Protein profile in Aspergillus nidulans recombinant strains overproducing heterologous enzymes

Filamentous fungi are robust cell factories and have been used for the production of large quantities of industrially relevant enzymes. However, the production levels of heterologous proteins still need to be improved. Therefore, this article aimed to investigate the global proteome profiling of Aspergillus nidulans recombinant strains in order to understand the bottlenecks of heterologous enzymes production. About 250, 441 and 424 intracellular proteins were identified in the control strain Anid_pEXPYR and in the recombinant strains Anid_AbfA and Anid_Cbhl respectively. In this context, the most enriched processes in recombinant strains were energy pathway, amino acid metabolism, ribosome biogenesis, translation, endoplasmic reticulum and oxidative stress, and repression under secretion stress (RESS). The global protein profile of the recombinant strains Anid_AbfA and Anid_Cbhl was similar, although the latter strain secreted more recombinant enzyme than the former. These findings provide insights into the bottlenecks involved in the secretion of recombinant proteins in A. nidulans, as well as in regard to the rational manipulation of target genes for engineering fungal strains as microbial cell factories.     

Glucose-induced production of a Penicillium purpurogenum xylanase by Aspergillus nidulans

The heterologous secretion of xylanase B from Penicillium purpurogenum using glucose as inducer was performed in Aspergillus nidulans. For this purpose, plasmid pEVXB, containing the xylanase B cDNA (including its own signal peptide) under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter, was constructed and used to transform A. nidulans. Analysis of transformed clones showed that several of them secreted extracellular xylanase activity when grown in a medium containing glucose. The clone showing the highest xylanase activity was chosen for further work. When this clone was grown on glucose, xylanase activity (0.72 U/ml), was detected in the culture supernatant. This was confirmed by a zymogram analysis and by the amplification of xynB cDNA from this clone. To our knowledge, this is the first example of the production of a xylanase from Penicillium in heterologous fungal hosts using glucose as inducer.     

<Emphasis Type="Italic">Streptomyces lividans</Emphasis> and <Emphasis Type="Italic">Brevibacterium lactofermentum</Emphasis> as heterologous hosts for the production of X<Subscript>22</Subscript> xylanase from <Emphasis Type="Italic">Aspergillus nidulans</Emphasis>

The Aspergillus nidulans gene xlnA coding for the fungal xylanase X₂₂ has been cloned and expressed in two heterologous bacterial hosts: Streptomyces lividans and Brevibacterium lactofermentum. Streptomyces strains yielded 10 units/ml of xylanase when the protein was produced with its own signal peptide, and 19 units/ml when its signal peptide was replaced by the one for xylanase Xys1 from Streptomyces halstedii. B. lactofermentum was also able to produce xylanase X₂₂, affording 6 units/ml upon using either the Aspergillus xlnA signal peptide or Streptomyces xysA. These production values are higher than those previously reported for the heterologous expression of the A. nidulans xlnA gene in Saccharomyces cerevisiae (1 unit/ml). Moreover, the X₂₂ enzyme produced by Streptomyces lividans showed oenological properties, indicating that this Streptomyces recombinant strain is a good candidate for the production of this enzyme at the industrial scale.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> signal peptidase recognizes and cleaves the signal sequence of xylanase from a newly isolated <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain R5

A gene encoding the xylanase from Bacillus subtilis strain R5 containing the native signal sequence was cloned and expressed in Escherichia coli. The heterologous expression of the gene resulted in the production of the recombinant protein in the cytoplasm as well as its secretion into the culture medium. The xylanase activity in the culture medium increased with time after induction up to 90% of the total activity in 14 h. Molecular mass and N-terminal amino acid sequence determinations of the purified recombinant xylanase revealed that the native signal peptide was cleaved off by E. coli signal peptidases between Ala28 and Ala29.     

Creation of a heterologous gene expression system on the basis of <Emphasis Type="Italic">Aspergillus awamori</Emphasis> recombinant strain

A heterologous gene expression system was created in a domestic Aspergillus awamori Co-6804 strain, which is a producer of glucoamylase. Vector pGa was prepared using promoter and terminator areas of the glucoamylase gene, and Aspergillus niger phytase, Trichoderma reesei endoglucanase, and Penicillium canescens xylanase genes were then cloned into pGa vector. Separation of enzyme samples using FPLC showed the amount of the recombinant proteins to be within the 0.6–14% range of total protein.     

Approaches for refining heterologous protein production in filamentous fungi

Fungi combine the advantages of a microbial system such as a simple fermentability with the capability of secreting proteins that are modified according to a general eukaryotic scheme. Filamentous fungi such as Aspergillus niger efficiently secrete genuine proteins but the secretion of recombinant proteins turned out be a difficult task. Aspergillus niger is an attractive organism because of its high secretion capacity and is frequently used as a model organism. Whereas high production yields can be obtained when homologous proteins are expressed, much lower amounts are obtained with the production of heterologous proteins. To fully exploit the potential of filamentous fungi, understanding of the molecular genetics, their physiology, and the glycosylation metabolism has to be investigated and clarified in more detail. This review summarizes recent developments in heterologous protein production by filamentous fungi and also generalizes the possibilities of improving the protein production by various genetic and bioprocessing approaches, thereby easing recognition of filamentous fungi as a relevant and reliable expression platform.     

MHF组蛋白折叠复合体组分编码基因MHF1过表达对重组酿酒酵母纤维素酶生产的影响

【背景】重组酿酒酵母可用于生产多种药用蛋白和工业酶等外源蛋白,但蛋白分泌水平低是限制其异源蛋白高效生产的重要因素。异源蛋白表达和分泌过程可能会对宿主细胞产生多种胁迫,因此,研究胁迫响应相关基因对重组酵母异源蛋白生产的影响具有重要意义。Mhf1p是MHF组蛋白折叠复合体的组分之一,与DNA损伤修复及维持基因组稳定性有关,但其对异源蛋白生产的作用尚不清楚。【目的】研究MHF1过表达对重组酿酒酵母蛋白生产的影响。【方法】在分泌表达纤维素酶的重组酿酒酵母菌株中利用基于CRISPR-Cas9的基因组编辑技术整合过表达MHF1,分析其对产酶的影响,并探讨影响产酶的分子机理。【结果】与出发菌株相比,过表达MHF1菌株的外切纤维素酶CBH酶活性提高了38%。对过表达MHF1的CBH生产菌株中蛋白合成和分泌途径相关基因转录水平进行检测,发现与对照菌株相比,CBH1基因和与分泌相关的SEC22、ERV29等基因在不同时间点呈现不同程度显著上调。【结论】MHF1过表达可促进酿酒酵母异源外切纤维素酶的生产,并影响外源酶基因和分泌途径基因的表达,可能通过对多基因的协同表达影响促进产酶。     

Cloning and expression of the chloroplast-encoded rbcL and rbcS genes from the marine diatom Cylindrotheca sp. strain N1

Both the rbcL and rbcS genes, encoding the large and small subunits, respectively, of ribulose 1,5-bisphosphate carboxylase/oxygenase, have been found to be encoded by chloroplast DNA in the marine diatom Cylindrotheca sp. N1. The rbcS gene in this diatom was found to be adjacent to the rbcL gene by a combination of: (i) Southern-blotting analyses, using heterologous probes; (ii) examination of recombinant proteins synthesized in Escherichia coli, directed by cloned rbcL/rbcS genes; and (iii) synthesis of enzymatically active heterologous Rubisco protein in vivo by recombinant DNA procedures using large subunits of Anacystis nidulans and small subunits of Cylindrotheca sp. N1. It appears that two copies of rbcL and rbcS genes are encoded by the chloroplast DNA of this diatom.     

Amino acid supplementation improves heterologous protein production by Saccharomyces cerevisiae in defined medium

Supplementation of a chemically defined medium with amino acids or succinate to improve heterologous xylanase production by a prototrophic Saccharomyces cerevisiae transformant was investigated. The corresponding xylanase production during growth on ethanol in batch culture and in glucose-limited chemostat culture were quantified, as the native ADH2 promoter regulating xylanase expression was derepressed under these conditions. The addition of a balanced mixture of the preferred amino acids, Ala, Arg, Asn, Glu, Gln and Gly, improved both biomass and xylanase production, whereas several other individual amino acids inhibited biomass and/or xylanase production. Heterologous protein production by the recombinant yeast was also improved by supplementing the medium with succinate. The production of heterologous xylanase during growth on ethanol or glucose could thus be improved by supplementing metabolic precursors in the carbon- or nitrogen-metabolism.     

Two distinct regions in the model protein Peb1 are critical for its heterologous transport out of <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Escherichia coli is frequently the first-choice host organism in expression of heterologous recombinant proteins in basic research as well as in production of commercial, therapeutic polypeptides. Especially the secretion of proteins into the culture medium of E. coli is advantageous compared to intracellular production due to the ease in recovery of the recombinant protein. Since E. coli naturally is a poor secretor of proteins, a few strategies for optimization of extracellular secretion have been described. We have previously reported efficient secretion of the diagnostically interesting model protein Peb1 of Campylobacter jejuni into the growth medium of Escherichia coli strain MKS12 (ΔfliCfliD). To generate a more detailed understanding of the molecular mechanisms behind this interesting heterologous secretion system with biotechnological implications, we here analyzed further the transport of Peb1 in the E. coli host.     

Implications of cellobiohydrolase glycosylation for use in biomass conversion

The cellulase producing ascomycete, Trichoderma reesei (Hypocrea jecorina), is known to secrete a range of enzymes important for ethanol production from lignocellulosic biomass. It is also widely used for the commercial scale production of industrial enzymes because of its ability to produce high titers of heterologous proteins. During the secretion process, a number of post-translational events can occur, however, that impact protein function and stability. Another ascomycete, Aspergillus niger var. awamori, is also known to produce large quantities of heterologous proteins for industry. In this study, T. reesei Cel7A, a cellobiohydrolase, was expressed in A. niger var. awamori and subjected to detailed biophysical characterization. The purified recombinant enzyme contains six times the amount of N-linked glycan than the enzyme purified from a commercial T. reesei enzyme preparation. The activities of the two enzyme forms were compared using bacterial (microcrystalline) and phosphoric acid swollen (amorphous) cellulose as substrates. This comparison suggested that the increased level of N-glycosylation of the recombinant Cel7A (rCel7A) resulted in reduced activity and increased non-productive binding on cellulose. When treated with the N-glycosidase PNGaseF, the molecular weight of the recombinant enzyme approached that of the commercial enzyme and the activity on cellulose was improved.     

毕赤酵母外源蛋白分泌途径的研究进展

毕赤酵母作为一种重要的表达外源蛋白的宿主,提高其外源蛋白的分泌量非常有必要。近年来很多学者报道了与毕赤酵母外源蛋白分泌相关的基因、蛋白质,同时毕赤酵母基因组的公布加快了这方面的研究进展。文章根据外源蛋白分泌的途径,分步骤地总结了涉及的基因和蛋白,有利于分析控制蛋白分泌效率的具体步骤,为构建更加高效的毕赤酵母表达系统提供参考。     

Analysis of the role of the gene bipA, encoding the major endoplasmic reticulum chaperone protein in the secretion of homologous and heterologous proteins in black Aspergilli

The function of the endoplasmic-reticulum-localized chaperone binding protein (BiP) in relation to protein secretion in filamentous fungi was studied. It was shown that the overproduction of several homologous and heterologous recombinant proteins by Aspergillus strains induces the expression of bipA, the BiP-encoding gene from Aspergillus niger and Aspergillus awamori. As this result could imply that BiP plays a role in protein overproduction, the effect of modulation of bipA gene expression on protein secretion was studied in several recombinant strains expressing glucoamylase (glaA) fusion genes. For overproduction of BiPA in these strains, extra copies of the bipA gene under the control of an inducible promoter were introduced. To allow analysis of the effect of a decreased bipA expression level on protein secretion, replacement of the wild-type gene for a bipA gene driven by the glaA promoter was attempted. However, this endeavour failed because of the lethality of this replacement. Although the final amount of secreted recombinant protein did not change significantly in strains with increased BiPA levels, increased levels of unprocessed fusion protein were detected in the total protein extracts of these strains. Received: 9 February 1998 / Received last revision: 26 May 1998 / Accepted: 14 June 1998     

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.     

Chaperone and foldase coexpression in the baculovirus-insect cell expression system

Conclusions The BEVS has become widely utilized for production of recombinant proteins. However, protein aggregation and inefficient processing often limit yields, especially for secreted and membrane proteins. Since many proteins of pharmaceutical interest require similar posttranslational processing steps, engineering the folding, assembly, and secretion pathway may enhance the production of a wide variety of valuable complex proteins. Efforts should be undertaken to coexpress the relevant chaperones or foldases at low levels in concert with the final product to ensure the ideal folding and assembly environment. In the future, expression of oligosaccharide modifying enzymes and secretion factors may further improve secretion rates of assembled proteins and provide heterologous proteins with altered glycoforms. Also significant is the use of BEVS as an in vivo eucaryotic laboratory to study the fundamental roles of differnt chaperones, foldases, and secretion factors. The coexpression of chaperones and foldases will complement other approaches such as the development of alternative insect cell lines, promoters, and signal peptides to optimize the baculovirus-insect cell expression system for generating high yields of valuable proteins.Abbreviations BEVS Baculovirus expression vector system - BiP immunoglobulin heavy chain binding protein - ELISA Enzyme-linked immunosorbent assay - ER Endoplasmic reticulum - GRP Glucose regulated protein - Hsp Heat shock protein - IgG Immunoglobulin G - PDI Protein Disulfide Isomerase - PPI Peptidyl-prolyl cis-trans isomerase - Sf-9 Spodoptera frugeperda     

<Emphasis Type="Italic">pspA</Emphasis> overexpression in <Emphasis Type="Italic">Streptomyces lividans</Emphasis> improves both Sec- and Tat-dependent protein secretion

Streptomyces is an interesting host for the secretory production of recombinant proteins because of its innate capacity to secrete proteins at high level in the culture medium. In this report, we evaluated the importance of the phage-shock protein A (PspA) homologue on the protein secretion yield in Streptomyces lividans. The PspA protein is supposed to play a role in the maintenance of the proton motive force (PMF). As the PMF is an energy source for both Sec- and Tat-dependent secretion, we evaluated the influence of the PspA protein on both pathways by modulating the pspA expression. Results indicated that pspA overexpression can improve the Tat-dependent protein secretion as illustrated for the Tat-dependent xylanase C and enhanced green fluorescent protein (EGFP). The effect on Sec-dependent secretion was less pronounced and appeared to be protein dependent as evidenced by the increase in subtilisin inhibitor (Sti-1) secretion but the lack of increase in human tumour necrosis factor (hTNFα) secretion in a pspA-overexpressing strain.     

Heterologous protein secretion by Lactobacillus plantarum using homologous signal peptides

Aims: To test seven selected putative signal peptides from Lactobacillus plantarum WCFS1 in terms of their ability to drive secretion of two model proteins in Lact. plantarum, and to compare the functionality of these signal peptides with that of well‐known heterologous signal peptides (Usp45, M6). Methods and Results: Signal peptide functionality was assessed using a series of modular derivatives of the pSIP vectors for peptide pheromone‐controlled high‐level gene expression in lactobacilli. Several of the constructs with homologous signal peptides yielded similar or higher reporter protein activities than constructs with heterologous signal peptides. Two of the homologous signal peptides (Lp_0373 and Lp_0600) appeared as especially promising candidates for directing secretion, as they were among the best performing with both reporter proteins. Conclusions: We have identified homologous signal peptides for high‐level secretion of heterologous proteins in Lact. plantarum. With the model proteins, some of these performed better than commonly used heterologous signal peptides. Significance and Impact of the Study: The homologous signal peptides tested out, in this study, could be useful in food‐grade systems for secretion of interesting proteins in Lact. plantarum. The constructed modular secretion vectors are easily accessible for rapid signal peptide screening.