Efficient production of secreted proteins by Aspergillus : progress, limitations and prospects

Filamentous fungi are widely used for the production of homologous and heterologous proteins but, compared to homologous proteins, the levels of production of heterologous proteins are usually low. During the last 5 years, the levels of production of heterologous proteins have been drastically improved by fusing the corresponding gene to the 3' end of a homologous gene, encoding a well-secreted protein such as glucoamylase. Nevertheless, little research has been carried out to determine the limitations that hamper heterologous protein production. Recently we have carried out a detailed analysis of the levels of production of several proteins and glucoamylase fusion proteins in defined recombinant Aspergillus awamori strains. In this review we will focus on the use of filamentous fungi for the production of heterologous, especially non-fungal, proteins. In particular, the effect of gene-fusion strategies will be reviewed. Furthermore, the remaining limitations in heterologous protein production and suggestions for improvement strategies for overproduction of these protein will be discussed. Received: 5 July 1996 / Accepted: 6 September 1996     

Ethanol production and fermentation characteristics of recombinant saccharomyces cerevisiae strains grown on starch

The production of ethanol from starch has been investigated in three genetically modified Saccharomyces cerevisiae strains (YPG/AB, YPG/MM, and YPB-G). Two of the three strains produce the Aspergillus awamori glucoamylase together with either the Bacillus subtilis (YPG/AB) or the mouse (YPG/MM) α-amylase as separately secreted polypeptides. YPB-G, on the other hand, secretes a bifunctional fusion protein that contains both the B. subtilis α-amylase and the A. awamori glucoamylase activities. Substrate utilization, biomass growth, and ethanol production were all studied in both starch- and glucose-containing media. Much higher growth rates were found when any of the three strains were grown on glucose. YPG/AB showed the most efficient utilization of starch for ethanol production with the lowest levels of reducing sugars accumulating in the medium. The superior performance of YPG/AB as compared to YPB-G was found to correlate with its higher level of α-amylase activity. The ethanol production levels of YPG/AB in starch- and glucose-containing media were found to be comparable. YPB-G, which secretes the bifunctional fusion protein, could produce ethanol in media with starch concentrations above 100 g l⁻¹ while YPG/MM did not produce ethanol from starch because of its negligible secretion of glucoamylase.     

Effects of a defective ERAD pathway on growth and heterologous protein production in <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Endoplasmic reticulum associated degradation (ERAD) is a conserved mechanism to remove misfolded proteins from the ER by targeting them to the proteasome for degradation. To assess the role of ERAD in filamentous fungi, we have examined the consequences of disrupting putative ERAD components in the filamentous fungus Aspergillus niger. Deletion of derA, doaA, hrdC, mifA, or mnsA in A. niger yields viable strains, and with the exception of doaA, no significant growth phenotype is observed when compared to the parental strain. The gene deletion mutants were also made in A. niger strains containing single- or multicopies of a glucoamylase–glucuronidase (GlaGus) gene fusion. The induction of the unfolded protein response (UPR) target genes (bipA and pdiA) was dependent on the copy number of the heterologous gene and the ERAD gene deleted. The highest induction of UPR target genes was observed in ERAD mutants containing multiple copies of the GlaGus gene. Western blot analysis revealed that deletion of the derA gene in the multicopy GlaGus overexpressing strain resulted in a 6-fold increase in the intracellular amount of GlaGus protein detected. Our results suggest that impairing some components of the ERAD pathway in combination with high expression levels of the heterologous protein results in higher intracellular protein levels, indicating a delay in protein degradation.     

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     

N-Glycan Modification in Aspergillus Species

The production by filamentous fungi of therapeutic glycoproteins intended for use in mammals is held back by the inherent difference in protein N-glycosylation and by the inability of the fungal cell to modify proteins with mammalian glycosylation structures. Here, we report protein N-glycan engineering in two Aspergillus species. We functionally expressed in the fungal hosts heterologous chimeric fusion proteins containing different localization peptides and catalytic domains. This strategy allowed the isolation of a strain with a functional α-1,2-mannosidase producing increased amounts of N-glycans of the Man₅GlcNAc₂ type. This strain was further engineered by the introduction of a functional GlcNAc transferase I construct yielding GlcNAcMan₅GlcNac₂ N-glycans. Additionally, we deleted algC genes coding for an enzyme involved in an early step of the fungal glycosylation pathway yielding Man₃GlcNAc₂ N-glycans. This modification of fungal glycosylation is a step toward the ability to produce humanized complex N-glycans on therapeutic proteins in filamentous fungi.     

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.     

Determination of the maximum product yield from glucoamylase-producing Aspergillus niger grown in the recycling fermentor

Aspergillus niger has been grown in glucose- and maltose-limited recycling cultures to determine the maximum growth yield, the maximum product yield for glucoamylase production, and the maintenance requirements at very slow specific growth rates. Using the linear equation for substrate utilization, and using the experimental data from both recycling experiments, both the maximum growth yield, Y_xsm, and the maximum product yield, Y_psm, could be determined. The values estimated were 157 g biomass per mol maltose for Y_xsm and 100 g protein per mol maltose for Y_psm. Expressed on a C₁-basis these values are 0.52 and 0.36 C-mole per C-mol for respectively Y_xsm and Y_psm. The found value for Y_psm is half the value found for alkaline serine protease production in Bacillus lichoniformis, and it can be concluded that formation of extracellular protein is more energy consuming in filamentous fungi than in prokaryotic organisms. Maintenance requirements are no significant factor during growth of Aspergillus niger, and reported maintenance requirements are most probably due to differentiation.     

High Level Secretion of Calf Chymosin Using a Glucoamylase-prochymosin Fusion Gene in Aspergillus oryzae

A recombinant chymosin was secreted at high levels using fusion genes with A. oryzae glucoamylase gene (glaA) and a wheat bran solid-state culture system. Two portions of the A. oryzae glucoamylase, one with almost the entire glucoamylase (GA1–603) lacking 9 amino acids at the carboxyl terminal, and the other (GA1–511) lacking the starch binding-domain, were fused in frame with prochymosin cDNA. Western blot analysis indicated that the mature chymosin was released from the secreted fusion protein by autocatalytic processing. The transformant harboring the GA1-511-prochymosin construct showed about 5-fold chymosin production of the transformant in which the chymosin gene was directly expressed under the control of the glaA promoter in submerged culture. Moreover, wheat bran solid-state culture gave about 500-fold higher yield of the chymosin (approximately 150 mg/kg wheat bran) compared with the submerged culture.     

米曲霉异源蛋白表达系统研究进展及展望

米曲霉作为一种重要的工业微生物,在异源蛋白表达方面已有广泛应用,受限于被表达蛋白的修饰及分泌过程,目前实际生产使用的基因供体主要局限于其他真菌,尤其是丝状真菌。当外源基因来源于植物、昆虫和哺乳动物时,米曲霉所生产的异源蛋白产量及生物活性往往不尽如人意。本文综述了米曲霉作为宿主表达异源蛋白的研究进展,包括其现有的遗传操作手段及异源表达方面的应用及探索,重点介绍了应用过程中面临的挑战和解决策略,另外,对米曲霉表达异源蛋白的应用前景及发展方向进行了展望。     

Protoplast Fusion in Streptomyces sp. for Increased Production of Laccase and Associated Ligninolytic Enzymes

Biodegradation of lignin by Streptomyces spp. results in the production of value-added chemicals such as Acid Precipitable Polymeric Lignins (APPLs), low molecular weight phenols, etc. To hasten the conversion metabolism through genetic manipulation, a preliminary attempt was made to standardize the methodology for isolation and regeneration of protoplasts. Protoplast fusion recombinants were developed and assayed for their ligninolytic activity, production of ligninolytic enzymes viz., peroxidase, laccase, polyphenol oxidase and crude protein. In comparison with the mutants and wild strain, fusion recombinant F₄ showed higher laccase activity and lower peroxidase activity. This attribute can be positively used for polymerization of free phenolics to polycondensates related to humic acids in soil or composting environments. This revised version was published online in July 2006 with corrections to the Cover Date.     

ChMCO1 of Cochliobolus heterostrophus is a new class of metallo-oxidase, playing an important role in DHN-melanization

A metallo-oxidase gene from a phytopathogenic filamentous fungus, Cochliobolus heterostrophus was cloned. Structural prediction of ChMco1 indicated that this protein lacks a transmembrane helix and is soluble, whereas other known fungal metallo-oxidases including Saccharomyces cerevisiae FET3 are localized to the cell membrane. The results of searches in fungal genomic databases and phylogenetic analysis of fungal metallo-oxidases revealed that ChMco1 and its allies are distinct homologues of Fet3 and unique to filamentous ascomycetous species including C. heterostrophus. We performed a functional analysis of ChMCO1 by generating null mutants for the ChMco1 gene. The ChMco1 null (∆ChMco1) mutants clearly had reduced melanization, although they showed normal growth and conidiation. Results also show that ∆ChMco1 mutants lost laccase activity. These results suggest that ChMCO1 is a novel class of metallo-oxidase that is necessary for laccase activity and melanization.     

A simple structured model for biomass and extracellular enzyme production with recombinant Saccharomyces cerevisiae YPB-G

A simple structured model is proposed for simulating batch cultivation data on growth, substrate utilization, and heterologous enzyme production of recombinant Saccharomyces cerevisiae YPB-G. The enzyme is a fusion protein displaying α-amylase and glucoamylase activities. Cell growth is modulated mainly by intracellular substrate and ethanol concentrations. Intracellular substrate concentration is evaluated by means of the extracellular substrate and biomass concentrations. Extracellular α-amylase and glucoamylase activities are taken to depend on biomass concentration. The nine parameters of the proposed model are determined using nonlinear estimation techniques, and the model is validated against experiments not used in parameter determination. The model developed simulates glucose consumption, cell mass, α-amylase and glucoamylase production in a batch system. Simulation and experimental results are found to be in good agreement. Journal of Industrial Microbiology & Biotechnology (2002) 29, 111–116 doi:10.1038/sj.jim.7000281 Received 07 January 2002/ Accepted in revised form 22 May 2002     

Modulating Endoplasmic Reticulum-Golgi Cargo Receptors for Improving Secretion of Carrier-Fused Heterologous Proteins in the Filamentous Fungus Aspergillus oryzae

Filamentous fungi are excellent hosts for industrial protein production due to their superior secretory capacity; however, the yield of heterologous eukaryotic proteins is generally lower than that of fungal or endogenous proteins. Although activating protein folding machinery in the endoplasmic reticulum (ER) improves the yield, the importance of intracellular transport machinery for heterologous protein secretion is poorly understood. Here, using Aspergillus oryzae as a model filamentous fungus, we studied the involvement of two putative lectin-like cargo receptors, A. oryzae Vip36 (AoVip36) and AoEmp47, in the secretion of heterologous proteins expressed in fusion with the endogenous enzyme α-amylase as the carrier. Fluorescence microscopy revealed that mDsRed-tagged AoVip36 localized in the Golgi compartment, whereas AoEmp47 showed localization in both the ER and the Golgi compartment. Deletion of AoVip36 and AoEmp47 improved heterologous protein secretion, but only AoVip36 deletion had a negative effect on the secretion of α-amylase. Analysis of ER-enriched cell fractions revealed that AoVip36 and AoEmp47 were involved in the retention of heterologous proteins in the ER. However, the overexpression of each cargo receptor had a different effect on heterologous protein secretion: AoVip36 enhanced the secretion, whereas AoEmp47 promoted the intracellular retention. Taken together, our data suggest that AoVip36 and AoEmp47 hinder the secretion of heterologous proteins by promoting their retention in the ER but that AoVip36 also promotes the secretion of heterologous proteins. Moreover, we found that genetic deletion of these putative ER-Golgi cargo receptors significantly improves heterologous protein production. The present study is the first to propose that ER-Golgi transport is a bottleneck for heterologous protein production in filamentous fungi.     

Disruption of ten protease genes in the filamentous fungus Aspergillus oryzae highly improves production of heterologous proteins

Proteolytic degradation by secreted proteases into the culture medium is one of the significant problems to be solved in heterologous protein production by filamentous fungi including Aspergillus oryzae. Double (tppA, and pepE) and quintuple (tppA, pepE, nptB, dppIV, and dppV) disruption of protease genes enhanced human lysozyme (HLY) and bovine chymosin (CHY) production by A. oryzae. In this study, we used a quintuple protease gene disruptant and performed successive rounds of disruption for five additional protease genes (alpA, pepA, AopepAa, AopepAd, and cpI), which were previously investigated by DNA microarray analyses for their expression. Gene disruption was performed by pyrG marker recycling with a highly efficient gene-targeting background (∆ligD) as previously reported. As a result, the maximum yields of recombinant CHY and HLY produced by a decuple protease gene disruptant were approximately 30% and 35%, respectively, higher than those produced by a quintuple protease gene disruptant. Thus, we successfully constructed a decuple protease gene disruptant possessing highly improved capability of heterologous protein production. This is the first report on decuple protease gene disruption that improved the levels of heterologous protein production by the filamentous fungus A. oryzae.     

The intra- and extracellular proteome of <Emphasis Type="Italic">Aspergillus niger</Emphasis> growing on defined medium with xylose or maltose as carbon substrate

Background  

The filamentous fungus Aspergillus niger is well-known as a producer of primary metabolites and extracellular proteins. For example, glucoamylase is the most efficiently secreted protein of Aspergillus niger, thus the homologous glucoamylase (glaA) promoter as well as the glaA signal sequence are widely used for heterologous protein production. Xylose is known to strongly repress glaA expression while maltose is a potent inducer of glaA promoter controlled genes. For a more profound understanding of A. niger physiology, a comprehensive analysis of the intra- and extracellular proteome of Aspergillus niger AB1.13 growing on defined medium with xylose or maltose as carbon substrate was carried out using 2-D gel electrophoresis/Maldi-ToF and nano-HPLC MS/MS.