferation and migration than wild-type control, which was associated with the upregulation of PI3K, p-Akt, mTOR, Bcl-2. In addition, overexpression of MUC1 in CIPp-MUC1 cells inhibited the suppressing activity of disulfiram on the growth and metastasis of tumor cells, as well as inhibiting the pro-apoptotic effect of disulfiram. In vivo studies, on the other side, showed more rapid tumor growth and stronger resistance to disulfiram treatment in CIPp-MUC1 xenograft mice than in wild-type control. In conclusion, our study demonstrated the importance of MUC1 in affecting the therapeutical efficiency of disulfiram against canine mammary tumors, indicating that the expression level of MUC1 should be considered for clinical use of disulfiram or other drugs targeting PI3K/Akt pathway.

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Modified Monosaccharides Content of Xanthan Gum Impairs Citrus Canker Disease by Affecting the Epiphytic Lifestyle of Xanthomonas citri subsp. citri

Microorganisms ◽

10.3390/microorganisms9061176 ◽

2021 ◽

Vol 9 (6) ◽

pp. 1176

Author(s):

Simone Cristina Picchi ◽

Laís Moreira Granato ◽

Maria Júlia Festa Franzini ◽

Maxuel Oliveira Andrade ◽

Marco Aurélio Takita ◽

...

Keyword(s):

Biofilm Formation ◽

Xanthan Gum ◽

Wild Type Strain ◽

Natural Infection ◽

Citrus Canker ◽

Wild Type ◽

Xanthomonas Citri ◽

Canker Disease ◽

Key Enzyme ◽

Citrus Canker Disease

Xanthomonas citri subsp. citri (X. citri) is a plant pathogenic bacterium causing citrus canker disease. The xanA gene encodes a phosphoglucomutase/phosphomannomutase protein that is a key enzyme required for the synthesis of lipopolysaccharides and exopolysaccharides in Xanthomonads. In this work, firstly we isolated a xanA transposon mutant (xanA::Tn5) and analyzed its phenotypes as biofilm formation, xanthan gum production, and pathogenesis on the sweet orange host. Moreover, to confirm the xanA role in the impaired phenotypes we further produced a non-polar deletion mutant (ΔxanA) and performed the complementation of both xanA mutants. In addition, we analyzed the percentages of the xanthan gum monosaccharides produced by X. citri wild-type and xanA mutant. The mutant strain had higher ratios of mannose, galactose, and xylose and lower ratios of rhamnose, glucuronic acid, and glucose than the wild-type strain. Such changes in the saccharide composition led to the reduction of xanthan yield in the xanA deficient strain, affecting also other important features in X. citri, such as biofilm formation and sliding motility. Moreover, we showed that xanA::Tn5 caused no symptoms on host leaves after spraying, a method that mimetics the natural infection condition. These results suggest that xanA plays an important role in the epiphytical stage on the leaves that is essential for the successful interaction with the host, including adaptive advantage for bacterial X. citri survival and host invasion, which culminates in pathogenicity.

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