Increased heterologous protein production in Aspergillus niger fermentation through extracellular proteases inhibition by pelleted growth

The dependence of filamentous fungal protease secretion on morphology was investigated by employing the recombinant Aspergillus niger strain AB4.1[pgpdAGLAGFP] which contains a gene for the glucoamylase-GFP (green fluorescence protein) fusion protein. Different inoculum levels were used to obtain different sizes of pellet or free mycelia. The extracellular protease activity of the cultures varied with the pellet size and decreased dramatically when the morphology was changed from free mycelia to pellets. The culture with an optimal pellet size of 1.6 mm was obtained from an inoculum of 4 x 10(6) spores/mL. It resulted in a specific protease activity of 158 units/L, only one-third of that in free mycelial growth, and a maximum specific GFP yield of 0.98 mg/g (cell mass) compared to 0. 29 mg/g for free mycelial growth with an inoculum of 10(7) spores/mL. The results indicate that this bioprocessing strategy can be effectively used to inhibit protease activity in filamentous fungal fermentation and thereby to enhance heterologous protein production.     

SParticle,an algorithm for the analysis of filamentous microorganisms in submerged cultures

Streptomycetes are filamentous bacteria that produce a plethora of bioactive natural products and industrial enzymes. Their mycelial lifestyle typically results in high heterogeneity in bioreactors, with morphologies ranging from fragments and open mycelial mats to dense pellets. There is a strong correlation between morphology and production in submerged cultures, with small and open mycelia favouring enzyme production, while most antibiotics are produced mainly in pellets. Here we describe SParticle, a Streptomyces Particle analysis method that combines whole slide imaging with automated image analysis to characterize the morphology of submerged grown Streptomyces cultures. SParticle allows the analysis of over a thousand particles per hour, offering a high throughput method for the imaging and statistical analysis of mycelial morphologies. The software is available as a plugin for the open source software ImageJ and allows users to create custom filters for other microbes. Therefore, SParticle is a widely applicable tool for the analysis of filamentous microorganisms in submerged cultures.     

Enhanced production of manganese peroxidase from immobilized <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> due to the increased autolysis of chlamydospore-like cells

The autolysis of chlamydospore-like cells in Phanerochaete chrysosporium immobilized in polyurethane foam correlated with the production of manganese peroxidase (MnP). The maximum specific activity of MnP was 1055 U g dry mycelium^–1 in the immobilized culture, compared with 260 U g dry mycelium^–1 in the submerged culture. Scattered mycelial pellets were formed in the immobilized culture in which almost all of the chlamydospore-like cells were subject to autolysis. However, highly crowded pellets were formed in the free culture, in which only the chlamydospore-like cells in the exterior were subject to autolysis. We propose that the enhanced production of MnP in immobilized cultures of P. chrysosporium is due to increased autolysis of the chlamydospore-like cells.     

A glucoamylase::GFP gene fusion to study protein secretion by individual hyphae of Aspergillus niger

Although Aspergillus niger is used as a host for heterologous protein production, yields are generally lower than those obtained for homologous proteins. Mechanisms of protein secretion and the secretory pathway in filamentous fungi are poorly characterised, although there is evidence to suggest that secretion occurs by a mechanism similar to that in other eukaryotes, but with proteins destined for secretion being directed to the hyphal tip. We report on a method using a glucoamylase: GFP gene fusion which allows us for the first time to monitor, in vivo, protein secretion in A. niger at the single hyphal level. A synthetic green fluorescent protein (sGFP(S65T)) was fused to truncated A. niger glucoamylase (GLA:499). Southern blot analysis of transformants confirmed that the gene fusion had successfully integrated into the A. niger genome. Confocal and fluorescence microscopy revealed that the GLA::GFP fusion protein is fluorescent in A. niger and appears to be directed to the hyphal tip. In young mycelia, hyphal cell wall fluorescence is apparent and immunogold labelling of GFP confirmed that GFP was partially localised within the hyphal cell wall. Using Western blotting, extracellular GLA::GFP was detected only in culture filtrates of young mycelia grown in a soya milk medium. The actin inhibitor latrunculin B was used to disrupt the secretion process, and its effects on the distribution of GLA::GFP were monitored.     

Production of bifunctional single-chain antibody-based fusion proteins in <Emphasis Type="Italic">Pichia </Emphasis><Emphasis Type="Italic">pastoris</Emphasis> supernatants

Recombinant antibody fusion constructs with heterologous functional domains are a promising approach to new therapeutic targeting strategies. However, expression of such constructs is mostly limited to cost and labor-intensive mammalian expression systems. Here we report on the employment of Pichia pastoris for the expression of heterologous antibody fusion constructs with green fluorescent protein, A33scFv::GFP, or with cytosine deaminase, A33scFv::CDy, their production in a biofermenter and a modified purification strategy. Combined, these approaches improved production yields by about thirty times over established standard protocols, with extracellular secretion of the fusion construct reaching 12.0 mg/l. Bifunctional activity of the fusion proteins was demonstrated by flow cytometry and an in-vitro cytotoxicity assay. With equal amounts of purified protein, the modified purification method lead to higher functional results. Our results demonstrate the suitability of methylotrophic Pichia expression systems and laboratory-scale bioreactors for the production of high quantities of bifunctionally active heterologous single-chain fusion proteins.     

Culture-Based Strategies for Reduction of Protease Activity in Filtrates from Aspergillus niger NRRL-3

Summary While Aspergillus strains are also being considered as potential hosts for production of extracellular heterologous proteins, the proteases produced by the host are highly problematic in that they typically modify and degrade the recombinant proteins. Culture-based approaches for minimization of protease activity in culture supernatants of Aspergillus niger NRRL-3 included reduction or elimination of peptide nitrogen in the medium, preferential use of a defined salts medium rather than a non-peptide nitrogen medium containing yeast-nitrogen base, supplementation of the medium with carboxymethylcellulose and cultivation at pH 6.5 rather than 7.5. In general, increased proteolytic activity was observed after maximum biomass was observed and biomass was declining suggesting the majority of protease activity was released by cell lysis. Carboxymethylcellulose shifted mycelial morphology from pelleted to filamentous. Mycelium lysis in the centre of pellets, with resultant release of intracellular proteases, would explain why filamentous cultures exhibited much lower proteolytic activity than pelleted cultures.     

Monitoring phase-specific gene expression in Histoplasma capsulatum with telomeric GFP fusion plasmids

Dimorphism is an essential feature of Histoplasma capsulatum pathogenesis, and much attention has been focused on characteristics that are unique to the saprophytic mycelial phase or the parasitic yeast phase. Recently, we identified a secreted calcium-binding protein, CBP, that is produced in large amounts by yeast cells but is undetectable in mycelial cultures. In this study, the green fluorescent protein (GFP) was established as a reporter in H. capsulatum to study regulation of CBP1 expression in cultures and in single cells grown under different conditions and inside macrophages. One GFP version that was optimized for human codon usage yielded highly fluorescent Histoplasma yeast cells. By monitoring GFP fluorescence during the transition from mycelia to yeast, we demonstrated that the CBP1 promoter is only fully active after complete morphological conversion to the yeast form, indicating for the first time that CBP1 is developmentally regulated rather than simply temperature regulated. Continuous activity of the CBP1 promoter during infection of macrophages supports the hypothesis that CBP secretion plays an important role for Histoplasma survival within the phagolysosome. Broth cultures of Histoplasma yeasts carrying a CBP–GFP protein fusion construct were able to secrete a full-length fluorescent fusion protein that remained localized within the phagolysosomes of infected macrophages. Additionally, a comparison of two Histoplasma strains carrying the CBP1 promoter fusion construct either epichromosomally or integrated into the chromosome revealed cell-to-cell variation in plasmid copy number due to uneven plasmid partitioning into daughter cells.     

Analysis of two distinct mycelial populations in liquid-grown Streptomyces cultures using a flow cytometry-based proteomics approach

Streptomycetes are proficient producers of enzymes and antibiotics. When grown in bioreactors, these filamentous microorganisms form mycelial pellets that consist of interconnected hyphae. We here employed a flow cytometry approach designed for large particles (COPAS) and demonstrate that liquid-grown Streptomyces cultures consist of two distinct populations of pellets. One population consists of mycelia with a constant mean diameter of approximately 260?μm, whereas the other population contains larger mycelia whose diameter depends on the strain, the age of the culture, and medium composition. Quantitative proteomics analysis revealed that 37 proteins differed in abundance between the two populations of pellets. Stress-related proteins and biosynthetic proteins for production of the calcium-dependent antibiotic were more abundant in the population of large mycelia, while proteins involved in DNA topology, modification, or degradation were overrepresented in the population of small mycelia. Deletion of genes for the cellulose synthase-like protein CslA and the chaplins affected the average size of the population of large pellets but not that of small pellets. Considering the fact that the production of enzymes and metabolites depends on pellet size, these results provide new leads toward rational strain design of Streptomyces strains tailored for industrial fermentations.     

丝状真菌异源蛋白表达系统研究进展

丝状真菌,俗称霉菌,在食品工业中被用于生产多种生物酶和有机酸。近年来,人们发现丝状真菌具有分泌量大、表达的蛋白有天然活性等特点,非常适合作为同源和异源重组蛋白的表达宿主,因此被广泛研究和探讨。简要综述了以黑曲霉为代表的几种常被用作蛋白表达宿主的丝状真菌的特点、应用中的主要问题和基本解决方案,以及近年关于丝状真菌表达系统的最佳培养条件和发酵条件的研究进展。     

Asexual sporulation signalling regulates autolysis of Aspergillus nidulans via modulating the chitinase ChiB production

Aims:  Elucidation of the regulation of ChiB production in Aspergillus nidulans .
Methods and Results:  Mutational inactivation of the A. nidulans chiB gene resulted in a nonautolytic phenotype. To better understand the mechanisms controlling both developmental progression and fungal autolysis, we examined a range of autolysis-associated parameters in A. nidulans developmental and/or autolytic mutants. Investigation of disorganization of mycelial pellets, loss of biomass, extra-/intracellular chitinase activities, ChiB production and chiB mRNA levels in various cultures revealed that, in submerged cultures, initialization of autolysis and stationary phase-induced ChiB production are intimately coupled, and that both processes are controlled by the FluG-BrlA asexual sporulation regulatory pathway. ChiB production does not affect the progression of apoptotic cell death in the aging A. nidulans cultures.
Conclusions:  The endochitinase ChiB plays an important role in autolysis of A. nidulans , and its production is initiated by FluG-BrlA signalling. Despite the fact that apoptosis is an inseparable part of fungal autolysis, its regulation is independent to FluG-initiated sporulation signalling.
Significance and Impact of the Study:  Deletion of chiB and fluG homologues in industrial filamentous fungal strains may stabilize the hyphal structures in the autolytic phase of growth and limit the release of autolytic hydrolases into the culture medium.     

Sorting of Streptomyces Cell Pellets Using a Complex Object Parametric Analyzer and Sorter

Streptomycetes are filamentous soil bacteria that are used in industry for the production of enzymes and antibiotics. When grown in bioreactors, these organisms form networks of interconnected hyphae, known as pellets, which are heterogeneous in size. Here we describe a method to analyze and sort mycelial pellets using a Complex Object Parametric Analyzer and Sorter (COPAS). Detailed instructions are given for the use of the instrument and the basic statistical analysis of the data. We furthermore describe how pellets can be sorted according to user-defined settings, which enables downstream processing such as the analysis of the RNA or protein content. Using this methodology the mechanism underlying heterogeneous growth can be tackled. This will be instrumental for improving streptomycetes as a cell factory, considering the fact that productivity correlates with pellet size.     

Role of N-terminal 28-amino-acid region of <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> lipase in directing proteins to secretory pathway of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

To develop a new approach for improving heterologous protein production in Aspergillus oryzae, we focused on the functional role of the N-terminal region of Rhizopus oryzae lipase (ROL). Several N-terminal deletion variants of ROL were expressed in A. oryzae. Interestingly, a segment of 28 amino acids from the C-terminal region of the propeptide (N28) was found to be critical for secretion of ROL into the culture medium. To further investigate the role of N28, the ROL secretory process was visualized in vivo using ROL-green fluorescent protein (GFP) fusion proteins. In cells producing ROL with N28, fluorescence observations showed that the fusion proteins are transported through endoplasmic reticulum (ER), Golgi, and cell wall, which is one of the typical secretory processes in a eukaryotic cell. Because the expression of the mature ROL-GFP fusion protein induced fluorescence accumulation without its translocation into the ER, N28 is considered to play a crucial role in protein transport. When N28 was inserted between the secretion signal and GFP, fluorescence observations showed that GFP, which is originally a cytoplasmic protein, was efficiently translocated into the ER of A. oryzae, resulting in an enhanced secretion of mature GFP after proteolytic cleavage of N28. These findings suggest that N28 facilitates protein translocation into ER and can be a promising candidate for improving heterologous protein production in A. oryzae.     

Optimization of a pellicular biocatalyst for penicillin G production by Penicillium chrysogenum

A previously developed immobilization technique involving latex coatings on solid particulate supports was investigated further for penicillin G production by Penicillium chrysogenum. Several modifications were found to decrease the germination lag time, including a higher spore concentration, a thinner latex layer, an increased latex porosity, and a decreased drying time. This approach enabled the development of immobilized mycelial pellets within 2-3 days from the onset of biocatalyst preparation and incubation.A continuous immobilized-cell airlift bioreactor produced penicillin G in a series of runs in which the production phase lasted up to 30 days. The productivity of this system was 3-6 times greater than the productivity of the corresponding free-cell shake flask fermentation.     

利用红色荧光蛋白分析里氏木霉合成纤维素酶的机理

以红色荧光蛋白作为报告蛋白研究了里氏木霉的纤维素酶合成机理。构建了里氏木霉的表达盒,通过该表达盒使红色荧光蛋白的基因整合到里氏木霉的基因组DNA上,并受纤维二糖水解酶基因启动子的调控,得到重组菌株T.reeseiTR2。在不同的条件下培养T.reeseiTR2,红色荧光蛋白的表达情况可以反映在不同条件下里氏木霉合成纤维素酶的情况。在诱导的情况下,红色荧光蛋白随时间变化的情况与培养液中纤维素酶活性的变化相似,培养至36h后可以观察到荧光,并且不断增强,到菌丝自溶时荧光减弱。另一方面,诱导后里氏木霉菌丝的各个部位均可以观察到荧光,而且分布均匀,表明菌丝的各个部位在纤维素酶合成过程中所起的作用相同。在非诱导的情况下,培养时间较长时也可以观察到较弱的荧光,表明在此条件下里氏木霉仍可以合成少量的纤维素酶,这一结果为解释纤维素诱导里氏木霉合成纤维素酶的机理提供了另一个试验依据。     

Classification and measurement of fungal pellets by automated image analysis

An automated image analysis method for classifying and measuring pellets of filamentous fungi growing in submerged fermentations has been developed. The method discriminates between pelleted mycelial growth and loose aggregates of dispersed hyphae. Pellets are classified into smooth and hairy types. In both cases, the core of the pellet is identified and its shape and size characterized. For hairy pellets the annular region is also characterized. The method was tested on pellets of Aspergillus niger ATCC 11414 grown in a defined medium in shake flasks. This rapid method makes practical extensive studies on the morphology of pellets in submerged fermentations and the influence of fermentation conditions on that morphology.     

Improved two-stage protein expression and purification via autoinduction of both autolysis and auto DNA/RNA hydrolysis conferred by phage lysozyme and DNA/RNA endonuclease

We report improved release of recombinant proteins in Escherichia coli, which relies on combined cellular autolysis and DNA/RNA autohydrolysis, conferred by the tightly controlled autoinduction of both phage lysozyme and the nonspecific DNA/RNA endonuclease from Serratia marcescens. Autoinduction occurs in a two-stage process wherein heterologous protein expression and autolysis enzymes are induced upon entry into stationary phase by phosphate depletion. Cytoplasmic lysozyme and periplasmic endonuclease are kept from inducing lysis until membrane integrity is disrupted. After cell harvest, the addition of detergent (0.1% Triton X-100) and a single 30 min freeze-thaw cycle results in >90% release of protein, green fluorescent protein. This cellular lysis is accompanied by complete oligonucleotide hydrolysis. The approach has been validated for shake flask cultures, high-throughput cultivation in microtiter plates, and larger scale stirred-tank bioreactors. This tightly controlled system enables robust growth and resistance to lysis in routine media when cells are propagated and autolysis/hydrolysis genes are only induced upon phosphate depletion.     

Genomic analysis reveals Lactobacillus sanfranciscensis as stable element in traditional sourdoughs

Background

The Ala-Pro-rich O-glycoprotein known as the 45/47 kDa or APA antigen from Mycobacterium tuberculosis is an immunodominant adhesin restricted to mycobacterium genus and has been proposed as an alternative candidate to generate a new vaccine against tuberculosis or for diagnosis kits. In this work, the recombinant O-glycoprotein APA was produced by the non-pathogenic filamentous bacteria Streptomyces lividans, evaluating three different culture conditions. This strain is known for its ability to produce heterologous proteins in a shorter time compared to M. tuberculosis.

Results

Three different shake flask geometries were used to provide different shear and oxygenation conditions; and the impact of those conditions on the morphology of S. lividans and the production of rAPA was characterized and evaluated. Small unbranched free filaments and mycelial clumps were found in baffled and coiled shake flasks, but one order of magnitude larger pellets were found in conventional shake flasks. The production of rAPA is around 3 times higher in small mycelia than in larger pellets, most probably due to difficulties in mass transfer inside pellets. Moreover, there are four putative sites of O-mannosylation in native APA, one of which is located at the carboxy-terminal region. The carbohydrate composition of this site was determined for rAPA by mass spectrometry analysis, and was found to contain different glycoforms depending on culture conditions. Up to two mannoses residues were found in cultures carried out in conventional shake flasks, and up to five mannoses residues were determined in coiled and baffled shake flasks.

Conclusions

The shear and/or oxygenation parameters determine the bacterial morphology, the productivity, and the O-mannosylation of rAPA in S. lividans. As demonstrated here, culture conditions have to be carefully controlled in order to obtain recombinant O-glycosylated proteins with similar "quality" in bacteria, particularly, if the protein activity depends on the glycosylation pattern. Furthermore, it will be an interesting exercise to determine the effect of shear and oxygen in shake flasks, to obtain evidences that may be useful in scaling-up these processes to bioreactors. Another approach will be using lab-scale bioreactors under well-controlled conditions, and study the impact of those on rAPA productivity and quality.     

Purification of human interleukin-2 fusion protein produced in insect larvae is facilitated by fusion with green fluorescent protein and metal affinity ligand

The fusion protein of green fluorescent protein (GFP) and human interleukin-2 (hIL-2) was produced in insect Trichoplusia ni larvae infected with recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV). This fusion protein was composed of a metal ion binding site (His)6 for rapid one-step purification using immobilized metal affinity chromatography (IMAC), UV-optimized GFP (GFPuv), enterokinase cleavage site for recovering hIL-2 from purified fusion protein, and hIL-2 protein. The additional histidine residues on fusion protein enabled the efficient purification of fusion protein based on immobilized metal affinity chromatography. In addition to advantages of GFP as a fusion marker, GFP was able to be used as a selectable purification marker; we easily determined the correct purified fusion protein sample fraction by simply detecting GFP fluorescence.     

Expression and purification of human interleukin-2 simplified as a fusion with green fluorescent protein in suspended Sf-9 insect cells

A fusion protein of human interleukin-2 (hIL-2) and green fluorescent protein (GFP) was expressed in insect Sf-9 cells infected with recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV). This fusion protein was comprised of a histidine affinity ligand for simplified purification using immobilized metal affinity chromatography (IMAC), UV-optimized GFP (GFPuv) as a marker, an enterokinase cleavage site for recovery of hIL-2 from the fusion, and the model product hIL-2. Successful production of hIL-2 as a fusion protein (approximately 52,000 Da) with GFPuv was obtained. GFPuv enabled rapid monitoring and quantification of the hIL-2 by simply checking the fluorescence, obviating the need for Western blot and/or ELISA assays during infection and production stages. There was no increased 'metabolic burden' due to the presence of GFPuv in the fusion product. The additional histidine residues at the N-terminus enabled efficient one-step purification of the fusion protein using IMAC. Additional advantages of GFP as a fusion marker were seen, particularly during separation and purification in that hIL-2 containing fractions were identified simply by illumination with UV light. Our results demonstrated that GFP was an effective non-invasive on-line marker for the expression and purification of heterologous protein in the suspended insect cell/baculovirus expression system.     

Production of lignin peroxidase by Phanerochaete chrysosporium immobilized on porous poly(styrene-divinylbenzene) carrier and its application to the degrading of 2-chlorophenol

Porous poly(styrene-divinylbenzene) carriers, for the immobilization of white rot fungus Phanerochaete chrysosporium have been prepared by the concentrated emulsion polymerization method. The concentrated emulsion consists of a mixture of styrene and divinylbenzene containing a suitable surfactant and an initiator as the continuous phase, and water as the dispersed phase. The polymerization of the monomers of the continuous phase generated the polymer carrier with a porcus structure. The white rot fungus Phanerochaete chrysosporium has been immobilized on porous poly(styrene-divinylbenzene) carriers and used for the batch production and the repeated batch production of lignin peroxidase in shake cultures based on a carbon-limited medium containing veratryl alcohol. The best results were achieved when a spore inoculum was used for immobilization instead of 1-day-old mycelial pellets, for both the batch production and the repeated batch production. The porous poly(styrene-divinylbenzene) immobilized Phanerochaete chrysosporium and freely suspended mycelial pellets were used as biocatalysts for the degradation of 2-chilorophenol in a 2-L bioreactor. The porous poly(styrene-divinylbenzene) particle (diameter congruent with 0.2 cm) immobilized spores exhibited a much higher activity in the degradation of 2-chlorophenol than the freely suspended mycelial pellets. (c) 1994 John Wiley & Sons, Inc.