scholarly journals Contributions of the Pre- and Pro-Regions of a Staphylococcus hyicus Lipase to Secretion of a Heterologous Protein by Bacillus subtilis

2009 ◽  
Vol 76 (3) ◽  
pp. 659-669 ◽  
Author(s):  
Thijs R. H. M. Kouwen ◽  
Allan K. Nielsen ◽  
Emma L. Denham ◽  
Jean-Yves F. Dubois ◽  
Ronald Dorenbos ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Signal Peptide ◽  
Stress Responses ◽  
Heterologous Protein ◽  
Surrounding Medium ◽  
Wall Stress ◽  
Secretory Proteins ◽  
Cell Factory ◽  
Heterologous Proteins ◽  
Established Cell

ABSTRACT Bacillus subtilis is a well-established cell factory for efficient secretion of many biotechnologically relevant enzymes that are naturally produced by it or related organisms. However, the use of B. subtilis as a host for production of heterologous secretory proteins can be complicated by problems related to inefficient translocation of the foreign proteins across the plasma membrane or to inefficient release of the exported proteins from the cell surface into the surrounding medium. Therefore, there is a clear need for tools that allow more efficient membrane targeting, translocation, and release during the production of these proteins. In the present study, we investigated the contributions of the pre (prelip) and pro (prolip) sequences of a Staphylococcus hyicus lipase to secretion of a heterologous protein, the alkaline phosphatase PhoA of Escherichia coli, by B. subtilis. The results indicate that the presence of the prolip-peptide, in combination with the lipase signal peptide (prelip), contributes significantly to the efficient secretion of PhoA by B. subtilis and that prelip directs PhoA secretion more efficiently than the authentic signal peptide of PhoA. Genome-wide transcriptional analyses of the host cell responses indicate that, under the conditions tested, no known secretion or membrane-cell wall stress responses were provoked by the production of PhoA with any of the pre- and pro-region sequences used. Our data underscore the view that the pre-pro signals of the S. hyicus lipase are very useful tools for secretion of heterologous proteins in B. subtilis.

scholarly journals Augmented peroxisomal ROS buffering capacity renders oxidative and thermal stress cross-tolerance in yeast

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Nai-Xin Lin ◽  
Rui-Zhen He ◽  
Yan Xu ◽  
Xiao-Wei Yu
Keyword(s):  
Thermal Stress ◽  
Production Efficiency ◽  
Heterologous Protein ◽  
Redox Balance ◽  
Industrial Applications ◽  
Ros Generation ◽  
Cell Factory ◽  
Heterologous Proteins ◽  
Cross Tolerance ◽  
Autophagy Pathway

Abstract Background Thermotolerant yeast has outstanding potential in industrial applications. Komagataella phaffii (Pichia pastoris) is a common cell factory for industrial production of heterologous proteins. Results Herein, we obtained a thermotolerant K. phaffii mutant G14 by mutagenesis and adaptive evolution. G14 exhibited oxidative and thermal stress cross-tolerance and high heterologous protein production efficiency. The reactive oxygen species (ROS) level and lipid peroxidation in G14 were reduced compared to the parent. Oxidative stress response (OSR) and heat shock response (HSR) are two major responses to thermal stress, but the activation of them was different in G14 and its parent. Compared with the parent, G14 acquired the better performance owing to its stronger OSR. Peroxisomes, as the main cellular site for cellular ROS generation and detoxification, had larger volume in G14 than the parent. And, the peroxisomal catalase activity and expression level in G14 was also higher than that of the parent. Excitingly, the gene knockdown of CAT encoding peroxisomal catalase by dCas9 severely reduced the oxidative and thermal stress cross-tolerance of G14. These results suggested that the augmented OSR was responsible for the oxidative and thermal stress cross-tolerance of G14. Nevertheless, OSR was not strong enough to protect the parent from thermal stress, even when HSR was initiated. Therefore, the parent cannot recover, thereby inducing the autophagy pathway and resulting in severe cell death. Conclusions Our findings indicate the importance of peroxisome and the significance of redox balance in thermotolerance of yeasts.

scholarly journals Impact of overproduced heterologous protein characteristics on physiological response in Yarrowia lipolytica steady-state-maintained continuous cultures

2020 ◽  
Vol 104 (22) ◽  
pp. 9785-9800
Author(s):  
Paulina Korpys-Woźniak ◽  
Piotr Kubiak ◽  
Wojciech Białas ◽  
Ewelina Celińska
Keyword(s):  
Steady State ◽  
Yarrowia Lipolytica ◽  
Heterologous Protein ◽  
Target Protein ◽  
Host Cells ◽  
Synthesis Process ◽  
Secretory Proteins ◽  
Heterologous Proteins ◽  
Glycosylation Pattern ◽  
The Impact

Abstract Overproduction of recombinant secretory proteins triggers numerous physiological perturbations. Depending on a given heterologous protein characteristics, the producer cell is faced with different challenges which lead to varying responses in terms of its physiology and the target protein production rate. In the present study, we used steady-state-maintained Yarrowia lipolytica cells to investigate the impact of different heterologous proteins on the physiological behavior of the host cells. Such an approach allowed to uncouple the impact of the overproduction of a particular protein from the phenomena that result from growth phase or are caused by the heterogeneity of the analyzed populations. Altogether, eight variants of recombinant strains, individually overproducing heterologous proteins of varying molecular weight (27–65 kDa) and reporting activity (enzymatic and fluorescent) were subjected to chemostat cultivations. The steady-state-maintained cells were analyzed in terms of the substrate utilization, biomass and metabolites production, as well as the reporter protein synthesis. Simplified distribution of carbon and nitrogen between the respective products, as well as expression analysis of the heterologous genes were conducted. The here-obtained data suggest that using a more transcriptionally active promoter results in channeling more C flux towards the target protein, giving significantly higher specific amounts and production rates of the target polypeptide, at the cost of biomass accumulation, and with no significant impact on the polyols production. The extent of the reporter protein’s post-translational modifications, i.e., the number of disulfide bonds and glycosylation pattern, strongly impacts the synthesis process. Specific responses in terms of the protein formation kinetics, the gene expression levels, and transcript-to-protein linearity were observed. Key Points • Eight expression systems, producing different reporter proteins were analyzed. • The cells were maintained in steady-state by continuous chemostat culturing. • Protein- and promoter-specific effects were observed.

scholarly journals Engineering of The Bacillus Subtilis PhoD Signal Peptide To Direct High-Level, Tat-Specific Export of A Single-Chain Antibody Fragment To The Periplasm in Eschericha Coli

2020 ◽  
Author(s):  
Chillel Jawara ◽  
Kirsty L Richards ◽  
Amber R Peswani ◽  
Kelly L Walker ◽  
Lara Nascimento ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Signal Peptide ◽  
Antibody Fragment ◽  
Signal Peptides ◽  
Heterologous Proteins ◽  
Single Chain ◽  
Single Chain Antibody ◽  
E Coli ◽  
Tat Pathway ◽  
Single Chain Antibody Fragment

Abstract Background: Numerous high-value proteins have been produced in E. coli, and a favoured strategy is to export the protein of interest to the periplasm by means of an N-terminal signal peptide. While the Sec pathway has been extensively used for this purpose, the Tat pathway has potential because it transports fully-folded heterologous proteins. Most studies on the Tat pathway have used the E. coli TorA signal peptide to direct export, because it is highly Tat-specific, unlike many Tat signal peptides which can also function as Sec signal peptides. However, the TorA signal peptide is prone to degradation in the cytoplasm, leading to reduced export rates in some cases. Here, we have tested a range of alternative signal peptides for their ability to direct Tat-dependent export of a single-chain antibody fragment (scFv). Results: We show that the signal peptides of E. coli AmiC, MdoD and YcbK direct efficient export of the scFv by both the Tat and Sec pathways, which may be a disadvantage when Tat-specific export is required. The same applies to the Tat signal peptide of Bacillus subtilis PhoD, which likewise directs efficient export by Sec. We engineered the PhoD signal peptide by introduction of a Lys or Asn residue in the C-terminal domain of the signal peptide, and we show that this substitution renders the signal peptide Tat-specific. These signal peptides, designated PhoDk and PhoDn, direct efficient export of scFv in shake flask and fed-batch fermentation studies, reaching export levels that are well above those obtained with the TorA signal peptide. Culturing in ambr250 bioreactors was used to fine-tune the growth conditions, and the net result was export of the scFv by the Tat pathway at levels of approximately 1g protein/L culture. Conclusions: The new PhoDn and PhoDk signal peptides have significant potential for the export of heterologous proteins by the Tat system.

scholarly journals Boosting heterologous protein production yield by adjusting global nitrogen and carbon metabolic regulatory networks inBacillus subtilis

2018 ◽  
Author(s):  
Haojie Cao ◽  
Julio Villatoro-Hernandez ◽  
Ruud Detert Oude Weme ◽  
Elrike Frenzel ◽  
Oscar P. Kuipers
Keyword(s):  
Bacillus Subtilis ◽  
Nutrient Intake ◽  
Regulatory Networks ◽  
Protein Production ◽  
Heterologous Protein ◽  
Heterologous Protein Production ◽  
Cell Factory ◽  
Regulatory Activity ◽  
Dna Binding Domains ◽  
Binding Domains

AbstractBacillus subtilisis extensively applied as a microorganism for the high-level production of heterologous proteins. Traditional strategies for increasing the productivity of this microbial cell factory generally focused on the targeted modification of rate-limiting components or steps. However, the longstanding problems of limited productivity of the expression host, metabolic burden and non-optimal nutrient intake, have not yet been solved to achieve production strain improvements. To tackle this problem, we systematically rewired the regulatory networks of the global nitrogen and carbon metabolism by random mutagenesis of the pleiotropic transcriptional regulators CodY and CcpA, to allow for optimal nutrient intake, translating into significantly higher heterologous protein production yields. Using a β-galactosidase expression and screening system and consecutive rounds of mutagenesis, we identified mutant variants of both CcpA and CodY that in conjunction increased production levels up to 290%. RNA-Seq and electrophoretic gel mobility shift analyses showed that amino acid substitutions within the DNA-binding domains altered the overall binding specificity and regulatory activity of the two transcription factors. Consequently, fine-tuning of the central metabolic pathways allowed for enhanced protein production levels. The improved cell factory capacity was further demonstrated by the successfully increased overexpression of GFP, xylanase and a peptidase in the double mutant strain.HighlightsThe global transcription machinery engineering (gTME) technique was applied to build mutational libraries of the pleiotropic regulators CodY and CcpA inBacillus subtilisSpecific point mutations within the DNA-binding domains of CodY and CcpA elicited alterations of the binding specificity and regulatory activityChanges in the transcriptome evoked the reprogramming of networks that gear the carbon and nitrogen metabolismThe rewired metabolic networks provided a higher building block capacity for heterologous protein production by adjusting the nutrient uptake and channeling its utilization for protein overexpression

In Silico Evaluation of Various Signal Peptides to Improve Secretion of Humulin Protein in E. coli Host

2020 ◽  
Vol 17 ◽  
Author(s):  
Soudabe Kavousipour ◽  
Mahadi Barazesh ◽  
Shiva Mohammadi ◽  
Meghdad Abdollahpour- Alitappeh ◽  
Shirzad Fallahi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Signal Peptide ◽  
In Silico ◽  
Signal Sequence ◽  
Expression Vectors ◽  
Secretory Proteins ◽  
Signal Peptides ◽  
Heterologous Proteins ◽  
Genetic Characteristics ◽  
E Coli

Background:: Escherichia coli host has been the workhorse for the production of heterologous proteins due to simplicity of use, low cost, availability of various expression vectors, and widespread knowledge on its genetic characteristics, but without a suitable signal sequence, this host cannot be used for production secretory proteins. Humulin is a form of insulin used to treat hyperglycemia caused by types 1 and 2 diabetes. To improve expression and make a straightforward production of Humulin protein, we chose a series of signal peptides. Objective:: aim our study to predict the most excellent signal peptides to express secretory Humulin in E. coli organisms. Method:: Therefore, to forecast the most excellent signal peptides for expression of Humulin in Escherichia coli, 47 signal sequences from bacteria organisms were elected and the most imperative elements of them were studied. Hence, signal peptide probability along with physicochemical features was evaluated by signal 4.1, and Portparam, PROSO II servers respectively. Later, the in-silico cloning in a known pET28a plasmid system also estimated the possibility of best signal peptide+ Humulin expression in E.Coli. Results:: The outcomes demonstrated among 47 signal peptides only 2 signal peptides can be suggested as suitable signal peptides. Conclusion:: Ultimately protein yebF precursor (YEBF_ECOLI) and protein yebF precursor (YEBF_YERP3) were suggested severally; as the most excellent signal peptides to express Humulin (With D scores 0.812 and 0.623 respectively). Although verification of these results want experimental analysis.

Recent Advances in Recombinant Protein Production by Bacillus subtilis

2020 ◽  
Vol 11 (1) ◽  
pp. 295-318 ◽  
Author(s):  
Kang Zhang ◽  
Lingqia Su ◽  
Jing Wu
Keyword(s):  
Bacillus Subtilis ◽  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Genetic Manipulation ◽  
Microbial Cell ◽  
Secretory Proteins ◽  
Cell Factory ◽  
Microbial Cell Factory ◽  
Recent Advances

Bacillus subtilis has become a widely used microbial cell factory for the production of recombinant proteins, especially those associated with foods and food processing. Recent advances in genetic manipulation and proteomic analysis have been used to greatly improve protein production in B. subtilis. This review begins with a discussion of genome-editing technologies and application of the CRISPR–Cas9 system to B. subtilis. A summary of the characteristics of crucial legacy strains is followed by suggestions regarding the choice of origin strain for genetic manipulation. Finally, the review analyzes the genes and operons of B. subtilis that are important for the production of secretory proteins and provides suggestions and examples of how they can be altered to improve protein production. This review is intended to promote the engineering of this valuable microbial cell factory for better recombinant protein production.

scholarly journals The YoaW signal peptide directs efficient secretion of different heterologous proteins fused to a StrepII-SUMO tag in Bacillus subtilis

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Janine Heinrich ◽  
Chris Drewniok ◽  
Eva Neugebauer ◽  
Harald Kellner ◽  
Thomas Wiegert
Keyword(s):  
Bacillus Subtilis ◽  
Signal Peptide ◽  
Heterologous Proteins

scholarly journals Signal Peptide and Propeptide Optimization for Heterologous Protein Secretion in Lactococcus lactis

2001 ◽  
Vol 67 (9) ◽  
pp. 4119-4127 ◽  
Author(s):  
Y. Le Loir ◽  
S. Nouaille ◽  
J. Commissaire ◽  
L. Brétigny ◽  
A. Gruss ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Protein Secretion ◽  
Signal Peptide ◽  
Heterologous Protein ◽  
Staphylococcal Nuclease ◽  
Heterologous Proteins ◽  
Heterologous Protein Secretion ◽  
Precursor Proteins ◽  
Secretion Efficiency

ABSTRACT Lactic acid bacteria are food-grade microorganisms that are potentially good candidates for production of heterologous proteins of therapeutical or technological interest. We developed a model for heterologous protein secretion in Lactococcus lactis using the staphylococcal nuclease (Nuc). The effects on protein secretion of alterations in either (i) signal peptide or (ii) propeptide sequences were examined. (i) Replacement of the native Nuc signal peptide (SPNuc) by that of L. lactis protein Usp45 (SPUsp) resulted in greatly improved secretion efficiency (SE). Pulse-chase experiments showed that Nuc secretion kinetics was better when directed by SPUsp than when directed by SPNuc. This SPUsp effect on Nuc secretion is not due to a better antifolding activity, since SPUsp:Nuc precursor proteins display enzymatic activity in vitro, while SPNuc:Nuc precursor proteins do not. (ii) Deletion of the native Nuc propeptide dramatically reduces Nuc SE, regardless of which SP is used. We previously reported that a synthetic propeptide, LEISSTCDA, could efficiently replace the native Nuc propeptide to promote heterologous protein secretion in L. lactis (Y. Le Loir, A. Gruss, S. D. Ehrlich, and P. Langella, J. Bacteriol. 180:1895–1903, 1998). To determine whether the LEISSTCDA effect is due to its acidic residues, specific substitutions were introduced, resulting in neutral or basic propeptides. Effects of these two new propeptides and of a different acidic synthetic propeptide were tested. Acidic and neutral propeptides were equally effective in enhancing Nuc SE and also increased Nuc yields. In contrast, the basic propeptide strongly reduced both SE and the quantity of secreted Nuc. We have shown that the combination of the native SPUsp and a neutral or acidic synthetic propeptide leads to a significant improvement in SE and in the quantity of synthesized Nuc. These observations will be valuable in the production of heterologous proteins in L. lactis.

scholarly journals In silico analysis of signal peptides for secretory production of a-amylase in Bacillus subtilis

2019 ◽  
pp. 113-124
Author(s):  
Marzieh Asadi ◽  
Morgaza Taheri-Anganeh ◽  
Zeinab Jamali ◽  
Seyyed Hossein Khatami ◽  
Cambyz Irajie ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Signal Peptide ◽  
In Silico ◽  
In Silico Analysis ◽  
Essential Elements ◽  
Secretory Proteins ◽  
Signal Peptides ◽  
Experimental Production ◽  
Secretory Production ◽  
Peptide Secretion

α-Amylases are important commercial enzymes and have a broad application in industrial processes and medicine. Gram-positive bacteria such as Bacillus subtilis are possible host organisms for α-amylases secretory production. Secretion of α-amylases to the culture medium versus intracellular production has several advantages such as prevention of inclusion bodies accumulation, higher product stability and solubility. Signal peptides are considered as one of the most essential elements for successful secretory synthesis of the recombinant proteins. Therefore, by the selection of an efficient signal peptide, secretion of the recombinant protein can be enhanced. The goal of this investigation was the in silico evaluation of several peptides to find the most suitable leader peptides for secretory production of α-amylase in B. subtilis. In present work, 30 signal peptides were selected, and numerous online servers such as SignalP, ProtParam, SOLpro, PRED-TAT and ProtComp was used for investigation of suitable signal peptides. According to in silico predictions all other signal peptides connected to α-amylase were stable and soluble except PPBD_BACSU. PPBD_BACSU because of having D-score below cut-off could not be recognized as a suitable signal peptide for α-amylase. Computational analysis identified QOX2_BACSU may direct protein into transmembrane location and was ignored. All 28 remained were predicted as secretory signal peptides which can excrete protein out of the bacteria. The signal peptides recommended by the present study are valuable for rational designing of secretory soluble α-amylase. Although, such information can be useful for future experimental production of these mentioned secretory proteins.

scholarly journals The Twin-Arginine Signal Peptide of Bacillus subtilis YwbN Can Direct either Tat- or Sec-Dependent Secretion of Different Cargo Proteins: Secretion of Active Subtilisin via the B. subtilis Tat Pathway

2008 ◽  
Vol 74 (24) ◽  
pp. 7507-7513 ◽  
Author(s):  
Marc A. B. Kolkman ◽  
René van der Ploeg ◽  
Michael Bertels ◽  
Maurits van Dijk ◽  
Joop van der Laan ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Signal Peptide ◽  
Heterologous Proteins ◽  
Independent Manner ◽  
Sec Pathway ◽  
Tat Pathway ◽  
Cargo Proteins ◽  
Dependent Protein ◽  
Application Potential ◽  
Tat System

ABSTRACT Proteins that are produced for commercial purposes in Bacillus subtilis are commonly secreted via the Sec pathway. Despite its high secretion capacity, the secretion of heterologous proteins via the Sec pathway is often unsuccessful. Alternative secretion routes, like the Tat pathway, are therefore of interest. Two parallel Tat pathways with distinct specificities have previously been discovered in B. subtilis. To explore the application potential of these Tat pathways, several commercially relevant or heterologous model proteins were fused to the signal peptides of the known B. subtilis Tat substrates YwbN and PhoD. Remarkably, the YwbN signal peptide directed secretion of active subtilisin, a typical Sec substrate, via the B. subtilis TatAyCy route. In contrast, the same signal peptide directed Tat-independent secretion of the Bacillus licheniformis α-amylase (AmyL). Moreover, the YwbN signal peptide directed secretion of SufI, an Escherichia coli Tat substrate, in a Tat-independent manner, most likely via Sec. Our results suggest that cytoplasmic protein folding prior to translocation is probably a major determinant of Tat-dependent protein secretion in B. subtilis, as is the case with E. coli. We conclude that future applications for the Tat system of B. subtilis will most likely involve commercially interesting proteins that are Sec incompatible.

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