Secretion Of Heterologous Proteins Research Articles

Establishment of Neurospora crassa as a host for heterologous protein production using a human antibody fragment as a model product

BackgroundFilamentous fungi are commonly used as production hosts for bulk enzymes in biotechnological applications. Their robust and quick growth combined with their ability to secrete large amounts of protein directly into the culture medium makes fungi appealing organisms for the generation of novel production systems. The red bread mold Neurospora crassa has long been established as a model system in basic research. It can be very easily genetically manipulated and a wealth of molecular tools and mutants are available. In addition, N. crassa is very fast growing and non-toxic. All of these features point to a high but so far untapped potential of this fungus for biotechnological applications. In this study, we used genetic engineering and bioprocess development in a design-build-test-cycle process to establish N. crassa as a production host for heterologous proteins.ResultsThe human antibody fragment HT186-D11 was fused to a truncated version of the endogenous enzyme glucoamylase (GLA-1), which served as a carrier protein to achieve secretion into the culture medium. A modular expression cassette was constructed and tested under the control of different promoters. Protease activity was identified as a major limitation of the production strain, and the effects of different mutations causing protease deficiencies were compared. Furthermore, a parallel bioreactor system (1 L) was employed to develop and optimize a production process, including the comparison of different culture media and cultivation parameters. After successful optimization of the production strain and the cultivation conditions an exemplary scale up to a 10 L stirred tank reactor was performed.ConclusionsThe data of this study indicate that N. crassa is suited for the production and secretion of heterologous proteins. Controlling expression by the optimized promoter Pccg1nr in a fourfold protease deletion strain resulted in the successful secretion of the heterologous product with estimated yields of 3 mg/L of the fusion protein. The fungus could easily be cultivated in bioreactors and a first scale-up was successful. The system holds therefore much potential, warranting further efforts in optimization.

Expression and secretion of fungal endoglucanase II and chimeric cellobiohydrolase I in the oleaginous yeast Lipomyces starkeyi

BackgroundLipomyces starkeyi is one of the leading lipid-producing microorganisms reported to date; its genetic transformation was only recently reported. Our aim is to engineer L. starkeyi to serve in consolidated bioprocessing (CBP) to produce lipid or fatty acid-related biofuels directly from abundant and low-cost lignocellulosic substrates.ResultsTo evaluate L. starkeyi in this role, we first conducted a genome analysis, which revealed the absence of key endo- and exocellulases in this yeast, prompting us to select and screen four signal peptides for their suitability for the overexpression and secretion of cellulase genes. To compensate for the cellulase deficiency, we chose two prominent cellulases, Trichoderma reesei endoglucanase II (EG II) and a chimeric cellobiohydrolase I (TeTrCBH I) formed by fusion of the catalytic domain from Talaromyces emersonii CBH I with the linker peptide and cellulose-binding domain from T. reesei CBH I. The systematically tested signal peptides included three peptides from native L. starkeyi and one from Yarrowia lipolytica. We found that all four signal peptides permitted secretion of active EG II. We also determined that three of these signal peptides worked for expression of the chimeric CBH I; suggesting that our design criteria for selecting these signal peptides was effective. Encouragingly, the Y. lipolytica signal peptide was able to efficiently guide secretion of the chimeric TeTrCBH I protein from L. starkeyi. The purified chimeric TeTrCBH I showed high activity against the cellulose in pretreated corn stover and the purified EG II showed high endocellulase activity measured by the CELLG3 (Megazyme) method.ConclusionsOur results suggest that L. starkeyi is capable of expressing and secreting core fungal cellulases. Moreover, the purified EG II and chimeric TeTrCBH I displayed significant and potentially useful enzymatic activities, demonstrating that engineered L. starkeyi has the potential to function as an oleaginous CBP strain for biofuel production. The effectiveness of the tested secretion signals will also benefit future secretion of other heterologous proteins in L. starkeyi and, given the effectiveness of the cross-genus secretion signal, possibly other oleaginous yeasts as well.

Novel signal peptides improve the secretion of recombinant Staphylococcus aureus Alpha toxinH35L in Escherichia coli

Secretion of heterologous proteins into Escherichia coli cell culture medium offers significant advantages for downstream processing over production as inclusion bodies; including cost and time savings, and reduction of endotoxin. Signal peptides play an important role in targeting proteins for translocation across the cytoplasmic membrane to the periplasmic space and release into culture medium during the secretion process. Alpha toxinH35L (ATH35L) was selected as an antigen for vaccine development against Staphylococcus aureus infections. It was successfully secreted into culture medium of E. coli by using bacterial signal peptides linked to the N-terminus of the protein. In order to improve the level of secreted ATH35L, we designed a series of novel signal peptides by swapping individual domains of modifying dsbA and pelB signal peptides and tested them in a fed-batch fermentation process. The data showed that some of the modified signal peptides improved the secretion efficiency of ATH35L compared with E. coli signal peptides from dsbA, pelB and phoA proteins. Indeed, one of the novel signal peptides improved the yield of secreted ATH35L by 3.5-fold in a fed-batch fermentation process and at the same time maintained processing at the expected site for signal peptide cleavage. Potentially, these new novel signal peptides can be used to improve the secretion efficiency of other heterologous proteins in E. coli. Furthermore, analysis of the synthetic signal peptide amino acid sequences provides some insight into the sequence features within the signal peptide that influence secretion efficiency.

Disruption of genes involved in CORVET complex leads to enhanced secretion of heterologous carboxylesterase only in protease deficient Pichia pastoris.

The methylotrophic yeast Pichia pastoris (Komagataella spp.) is a popular microbial host for the production of recombinant proteins. Previous studies have shown that mis-sorting to the vacuole can be a bottleneck during production of recombinant secretory proteins in yeast, however, no information was available for P. pastoris. In this work the authors have therefore generated vps (vacuolar protein sorting) mutant strains disrupted in genes involved in the CORVET (class C core vacuole/endosome tethering) complex at the early stages of endosomal sorting. Both Δvps8 and Δvps21 strains contained lower extracellular amounts of heterologous carboxylesterase (CES) compared to the control strain, which could be attributed to a high proteolytic activity present in the supernatants of CORVET engineered strains due to rerouting of vacuolar proteases. Serine proteases were identified to be responsible for this proteolytic degradation by liquid chromatography-mass spectrometry and protease inhibitor assays. Deletion of the major cellular serine protease Prb1 in Δvps8 and Δvps21 strains did not only rescue the extracellular CES levels, but even outperformed the parental CES strain (56 and 80% higher yields, respectively). Further deletion of Ybr139W, another serine protease, did not show a further increase in secretion levels. Higher extracellular CES activity and low proteolytic activity were detected also in fed batch cultivation of Δvps21Δprb1 strains, thus confirming that modifying early steps in the vacuolar pathway has a positive impact on heterologous protein secretion.

A Secretion-Amplification Role for Salmonella enterica Translocon Protein SipD.

The bacterial type III secretion system (T3SS) is an important target for enabling high-titer production of proteins of biotechnological interest as well as for synthetic biology applications that rely on protein delivery to host cells. The T3SS forms a membrane-embedded needle complex that is capped by the translocon proteins and extends into the extracellular space. The needle tip complex in Salmonella enterica consists of three translocon proteins: SipB, SipC, and SipD. It is known that knocking out sipD disrupts T3SS regulation to cause constitutive secretion of native proteins. However, we discovered that complementation of SipD in trans via exogenous addition to T3SS-expressing cultures further improves heterologous protein secretion titers, suggesting a previously unknown but important role for this protein. Building on this knowledge, we have engineered a hyper-secreting strain of S.enterica for a greater than 100-fold improvement in the production of a variety of biotechnologically valuable heterologous proteins that are challenging to produce, such as toxic antimicrobial peptides and proteolysis-prone biopolymer proteins. We determined that transcription by several T3SS promoters is upregulated with the addition of SipD, that the N-terminal domain of SipD is sufficient to observe the increased secretion phenotype, and that the effect is post-transcriptional and post-translational. These results lend support to the use of bacterial secretion as a powerful protein production strategy, and the hypothesis that translocon proteins contribute to type III secretion regulation.

An Artificial Design Technique to Optimize Signal Peptide

To determine optimal artificial signal peptide candidates for the possibility of creating high levels of secretion of heterologous proteins, substitution and redesign of amino acid sequences in the H-domain of the signal peptide was theoretically attempted. The method was based on comprehensive score matrix and Markov transfer matrix, which can make the artificial sequences maintain the structural characteristics and original polarity of signal peptides. For the artificial sequence, the feature vector of Structural Fusion Degree (SFD) is first extracted to quantitatively describe the compatibility of artificial cleaved region, then by comparing with highly secreted natural samples; tendencies of specific substitutions in the amino acid sequence can be identified at certain locations. These substitutions may represent the key amino acids that influence the secretion and expression levels of heterologous proteins.

Beyond amino acids: Use of the Corynebacterium glutamicum cell factory for the secretion of heterologous proteins

The Gram-positive soil bacterium Corynebacterium glutamicum has a long tradition in industry as a potent cell factory for the production of various amino acids. Besides this, in the last few years it became increasingly clear that this microorganism can also efficiently be used as a host organism for the expression and secretion of biotechnologically or pharmaceutically relevant heterologous proteins. In this review, first a short overview is given on the two main protein export pathways (Sec and Tat) of C. glutamicum that can be exploited for the transport of heterologous target proteins across the cytoplasmic membrane. Subsequently, the current knowledge on the successful use of C. glutamicum for the secretory production of an already impressive variety of heterologous proteins derived from different pro- and eukaryotic sources is summarized, whereby a special emphasis is given on the various optimization strategies and tools that have recently been developed and that now can be used to establish and further improve C. glutamicum as a secretory expression platform for the production of any desired heterologous target protein.

Recombinant production of the antibody fragment D1.3 scFv with different Bacillus strains

BackgroundDifferent strains of the genus Bacillus are versatile candidates for the industrial production and secretion of heterologous proteins. They can be cultivated quite easily, show high growth rates and are usually non-pathogenic and free of endo- and exotoxins. They have the ability to secrete proteins with high efficiency into the growth medium, which allows cost-effective downstream purification processing. Some of the most interesting and challenging heterologous proteins are recombinant antibodies and antibody fragments. They are important and suitable tools in medical research for analytics, diagnostics and therapy. The smallest conventional antibody fragment with high-affinity binding to an antigen is the single-chain fragment variable (scFv). Here, different strains of the genus Bacillus were investigated using diverse cultivation systems for their suitability to produce and secret a recombinant scFv.ResultsExtracellular production of lysozyme-specific scFv D1.3 was realized by constructing a plasmid with a xylose-inducible promoter optimized for Bacillus megaterium and the D1.3scFv gene fused to the coding sequence of the LipA signal peptide from B. megaterium. Functional scFv was successfully secreted with B. megaterium MS941, Bacillus licheniformis MW3 and the three Bacillus subtilis strains 168, DB431 and WB800N differing in the number of produced proteases. Starting with shake flasks (150 mL), the bioprocess was scaled down to microtiter plates (1250 µL) as well as scaled up to laboratory-scale bioreactors (2 L). The highest extracellular concentration of D1.3 scFv (130 mg L−1) and highest space–time-yield (8 mg L−1 h−1) were accomplished with B. subtilis WB800N, a strain deficient in eight proteases. These results were reproduced by the production and secretion of a recombinant penicillin G acylase (Pac).ConclusionsThe genus Bacillus provides high potential microbial host systems for the secretion of challenging heterologous proteins like antibody fragments and large proteins at high titers. In this study, the highest extracellular concentration and space–time-yield of a recombinant antibody fragment for a Gram-positive bacterium so far was achieved. The successful interspecies use of the here-designed plasmid originally optimized for B. megaterium was demonstrated by two examples, an antibody fragment and a penicillin G acylase in up to five different Bacillus strains.

Erratum to: Cloning and Expression of Metagenomic DNA in Streptomyces lividans and Subsequent Fermentation for Optimized Production.

The choice of an expression system for the metagenomic DNA of interest is of vital importance for the detection of any particular gene or gene cluster. Most of the screens to date have used the gram-negative bacterium Escherichia coli as a host for metagenomic gene libraries. However, the use of E. coli introduces a potential host bias since only 40 % of the enzymatic activities may be readily recovered by random cloning in E. coli. To recover some of the remaining 60 %, alternative cloning hosts such as Streptomyces spp. have been used. Streptomycetes are high-GC gram-positive bacteria belonging to the Actinomycetales and they have been studied extensively for more than 15 years as an alternative expression system. They are extremely well suited for the expression of DNA from other actinomycetes and genomes of high GC content. Furthermore, due to its high innate, extracellular secretion capacity, Streptomyces can be a better system than E. coli for the production of many extracellular proteins. In this article an overview is given about the materials and methods for growth and successful expression and secretion of heterologous proteins from diverse origin using Streptomyces lividans has a host. More in detail, an overview is given about the protocols of transformation, type of plasmids used and of vectors useful for integration of DNA into the host chromosome, and accompanying cloning strategies. In addition, various control elements for gene expression including synthetic promoters are discussed, and methods to compare their strength are described. Integration of the gene of interest under the control of the promoter of choice into S. lividans chromosome via homologous recombination using pAMR4-based system is explained. Finally a basic protocol for benchtop bioreactor experiments which can form the start in the production process optimization and upscaling is provided.

New Progress regarding the Use of Lactic Acid Bacteria as Live Delivery Vectors, Treatment of Diseases and Induction of Immune Responses in Different Host Species Focusing on <i>Lactobacillus</i> Species

Lactobacillus species are non-spore-forming, gram-positive bacteria and lactic acid producing bacteria (LAPB) that naturally inhabit the human and animal gastrointestinal and mouth organs. The aim of this review was to evaluate the new progress regarding the use of Lactobacillus species as live delivery vectors, prevention, and treatment of pathogenic and metabolic diseases. Lactobacillus strains of probiotics have been extensively studied and have confirmed that they can absolutely improve performance as live delivery vectors, a treatment option of various diseases such as: Hemorrhagic cecal coccidiosis in young poultry, hypertension, avian flu, obesity, diabetes, Derzsy’s disease or parvovirus infection, human immunodeficiency virus infections, irritable bowel syndrome, gastrointestinal disorders, Fungal infections, vaginal eubiosis, fish and shellfish species diseases. We give you an idea about that Lactobacillus species have been proficient in preventing and treating both disorders in animal models and some are used for clinical trials. We present the most current studies on the use of Lactobacillus strains that had an impact on an effective immune response to a specific antigen because a variety of antigens have been expressed. Therefore Lactobacillus strains can be considered as good candidates because of its potential for diseases treatment and vaccine development as heterologous protein secretion to date.

New Progress Regarding the Use of Lactic Acid Bacteria as Live Delivery Vectors, Treatment of Diseases and Induction of Immune Responses in Different Host Species Focusing on Lactobacillus Species

Lactobacillus species are non-spore-forming, Gram-positive bacteria and lactic acid producing bacteria (LAPB) that naturally inhabits the human and animal gastrointestinal and mouth organs. The aim of this review was to evaluate the new progress regarding the use of Lactobacillus species as live delivery vectors, prevention and treatment of pathogenic and metabolic diseases. Lactobacillus strains as probiotics have been extensively studied and have confirmed that they can absolutely improve performance as live delivery vectors, a treatment option of various diseases such as: hemorrhagic cecal coccidiosis in young poultry, Hypertension, avian flu, obesity, diabetes, Derzsy's disease or parvovirus infection, human immunodeficiency virus infections, irritable bowel syndrome, gastrointestinal disorders, Fungal infections, vaginal eubiosis, fish and shellfish species diseases. We give you an idea about that Lactobacillus species have been proficient in preventing and treating both disorders in animal models and some used for clinical trials. We present the most current studies on the use of Lactobacillus strains that had an impact on an effective immune response to a specific antigen because a variety of antigens have been expressed. Therefore Lactobacillus strains can be considered as good candidates because of its potential for diseases treatment and vaccine development as heterologous protein secretion to date.

The Three Streptomyces lividans HtrA-Like Proteases Involved in the Secretion Stress Response Act in a Cooperative Manner.

Overproduction of Sec-proteins in S. lividans accumulates misfolded proteins outside of the cytoplasmic membrane where the accumulated proteins interfere with the correct functioning of the secretion machinery and with the correct cell functionality, triggering the expression in S. lividans of a CssRS two-component system which regulates the degradation of the accumulated protein, the so-called secretion stress response. Optimization of secretory protein production via the Sec route requires the identification and characterisation of quality factors involved in this process. The phosphorylated regulator (CssR) interacts with the regulatory regions of three genes encoding three different HtrA-like proteases. Individual mutations in each of these genes render degradation of the misfolded protein inoperative, and propagation in high copy number of any of the three proteases encoding genes results on indiscriminate alpha-amylase degradation. None of the proteases could complement the other two deficiencies and only propagation of each single copy protease gene can restore its own deficiency. The obtained results strongly suggest that the synthesis of the three HtrA-like proteases needs to be properly balanced to ensure the effective degradation of misfolded overproduced secretory proteins and, at the same time, avoid negative effects in the secreted proteins and the secretion machinery. This is particularly relevant when considering the optimisation of Streptomyces strains for the overproduction of homologous or heterologous secretory proteins of industrial application.

Cloning and Expression of Metagenomic DNA in Streptomyces lividans and Subsequent Fermentation for Optimized Production.

The choice of an expression system for the metagenomic DNA of interest is of vital importance for the detection of any particular gene or gene cluster. Most of the screens to date have used the gram-negative bacterium Escherichia coli as a host for metagenomic gene libraries. However, the use of E. coli introduces a potential host bias since only 40 % of the enzymatic activities may be readily recovered by random cloning in E. coli. To recover some of the remaining 60 %, alternative cloning hosts such as Streptomyces spp. have been used. Streptomycetes are high-GC gram-positive bacteria belonging to the Actinomycetales and they have been studied extensively for more than 15 years as an alternative expression system. They are extremely well suited for the expression of DNA from other actinomycetes and genomes of high GC content. Furthermore, due to its high innate, extracellular secretion capacity, Streptomyces can be a better system than E. coli for the production of many extracellular proteins. In this article an overview is given about the materials and methods for growth and successful expression and secretion of heterologous proteins from diverse origin using Streptomyces lividans has a host. More in detail, an overview is given about the protocols of transformation, type of plasmids used and of vectors useful for integration of DNA into the host chromosome, and accompanying cloning strategies. In addition, various control elements for gene expression including synthetic promoters are discussed, and methods to compare their strength are described. Integration of the gene of interest under the control of the promoter of choice into S. lividans chromosome via homologous recombination using pAMR4-based system is explained. Finally a basic protocol for benchtop bioreactor experiments which can form the start in the production process optimization and upscaling is provided.

Use of Transcriptional Control to Increase Secretion of Heterologous Proteins in T3S Systems.

Heterologous proteins can be produced in a bacterial host and purified from the cellular constituents. Secretion of the protein of interest to the extracellular space simplifies the purification process and is thought to alleviate toxicity problems associated with intracellular accumulation of the protein of interest. In this protocol, we describe a strategy to engineer protein secretion in a bacterial culture using transcriptional control. The transcription factor HilA is inducibly produced to control production of the secretion machine, and in turn signals the production and secretion of a protein of interest. This allows for high titer of secreted protein in optimized culturing conditions and the effect is observed with all proteins tested.

Enhancing heterologous protein expression and secretion in HEK293 cells by means of combination of CMV promoter and IFNα2 signal peptide

Efficient production and secretion of recombinant proteins in mammalian cell lines relies in a combination of genetic, metabolic and culture strategy factors. The present work assesses the influence of two key genetic components of expression vectors (promoter and signal peptide) on protein production and secretion effciency in HEK293 cells expressing eGFP as a reporter protein. Firstly, the strength of 3 different promoters was evaluated using transient expression methods. Flow cytometry analysis revealed that the highest level of intracellular protein expression was found when eGFP was under the control of CMV promoter, being 3-times higher in comparison to the rest of the promoters tested. Secondly, 5 different signal peptides were assessed in stable transfected cell lines. Spectrofluorometry was used to determine intra- and extracellular protein expression levels in terms of fluorescence, and the results were further confirmed by SDS-PAGE. The highest secretion efficiency was found for human IFNα2 signal peptide, achieving up to 2-fold increase in the amount of secreted protein compared to other signal peptides. The results showed that the combination of CMV promoter and IFNα2 signal peptide resulted highly efficient for recombinant protein production in HEK293 cells.

Effects of co-overexpression of secretion helper factors on the secretion of a HSA fusion protein (IL2-HSA) in pichia pastoris.

Pichia pastoris is generally considered as an expression host for heterologous proteins with the coding gene under control of the alcohol oxidase 1 (AOX1) promoter. The secretion of heterologous proteins in P. pastoris can be potentially affected by many factors. Based on our previous results, the secretion levels of human albumin (HSA) fusion protein IL2-HSA were only around 500 mg/L or less in fermentor cultures, which decreased more than 50% compared with that of HSA (>1 g/L). In this study, we selected five potential secretion helper factors, in which Ero1, Pdi1 and Kar2 were involved in protein folding and Sec1 and Sly1 were involved in vesicle trafficking. We evaluated the possible effects of individual overexpression of these secretion helper factors on the secretion of IL2-HSA in P. pastoris. Constitutive overexpression of the five selected secretion factors did not have an obvious negative effect on cell growth of the IL2-HSA secreting strain. Individual co-overexpression of Ero1, Kar2, Pdi1, Sec1 and Sly1 improved the secretion level of IL2-HSA to ~2.3-, 1.9-, 2.2-, 2.5- and 1.9-fold that in the control strain respectively in shake flasks. We evaluated the changes in mRNA and protein levels of the intracellular IL2-HSA, as well as the secretion helper factor genes in the co-overexpressing strains. Our results indicated that manipulating the expression level of ER resident protein Pdi1, Ero1, Kar2 and SM protein Sec1 and Sly1 could improve the secretion level of IL2-HSA fusion protein in P. pastoris, which provided new candidates for combinatorial engineering in future study. Copyright © 2016 John Wiley & Sons, Ltd.

Secretion of M2e:HBc fusion protein by Lactobacillus casei using Cwh signal peptide.

The ability to serve as a delivery vehicle for various interesting biomolecules makes lactic acid bacteria (LAB) very useful in several applications. In the medical field, recombinant LAB expressing pathogenic antigens at different cellular locations have been used to elicit both mucosal and systemic immune responses. Expression–secretion vectors (ESVs) with a signal peptide (SP) are pivotal for protein expression and secretion. In this study, the genome sequence of Lactobacillus casei ATCC334 was explored for new SPs using bioinformatics tools. Three new SPs of the proteins Cwh, SurA and SP6565 were identified and used to construct an ESV based on our Escherichia coli–L. casei shuttle vector, pRCEID-LC13.9. Functional testing of these constructs with the green fluorescence protein (GFP) gene showed that they could secrete the GFP. The construct with CwhSP showed the highest GFP secretion. Consequently, CwhSP was selected to develop an ESV construct carrying a synthetic gene encoding the extracellular domain of the matrix 2 protein fused with the hepatitis B core antigen (M2e:HBc). This ESV was shown to efficiently express and secrete the M2e:HBc fusion protein. The identified SPs and the developed ESVs can be exploited for expression and secretion of homologous and heterologous proteins in L. casei.

Highly active promoters and native secretion signals for protein production during extremely low growth rates in Aspergillus niger.

BackgroundThe filamentous ascomycete Aspergillus niger is used in many industrial processes for the production of enzymes and organic acids by batch and fed-batch cultivation. An alternative technique is continuous cultivation, which promises improved yield and optimized pipeline efficiency.ResultsIn this work, we have used perfusion (retentostat) cultivation to validate two promoters that are suitable for A. niger continuous cultivation of industrially relevant products. Firstly, promoters of genes encoding either an antifungal protein (Panafp) or putative hydrophobin (PhfbD) were confirmed as active throughout retentostat culture by assessing mRNA and protein levels using a luciferase (mluc) reporter system. This demonstrated the anafp promoter mediates a high but temporally variable expression profile, whereas the hfbD promoter mediates a semi-constant, moderate-to-high protein expression during retentostat culture. In order to assess whether these promoters were suitable to produce heterologous proteins during retentostat cultivation, the secreted antifungal protein (AFP) from Aspergillus giganteus, which has many potential biotechnological applications, was expressed in A. niger during retentostat cultivation. Additionally, this assay was used to concomitantly validate that native secretion signals encoded in anafp and hfbD genes can be harnessed for secretion of heterologous proteins. Afp mRNA and protein abundance were comparable to luciferase measurements throughout retentostat cultivation, validating the use of Panafp and PhfbD for perfusion cultivation. Finally, a gene encoding the highly commercially relevant thermal hysteresis protein (THP) was expressed in this system, which did not yield detectable protein.ConclusionBoth hfbD and anafp promoters are suitable for production of useful products in A. niger during perfusion cultivation. These findings provide a platform for further optimisations for high production of heterologous proteins with industrial relevance.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0543-2) contains supplementary material, which is available to authorized users.

Mapping N-linked glycosylation of carbohydrate-active enzymes in the secretome of Aspergillus nidulans grown on lignocellulose.

BackgroundThe genus Aspergillus includes microorganisms that naturally degrade lignocellulosic biomass, secreting large amounts of carbohydrate-active enzymes (CAZymes) that characterize their saprophyte lifestyle. Aspergillus has the capacity to perform post-translational modifications (PTM), which provides an additional advantage for the use of these organisms as a host for the production of heterologous proteins. In this study, the N-linked glycosylation of CAZymes identified in the secretome of Aspergillus nidulans grown on lignocellulose was mapped.ResultsAspergillus nidulans was grown in glucose, xylan and pretreated sugarcane bagasse (SCB) for 96 h, after which glycoproteomics and glycomics were carried out on the extracellular proteins (secretome). A total of 265 proteins were identified, with 153, 210 and 182 proteins in the glucose, xylan and SCB substrates, respectively. CAZymes corresponded to more than 50 % of the total secretome in xylan and SCB. A total of 182 N-glycosylation sites were identified, of which 121 were detected in 67 CAZymes. A prevalence of the N-glyc sequon N-X-T (72.2 %) was observed in N-glyc sites compared with N-X-S (27.8 %). The amino acids flanking the validated N-glyc sites were mainly composed of hydrophobic and polar uncharged amino acids. Selected proteins were evaluated for conservation of the N-glyc sites in Aspergilli homologous proteins, but a pattern of conservation was not observed. A global analysis of N-glycans released from the proteins secreted by A. nidulans was also performed. While the proportion of N-glycans with Hex5 to Hex9 was similar in the xylan condition, a prevalence of Hex5 was observed in the SCB and glucose conditions.ConclusionsThe most common and frequent N-glycosylated motifs, an overview of the N-glycosylation of the CAZymes and the number of mannoses found in N-glycans were analyzed. There are many bottlenecks in protein production by filamentous fungi, such as folding, transport by vesicles and secretion, but N-glycosylation in the correct context is a fundamental event for defining the high levels of secretion of target proteins. A comprehensive analysis of the protein glycosylation processes in A. nidulans will assist with a better understanding of glycoprotein structures, profiles, activities and functions. This knowledge can help in the optimization of heterologous expression and protein secretion in the fungal host.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0580-4) contains supplementary material, which is available to authorized users.

OmpA signal peptide leads to heterogenous secretion of B. subtilis chitosanase enzyme from E. coli expression system.

The production of secreted recombinant proteins from E. coli is pivotal to the biotechnological industry because it reduces the cost of downstream processing. Proteins destined for secretion contain an N-terminal signal peptide that is cleaved by secretion machinery in the plasma membrane. The resulting protein is released in an active mature form. In this study, Bacillus subtilis chitosanase (Csn) was used as a model protein to compare the effect of two signal peptides on the secretion of heterologous recombinant protein. The results showed that the E. coli secretion machinery could recognize both native bacillus and E. coli signal peptides. However, only the native bacillus signal peptide could generate the same N-terminal sequence as in the wild type bacteria. When the recombinant Csn constructs contained the E. coli OmpA signal peptide, the secreted enzymes were heterogeneous, comprising a mixed population of secreted enzymes with different N-terminal sequences. Nevertheless, the E. coli OmpA signal peptide was found to be more efficient for high expression and secretion of bacillus Csn. These findings may be used to help engineer other recombinant proteins for secretory production in E. coli.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-2893-y) contains supplementary material, which is available to authorized users.