Rational design of a novel propeptide for improving active production of Streptomyces griseus trypsin in Pichia pastoris.
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ABSTRACT:
SUBMITTER: Ling Z
PROVIDER: S-EPMC3675915 | biostudies-literature | 2013 Jun
REPOSITORIES: biostudies-literature
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Rational design of a novel propeptide for improving active production of Streptomyces griseus trypsin in Pichia pastoris.
Ling Zhenmin Z Liu Yi Y Teng Shaolei S Kang Zhen Z Zhang Jingjing J Chen Jian J Du Guocheng G
Applied and environmental microbiology 20130405 12
Applying in silico simulations and in vitro experiments, the amino acid proline was proved to have a profound influence on Streptomyces griseus trypsinogen, and the hydrogen bond between H(57) and D(102) was found to be crucial for trypsin activity. By introducing an artificial propeptide, IVEF, the titer of trypsin was increased 6.71-fold. ...[more]
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Project description:BackgroundTrypsin has many applications in food and pharmaceutical manufacturing. Although commercial trypsin is usually extracted from porcine pancreas, this source carries the risks of infectivity and immunogenicity. Microbial Streptomyces griseus trypsin (SGT) is a prime alternative because it possesses efficient hydrolysis activity without such risks. However, the remarkable hydrolysis efficiency of SGT causes autolysis, and five autolysis sites, R21, R32, K122, R153, and R201, were identified from its autolysate.ResultsThe tbcf (K101A, R201V) mutant was screened by a directed selection approach for improved activity in flask culture (60.85 ± 3.42 U mL-1, increased 1.5-fold). From the molecular dynamics simulation, in the K101A/R201V mutant the distance between the catalytical residues D102 and H57 was shortened to 6.5 Å vs 7.0 Å in the wild type, which afforded the improved specific activity of 1527.96 ± 62.81 U mg-1. Furthermore, the production of trypsin was increased by 302.8% (689.47 ± 6.78 U mL-1) in a 3-L bioreactor, with co-overexpression of chaperones SSO2 and UBC1 in Pichia pastoris.ConclusionsSGT protein could be a good source of trypsin for insulin production. As a result of the hydrolysates analysis and direct selection, the activity of the tbcf (K101A, R201V) mutant increased 1.5-fold. Furthermore, the production of trypsin was improved threefold by overexpressing chaperone protein in Pichia pastoris. Future studies should investigate the application of SGT to insulin and pharmaceutical manufacturing.
| S-EPMC7155311 | biostudies-literature
Project description:N-terminal sequences play crucial roles in regulating expression, translation, activation and enzymatic properties of proteins. To reduce cell toxicity of intracellular trypsin and increase secretory expression, we developed a novel auto-catalyzed strategy to produce recombinant trypsin by engineering the N-terminus of mature Streptomyces griseus trypsin (SGT). The engineered N-terminal peptide of SGT was composed of the thioredoxin, glycine-serine linker, His6-tag and the partial bovine trypsinogen pro-peptide (DDDDK). Furthermore, we constructed a variant TLEI with insertion of the artificial peptide at N-terminus and site-directed mutagenesis of the autolysis residue R145. In fed-batch fermentation, the production of extracellular trypsin activity was significantly improved to 47.4 ± 1.2 U·ml(-1) (amidase activity, 8532 ± 142.2 U·ml(-1) BAEE activity) with a productivity of 0.49 U·ml(-1)·h(-1), which was 329% greater than that of parent strain Pichia pastoris GS115-SGT. This work has significant potential to be scaled-up for microbial production of SGT. In addition, the N-terminal peptide engineering strategy can be extended to improve heterologous expression of other toxic enzymes.
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Project description:BackgroundHigh thermostability is required for trypsin to have wider industrial applications. Target mutagenesis at flexible regions has been proved to be an efficient protein engineering method to enhance the protein thermostability.ResultsThe flexible regions in porcine trypsin were predicted using the methods including molecular dynamic simulation, FlexPred, and FoldUnfold. The amino acids 78-90 was predicted to be the highly flexible region simultaneously by the three methods and hence selected to be the mutation target. We constructed five variants (D3, D5, D7, D9, and D11) by truncating the region. And the variant D9 showed higher thermostability, with a 5 °C increase in Topt, 5.8 °C rise in [Formula: see text], and a 4.5 °C rise in Tm, compared to the wild-type. Moreover, the half-life value of the variant D9 was also found to be dramatically improved by 46 min. Circular dichroism and intrinsic fluorescence indicated that the structures had no significant change between the variant D9 and the wild-type. The surface hydrophobicity of D9 was measured to be lower than that of wild-type, indicating the increased hydrophobic interaction, which could have contributed to the improved thermostability of D9.ConclusionsThese results showed that the thermostability of variant D9 was increased. The variant D9 could be expected to be a promising tool enzyme for its wider industrial applications. The method of truncating the flexible region used in our study has the potential to be used for enhancing the thermostability of other proteins.
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Project description:AdpA is the key transcriptional activator for a number of genes of various functions in the A-factor regulatory cascade in Streptomyces griseus, forming an AdpA regulon. Trypsin-like activity was detected at a late stage of growth in the wild-type strain but not in an A-factor-deficient mutant. Consistent with these observations, two trypsin genes, sprT and sprU, in S. griseus were found to be members of the AdpA regulon; AdpA activated the transcription of both genes by binding to the operators located at about -50 nucleotide positions with respect to the transcriptional start point. The transcription of sprT and sprU, induced by AdpA, was most active at the onset of sporulation. Most trypsin activity exerted by S. griseus was attributed to SprT, because trypsin activity in an sprT-disrupted mutant was greatly reduced but that in an sprU-disrupted mutant was only slightly reduced. This was consistent with the observation that the amount of the sprT mRNA was much greater than that of the sprU transcript. Disruption of both sprT and sprU (mutant DeltasprTU) reduced trypsin activity to almost zero, indicating that no trypsin genes other than these two were present in S. griseus. Even the double mutant DeltasprTU grew normally and developed aerial hyphae and spores over the same time course as the wild-type strain.
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Project description:Rennet, a complex of enzymes found in the stomachs of ruminants, is an important component for cheese production. In our study, we described that yak chymosin gene recombinant Pichia pastoris strain could serve as a novel source for rennet production. Yaks total RNA was extracted from the abomasum of an unweaned yak. The yak preprochymosin, prochymosin, and chymosin genes from total RNA were isolated using gene specific primers based on cattle chymosin gene sequence respectively and analyzed their expression pattern byreal time-polymerase chain reaction. The result showed that the chymosin gene expression level of the sucking yaks was 11.45 times higher than one of adult yaks and yak chymosin belongs to Bovidae family in phylogenetic analysis. To express each, the preprochymosin, prochymosin, and chymosin genes were ligated into the expression vector pPICZ?A, respectively, and were expressed in Pichia pastoris X33. The results showed that all the recombinant clones of P. pastoris containing the preprochymosin, prochymosin or chymosin genes could produce the active form of recombinant chymosin into the culture supernatant. Heterologous expressed prochymosin (14.55 Soxhlet unit/mL) had the highest enzyme activity of the three expressed chymosin enzymes. Therefore, we suggest that the yak chymosin gene recombinant Pichia pastoris strain could provide an alternative source of rennet production.
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Project description:?-Lactamase inhibitory protein (BLIP), a low molecular weight protein from Streptomyces clavuligerus, has a wide range of potential applications in the fields of biotechnology and pharmaceutical industry because of its tight interaction with and potent inhibition on clinically important class A ?-lactamases. To meet the demands for considerable amount of highly pure BLIP, this study aimed at developing an efficient expression system in eukaryotic Pichia pastoris (a methylotrophic yeast) for production of BLIP. With methanol induction, recombinant BLIP was overexpressed in P. pastoris X-33 and secreted into the culture medium. A high yield of ~?300 mg/L culture secretory BLIP recovered from the culture supernatant without purification was found to be >?90% purity. The recombinant BLIP was fully active and showed an inhibition constant (Ki) for TEM-1 ?-lactamase (0.55?±?0.07 nM) comparable to that of the native S. clavuligerus-expressed BLIP (0.5 nM). Yeast-produced BLIP in combination with ampicillin effectively inhibited the growth of ?-lactamase-producing Gram-positive Bacillus. Our approach of expressing secretory BLIP in P. pastoris gave 71- to 1200-fold more BLIP with high purity than the other conventional methods, allowing efficient production of large amount of highly pure BLIP, which merits fundamental science studies, drug development and biotechnological applications.
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Project description:The research communities studying microbial model organisms, such as Escherichia coli or Saccharomyces cerevisiae, are well served by model organism databases that have extensive functional annotation. However, this is not true of many industrial microbes that are used widely in biotechnology. In this Opinion piece, we use Pichia (Komagataella) pastoris to illustrate the limitations of the available annotation. We consider the resources that can be implemented in the short term both to improve Gene Ontology (GO) annotation coverage based on annotation transfer, and to establish curation pipelines for the literature corpus of this organism.
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Project description:BackgroundPhytase is used as an animal feed additive that degrades phytic acid and reduces feeding costs and pollution caused by fecal excretion of phosphorus. Some phytases have been expressed in Pichia pastoris, among which the phytase from Citrobacter amalonaticus CGMCC 1696 had high specific activity (3548 U/mg). Improvement of the phytase expression level will contribute to facilitate its industrial applications.MethodsTo improve the phytase expression, we use modification of P AOX1 and the α-factor signal peptide, increasing the gene copy number, and overexpressing HAC1 (i) to enhance folding and secretion of the protein in the endoplasmic reticulum. The genetic stability and fermentation in 10-L scaled-up fed-batch fermenter was performed to prepare for the industrial production.ResultsThe phytase gene from C. amalonaticus CGMCC 1696 was cloned under the control of the AOX1 promoter (P AOX1 ) and expressed in P. pastoris. The phytase activity achieved was 414 U/mL. Modifications of P AOX1 and the α-factor signal peptide increased the phytase yield by 35 and 12%, respectively. Next, on increasing the copy number of the Phy gene to six, the phytase yield was 141% higher than in the strain containing only a single gene copy. Furthermore, on overexpression of HAC1 (i) (i indicating induced), a gene encoding Hac1p that regulates the unfolded protein response, the phytase yield achieved was 0.75 g/L with an activity of 2119 U/mL, 412% higher than for the original strain. The plasmids in this high-phytase expression strain were stable during incubation at 30 °C in Yeast Extract Peptone Dextrose (YPD) Medium. In a 10-L scaled-up fed-batch fermenter, the phytase yield achieved was 9.58 g/L with an activity of 35,032 U/mL.DiscussionThe production of a secreted protein will reach its limit at a specific gene copy number where further increases in transcription and translation due to the higher abundance of gene copies will not enhance the secretion process any further. Enhancement of protein folding in the ER can alleviate bottlenecks in the folding and secretion pathways during the overexpression of heterologous proteins in P. pastoris.ConclusionsUsing modification of P AOX1 and the α-factor signal peptide, increasing the gene copy number, and overexpressing HAC1 (i) to enhance folding and secretion of the protein in the endoplasmic reticulum, we have successfully increased the phytase yield 412% relative to the original strain. In a 10-L fed-batch fermenter, the phytase yield achieved was 9.58 g/L with an activity of 35,032 U/mL. Large-scale production of phytase can be applied towards different biocatalytic and feed additive applications.
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Project description:α-Galactosidases are broadly used in feed, food, chemical, pulp, and pharmaceutical industries. However, there lacks a satisfactory microbial cell factory that is able to produce α-galactosidases efficiently and cost-effectively to date, which prevents these important enzymes from greater application. In this study, the secretory expression of an Aspergillus niger α-galactosidase (AGA) in Pichia pastoris was systematically investigated. Through codon optimization, signal peptide replacement, comparative selection of host strain, and saturation mutagenesis of the P1' residue of Kex2 protease cleavage site for efficient signal peptide removal, a mutant P. pastoris KM71H (Muts) strain of AGA-I with the specific P1' site substitution (Glu to Ile) demonstrated remarkable extracellular α-galactosidase activity of 1299 U/ml upon a 72 h methanol induction in 2.0 L fermenter. The engineered yeast strain AGA-I demonstrated approximately 12-fold higher extracellular activity compared to the initial P. pastoris strain. To the best of our knowledge, this represents the highest yield and productivity of a secreted α-galactosidase in P. pastoris, thus holding great potential for industrial application.
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Project description:Comparison of transcription profile of Pichia pastoris cells grown on Glucose medium with Pichia pastoris cells grown on Methanol/Glycerol medium, the fermentations were done in a chemostat.
2015-08-31 | GSE67690 | GEO