Indian Journal of Microbiology Vol 46, No. 4, December 2006, pp 307-324

Review

Fungi as cell factories: Hype, reality and hope R. Maheshwari Formerly, Department of Biochemistry, Indian Institute of Science, Bangalore 560012 The extant diversity of fungi and their intraspecies genetic variability offers the scope of finding useful strains producing enzymes, antibiotics or metabolites of scientific or practical importance. Genetic and molecular techniques allow a gene from one organism to be transferred to a safe domesticated fungal species and engineer precise changes in its regulatory DNA sequences to increase expression of the transgene. Cultivation of fungus in industrial-sized fermentors facilitates a desired substance to be produced for commercial use. A few species produce homologous proteins in significant amounts but the expression of heterologous proteins of mammalian origin for practical use is generally low. Among the perceived uses of fungi as cell factories is production of human antibodies. For increasing the secretion of a heterologous protein, an understanding is required of its structure and of the regulatory genes controlling expression of the protein. Furthermore a detailed study of the architecture of the fungal cell wall over period of mycelial growth is required since proteins finally exit through the cell wall. Keywords: Fungi, protein secretion, recombinant protein, antibody production, biotechnology.

Fungi have evolved as chemical factories, secreting in their environment several classes of enzymes which break down polymeric constituents of dead organic matter into soluble forms for absorption and utilization as sources of carbon and energy. An adjunct to the success of their absorptive mode of nutrition is the production of antagonistic chemicals which enable fungi to survive amidst microflora and microfauna comprising of competitors, parasites and predators. For example, yeasts produce alcohol - a toxic end-product of anaerobic metabolism - not for human use but to arrest the fouling of their “sugary habitats” by microbial competitors. The diversity of fungi, estimated to comprise over 1.5 million species1, and the intraspecies genetic variation offers a huge resource for finding a potentially useful strain that produces a required enzyme for biotransformation of a particular substrate or a metabolite of value. Genetic and recombinant DNA techniques allow a strain to be improved by specific alterations to overexpress the transgene and the product to be produced for commercial application. This review will assess fungi as cell factories and consider on anvil the issue of hype, reality and hope from the perspective of fungal biology. *e-mail: [email protected] Tel: 91 80 23341045

Why fungi? In biotechnology unicellular yeasts and multicellular filamentous molds, rather than plant and animal cells, offer greater possibilities for production of a desired substance by fermentation - the term fermentation in biotechnology refers to the process of growing microorganisms in large-scale to produce a biochemical product. The reasons for the selection of fungi are: 1. Their adaptability to diverse environmental conditions provides greater opportunity of finding and selecting an appropriate strain for use. 2. Scaling-up of process is easier because of their rapid growth rate. For example Saccharomyces cerevisiae (budding yeast) has a doubling rate (0.5 h) in liquid medium almost matching the bacterium E. coli; and the molds Aspergillus and Neurospora double their mass in about 2.22.7 hours at 25ºC. Thus a fungus-based process is completed in a shorter time (usually one week or less) compared to animal cells (usually weeks) or plant cells (months). 3. Selection of exceptional high-producing variants among hundreds or thousands of colonies is facilitated by colour tests or replica plating on

308 Indian J Microbiol, December 2006

selective media. Special media also allow colonial growth of some filamentous fungi for plate assays. 4. Transformation of cells is easier. In yeast, the transforming DNA is integrated at homologous site in the chromosome although it is commonly ectopic in filamentous molds. 5. The large surface area of plasma membrane provides for increased sites for protein secretion. 6. Expression cloning combines the advantageous features of unicellular yeast and the multicellular molds, facilitating construction of strains secreting higher levels of a desired enzyme. 7. Post-translational processing (glycosylation, phosphorylation or acetylation) allows active heterologous proteins to be produced 2. 8. Large-scale cultivation in defined media containing simple carbohydrate and nitrogen compound eliminates unknown influence of constituents of complex substrates. 9. Inducible synthesis of some proteins allows their production with minimum background proteins. 10. Useful strains can be permanently preserved as spores. The spores also serve as material for mutagenesis for further strain improvement. 11. Spontaneous hyphal fusions between geneticallyrelated strains allow construction of heterokaryotic mycelium for large-scale production of therapeutic antibody. Architectural and functional marvels of hypha A fungal factory is comprised of longitudinally joined hyphal compartments. Inside these compartments, extending over considerable physical distance, are longitudinal arrays of actin bundles that act as tracks for the intracellular movement and positioning of organelles. The plasma membrane has transmembrane protein pumps for uptake of nutrients inside against concentration gradients. The compartments are replete with nuclei, endoplasmic reticulum and Golgi which bud out thousands of secretory vesicles every second for fusing with the plasma membrane for exocytosis. The hypha is polarized, i.e., it is spatially coordinated into an apical region characterized by rapid uptake of ions (phosphate, potassium and ammonium) and nutrients (sugars and amino acids) and different sites for release

of proteins, toxins, antibiotics and pigments. The hyphal extension is driven by hydrostatic (turgor) pressure of 4-5 bar3. To contain this internal pressure, the hypha is surrounded by a rigid wall, yet is elastic enough for it to extend rapidly. It is selectively porous to allow digestive enzymes to exit4,5, but at the same time retain some molecules. From the viewpoint of fungal based fermentation process, a feature of hypha that strikes as of special significance is the presence of septa (Fig.1). Culturing aerobic fungi requires that air be bubbled through the culture medium with constant vigorous stirring. This subjects hyphae to shear; however, like rungs of a ladder, septa provide strength to elongated hyphae (generally 5-10 µm in diameter), limiting shearing and reducing the ‘bleeding’ of protoplasm from any severed ends. Not surprisingly, most fungi used in fermentation are septate. However, the septum is centrally perforated; the pore being minute, approximately 0.5µm. The perforated septa allow aged or dysfunctional organelles to be replaced by migration of healthy (functional) organelles from other compartments. It also allows aged organelles to be partitioned into separate (distal) region of hypha for recycling. With protoplasm rapidly moving forwards or backwards (approximately at a rate of 4-6 cm per hour) through septal pores, the entire mycelium comprising of hyphae interlinked by short lateral branches (bridges) is converted into one single intercommunicating unit in which metabolic activities are synchronized. The interconnection (cell fusion) among hyphae allows related strains to be fused into a single heterokaryotic mycelium containing genetically distinct nuclei. Heterokaryotic mycelium proffer the production of human antibody (described later). The septal pores can be plugged by a proteinaceous material called Woronin body, enabling parts of a hypha or parts of mycelium to take up specialized functions. For example, parts of single hypha or portion of mycelium can be isolated and their metabolism shifted to pathways for generating and maintaining a reducing environment for housing and functioning of redox-sensitive enzymes6 and/or formation of special products by shunt metabolic pathways. Finally, a puzzling feature of fungi that merits comment is the unique multinuclear condition of

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Fig. 1. Enlarged view of hyphae. The hypha is divided by perforated transverse walls (septa). The compartments (cell) contain several haploid nuclei.

molds. Although the role of nucleus in heredity is well established, the supernumerary nuclei do not contribute to phenotype7. Rather, since in nature fungi grow under limiting conditions of nutrients; supernumerary nuclei may be a repository of scarce phosphorus and nitrogen in the protected form of nucleotides in DNA. When nutrient availability is limited, the supernumerary nuclei, partitioned into separate hyphal compartments, can be degraded by regulated autophagy 8 and DNA recycled by phosphodiesterases9,10, making available phosphorus and nitrogen for translocation to apical, metabolic region for synthesis of membrane and organelles, and the hypha to prolong its functional state. Thus over the course of evolution hypha has been shaped into a factory of great metabolic potential,

with fine coordination of its component compartments, and minimization on expenditure of energy. Exploiting genetic variation RFLP or RAPD or protein polymororphism analyses have revealed high degree of intraspecies variation in fungal populations, providing for selection of genetic variants for specific uses. A relevant example is Trichoderma viride QM-9414 isolated from moldy cotton fabrics in Solomon Islands. It was selected as the best cellulase-producing strain and its productivity improved by further mutagen treatments11. A mutant named T. reesei (to honour Elwyn T. Reese for his pioneering research on enzymatic degradation of cellulose) secreted 30- 40 gram protein per liter culture medium, of which 60%

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is just one specific cellulase protein, namely cellobiohydrolase12. This yield of enzyme protein has not yet been matched for any protein by recombinant DNA methodology – a pointer that classical methods of selection and improvement of strain by random mutagenesis remain the mainstay of biotechnology. Although the genetic changes behind cellulase overproduction by the mutant Trichoderma reesei strain are not known, nonetheless this work has had much impact: (1) It indicated the amount (or the limit) of protein that a fungus is capable of producing/ secreting. (2) It provided a major impetus for microbial culture collections, particularly for isolation and recognition of some potent polysaccharide-degrading enzymes13. (3) It galvanized research on bioconversion of lignocellulose - “the most abundant biological material on earth”. This was an impetus for research on enzymatic bioconversion of lignocellulosic material into glucose to produce fuel ethanol by yeast fermentation14. (4) It led to identification of several other sources of polymer-degrading enzymes, their purification and investigations of mechanisms of their action. (5) It recognized T. reesei as a potentially efficient expression host for recombinant proteins and stimulated research on production of foreign (heterologous) proteins by fungal fermentation process. Its genome has been sequenced (http:// www.genencor.com/wt/gcor/pr_1059584144) and, hopefully, the reasons for the improved production of cellulase will be discerned. The ‘perfect host’ system A fungal factory capitalizes on simple requirements of raw materials - a carbohydrate as source of carbon and energy, inorganic salts as sources of nitrogen, phosphorus and sulphur, trace minerals as micronutrients, and sometimes vitamins. These are easily satisfied by commercial grade compounds such as sucrose and urea, or materials available in bulk such as sugar cane or beet molasses, corn steep liquor, starch, soybean meal. Recombinant DNA techniques allow regulatory properties of a strain to be modified for overproduction (>10 -15 % of total cellular protein) of a particular protein. Secretion is important - the product can be recovered for downstream processing by simple filtration. The brewing industry is attempting to genetically engineer yeast strains which will aggregate (flocculate) and

settle down, yielding bright beer and wine. Fungi have been regarded as the “perfect hosts” for production of proteins and useful metabolites15. Fungal fermentation Since the hypha is only a single cell thick, even small changes in culture conditions influence its physiology and affect the yield. J.W. Foster, a pioneer investigator of fungal metabolism, cautioned16: “Indeed it is a common event to have an organism produce no detectable amount of a particular metabolic product, and yet under different cultural conditions, produce that very substance abundantly. Finally, there is the situation in which, on the one hand, one kind of product is produced, and, on the other hand, a totally different product.” Different trials in the same laboratory can affect reproducibility of results. For example, in a pioneering study of penicillin production by Penicillium notatum, Backus and Stauffer17 found antibiotic yield by surface-grown cultures to be markedly affected by factors of which the worker may be ignorant of, such as the “tightness of cotton plug” in the culture flasks/bottles (implying aeration). In 1960, E. W. Buxton18 sounded a warning that fungi are “a mutable and treacherous tribe”. This allegation could have been due to lack of strict control of physical and chemical conditions of growth rather than their inherent finicky behaviour. Fungi are increasingly grown in stainless-steel vessels fitted with ancillary machinery for in situ sterilization of culture media; for adding inoculum and antifoaming agents; for adding nutrients at desired rate and time; for supply of sterile air; exhaust for carbon dioxide; stirrer for good-mixing; constant measurement and control of pH, dissolved oxygen concentration, of temperature, and for intermittent removal of samples for monitoring process, in hygienic and aesthetically pleasing surroundings (Fig. 2). The production of many enzymes is regulated by temperature, dissolved oxygen or/and pH. As an example, by maintaining pH at 6, recombinant glucoamylase production was enhanced over 10-fold compared to that without pH control19. A system for the regulation of gene expression by ambient (extracellular) pH has been identified. This system consists of the products of the pacC and palA, B, C, F, H, and I genes. While pacC encodes a zinc finger

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Fig. 2. Fermentor and auxiliary equipment for online control of process parameters. Photo courtesy of Bioengineering /Spinco.

transcription factor, the pal genes encode components of an ambient pH signal transduction pathway20. A lesson from penicillin fermentation was that secretion of a product is neither related to growth rate nor the biomass yield: lactose-a poor carbon source- gave higher titers of penicillin. The production of recombinant glucoamylase by cultures of Aspergillus niger was increased by continuous dilution of medium with mineral salt medium and slow addition of peptone and glucose21. Fermentation processes are becoming increasingly sophisticated by programmed profiling of medium addition/dilution, temperature, and pH and dissolved oxygen to manipulate growth rate and biomass formation, release enzyme and inactivate protease22. Impact of recombinant DNA technology The small genomic size of fungi - approximately 10-40 Mb - has speeded up genome sequencing23,24,25,26 in the expectation that genes will be manipulated in systematic manner to create superior alleles for improved yields (https://

fungalgenomics.concordia.ca/fungi/Anig.php). The genetic material in both yeast and molds can be modified by random or site-directed methods; mutants can be isolated; genes can be cloned through their ability to complement mutant phenotypes; and an endogenous gene replaced with an engineered derivative. Shuttle plasmid vectors were constructed which can replicate both in E. coli, yeast or molds, enabling genes cloned in a bacterium or yeast to be returned for its high-level expression in a filamentous fungus and secretion in greater quantity and higher purity (see below). Expression cloning Expression cloning simplifies the time-consuming process of purifying desired enzyme from the mixture as enzyme is over-produced by an efficient strain of filamentous fungus. Often concentration of culture filtrates by simple freeze-drying may suffice practical needs. This method combines the advantageous features of unicellular yeast and multicellular, filamentous mold, i.e., the ability of yeast to express

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heterologous fungal cDNA, its ability to be grown as colonies on screening plates for visual detection of enzyme activity27, 28 and the gene from a selected clone to be expressed in mold for higher secreted amount of enzyme. As an example, pectinases have industrial applications for clarification of fruit juices that contain highly methyl-esterified pectin. E. coli shuttle vector was used to transform yeast as intermediate host and the secreting colonies visually identified by replica plating27. The gene encoding enzyme from a selected yeast colony (donor) was subcloned and transferred into a safe, filamentous fungus for large-scale production (secretion) of

enzyme. The nonhomologous pectin methyl esterase together with polygalacturonases caused a rapid depolymerzation of pectin29.The steps in the expression cloning are given in scheme shown in Fig. 3. Examples of non-homologous protein production The examples below illustrate how molds began to be used for production of foreign proteins. Chymosin According to a legend, an Arab nomad with a saddlebag of milk to sustain him on a journey was

mRNA ↓ cDNA library in E. coli ↓ 50 pools (5000 transformants/pool) ↓ Transformation of yeast (25,000/pool) using a shuttle vector ↓ Screening sub-libraries (200 plates with 500 colonies) ↓ Rescreening of positive clones ↓ Isolation of DNA ↓ Transformation of E. coli ↓ Characterization of DNA by nucleotide sequencing for presence of cloned gene ↓ Cloning of gene in a filamentous fungus secreting protein in high amounts

Fig. 3. Scheme for expression cloning.

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crossing desert riding a horse. After several hours when he stopped to quench his thirst, he found that the milk had separated into a pale watery liquid and solid white lump (cheese). Taking cue from this legend, the ancient Romans recognized a link between saddlebag made from the stomach of a suckling calf and the transformation of milk into cheese. Therefore, for making cheese, an enzyme preparation called rennet, made from the lining of the stomach of suckling calves began to be added. It is now known to contain a specific protease enzyme called chymosin (rennin) which breaks protein casein in milk, causing casein to clump into a solid gel. Protests by animal rights activists that it is inhumane to kill newly born calves and shortages of obtaining calf rennet led to search for substitutes. The Japanese microbiologists perceived that since microorganisms secrete a variety of digestive enzymes, some microorganism-derived enzyme could be used as a substitute. From the isolation and screening of several hundred soil microorganisms, a thermophilic fungus, Mucor pusillus, was obtained which had high milk-clotting activity30. Genetic engineering was used to transfer the gene to a historically safe fungus. Since koji mold (Fig. 4) mostly A. oryzae and A. sojae but may include A.

awamori and A. kawachii (black koji-mold) -has long been used in Japan for production of fermented food and beverages, and information on its cultivation methods is available, it is presently the most widely used filamentous fungus. A recombinant A. oryzae strain was constructed in which the chymosin gene was placed under the control of promoter of glucoamylase, a well-secreted protein. The recombinant host strain produced heterologous Mucor (Rhizomucor) miehei protease in excess of 3 g/liter31. This yield compares favourably with that of recombinant proteins in milk produced by transgenic animals (2-10 g/liter) (http://www.gtc-bio.com/science/ questions.html). Chymosin identical to calf rennet is being produced commercially by yeast or filamentous fungi by transformation with a plasmid containing an artificially synthesized chymosin gene. This pure form of “vegetarian cheese” or Chy-Max®, was the first product of recombinant DNA technology in the U.S. food supply. This illustrates that chance observations often form the basis of exploitation of fungi in industries. For example, the antibiotic industry is based on chance discovery of penicillin produced by Penicillium notatum in contaminated plate of bacterial culture. Recently, the fungus Aspergillus awamori

Fig. 4. Koji mold, Aspergillus oryzae. Left, mycelium growing on a grain of steamed rice. Photo courtesy of John Gauntner. Right, colonies growing on agar medium in a Petri dish. The colonies are sporulating at the center. Photo courtesy of Novozymes Inc.

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has been used to produce thaumatin - a protein in the katemfe fruit (Thaumatococcus daniellii) growing in West Africa. This protein which on weight basis is 3000 times sweeter than sucrose has been expressed in Pichia pastoris with a yield of 5-7 mg/liter – higher than in the transgenic plants32. Lipase and protease The original idea of using enzymes in detergents was described in 1913 by the chemist Otto Rohm, who along with Otto Haas, founded Rohm and Haas -one of the world’s largest specialty chemical producing company. Rohm patented the use of crude pancreatic extracts in laundry pre-soak composition to improve the removal of greasy food stains (www.novozymes.com/cgi-bin/bvisapi.dll/biotimes/ one_article.jsp?id=11394&lang=en - 41k -). In 1994, Novozyme Inc. launched the first recombinant lipase, LipolaseTM, obtained by cloning lipase gene from a thermophilic fungus Thermomyces lanuginosus into Aspergillus oryzae wherein it produced 1000-fold more enzyme protein. It was used in formulation of detergents for hot-water machine wash for garments and dishes and is highly effective in removing oil stain. Several companies manufacture lipase fortifiedhousehold detergents. Similarly alkaline proteases are increasingly used as additives in detergents for removal of blood stains. The search is now for psychrophilic fungi as sources of lipases and proteases for use in detergent formulations for cold water wash. Lactoferrin Lactoferrin, an iron-binding glycoprotein present in human milk plays protective role against microbial and viral infection. Expression of human lactoferrin (hLF), a 78 kD glycoprotein, was achieved by placing the cDNA under the control of the A. oryzae áamylase promoter33. Using this system, hLF is expressed and secreted into the growth medium at levels up to 25 mg/l. Subsequently a modification of this production system combined with a classical strain improvement program enabled production of recombinant hLF in excess of 2 g/l in Aspergillus awamori as a glucoamylase fusion polypeptide which was secreted into the growth medium and processed to mature hLF by an endogenous KEX-2 peptidase 34 . The recombinant lactoferrin was indistinguishable from human milk lactoferrin with respect to its size,

immunoreactivity, and iron-binding capacity. It retained full biological activity in terms of its ability to bind iron and human enterocyte receptors. The recombinant protein functioned as a potent broad spectrum antimicrobial protein. Lactoferrin is the largest heterologous protein and the first mammalian glycoprotein expressed in the Aspergillus system. Miscellaneous During 1970s, Phillips Petroleum developed Pichia pastoris as a source of single cell protein as it can be grown economically in large scale in a completely defined growth medium containing methanol as a sole carbon source. P. pastoris has emerged as an alternative expression host for production of vaccines against bacterial toxins35. This yeast ferments glucose and related sugars even in the presence of air (absence of Crabtree effect) and hence it is termed as non-conventional yeast. It has a strong, methanol-induced alcohol oxidase promoter (AOX1). The alcohol oxidase -the enzyme which catalyzes the fist step in the metabolism of methanolcan constitute as much as 35% of the soluble protein in the cell. The promoter region of the gene encoding this enzyme is being used in gene-fusion approach to express foreign genes with yields from milligram to gram quantity of protein 35,36,37 . The posttranslational glycosylation is similar to that in mammalian-proteins. It is being advocated for heterologous protein secretion35. The production of high (14.8 g/liter) amount of animal protein (gelatin) has led many companies to adopt the Pichia expression system for production of heterologous proteins2. Seeds constitute the main diet of poultry and pigs. There is therefore much interest in improvement of animal feed using phytase. Supplementation of feed with phytase increases availability of phosphorus in the seed which store it chiefly as phytic acid (myoinositol hexakisphosphate). The E. coli phytase gene is reportedly ‘highly’ expressed in Pichia pastoris under the control of AOX1 promoter. Replacement of culture medium with fresh medium to remove repressing glycerol and metabolic wastes prior to methanol induction gave the highest level phytase expression38.

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The most recently developed method of manufacturing human insulin uses recombinant DNA technology with baker’s yeast as the host cell, offering potentially limitless supplies of insulin structurally identical to that made by the human pancreas. The above examples show that not only gene products from fungi but also from quite unrelated organisms have been successfully produced in fungi. However, yields of heterologous proteins by fungi (Table 1) are, for unknown reasons, several orders of magnitude lower than of homologous proteins even when the same expression signals were used.

Improving yield Increase in copy number of genes Several approaches are being tried to obtain reproducible higher yields. The results of inserting extra copies of the desired gene have been disappointing. For example, Mellon and Casselton52 (1988) found that as many as eight copies of the gene encoding isocitrate lyase gave only 25% activity compared to the wild type Coprinus cinereus. Analysis of Aspergillus niger transformed with glaA gene also showed no correlation between the

Table 1. Yields of some proteins and metabolites from fungi. Product/Source

Production (host) fungus

Yield (per liter)

Reference no

Aspergillus niger

1.3 g

39

Trichoderma reesei

30-40 g

12

Glucoamylase (Ho)

Aspergillus niger

20 g

15

Lipase (He) Thermomyces lanuginosus

Aspergillus oryzae

NA

www.novozymes.com

Aspergillus oryzae

NA

40

Acremonium chrysogenum

4g

41

Aspergillus oryzae

20 mg

42

Aspergillus oryzae

5 mg

43

Aspergillus niger

100 mg

44

Lactoferrin (He) Human

Aspergillus niger

25 mg

45

Interleukin-6 (He) Human

Aspergillus niger

150 mg

46

Aspergillus nidulans

4.8 mg

47

Pichia pastoris

~ 3 mg

48

Human Insulin

Pichia pastoris

1.5 g

49

Citric acid

Aspergillus niger

130-150 g

50

Penicillin

Penicillium chrysogenum

40-50 g

51

Acremonium chrysogenum

20-25 g

51

Calf chymosin (He) Cellulase (Ho)

Rhizomucor miehei Protease (alkaline) (He) Fusarium Protease (acid) (He) Mucor pusillus Mn Peroxidase (He) Phanerochaete chrysosporium Aspergillus niger (He)

Human Chorionic Gonadotropin (He)

Cephalosporin

Abbreviation: Ho, homologous production; He, heterologous production, NA, data not available.

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copy number of transforming gene and its level of glucoamylase 53. Since journals are reluctant to publish negative results, one must suppose that failures to increase yields by increasing gene dosage have outnumbered successes. In view of the discovery of gene-silencing processes in fungi such as “RIP, repeat-induced point mutation” 54 and “quelling”55 it is now understood why overexpression of a gene product by increasing copy number has failed, as in the plants. Cultural manipulation of morphology The few cases where this has been studied show that morphology can vary from one product to another. The gross colony morphology of filamentous fungi in submerged cultures is broadly of three types: freely dispersed hyphae, pellets of densely interwoven hyphae, or clumped mycelia, with zero concentration of oxygen in center 56, 57. A fungus can have all three different morphologies but the form most suitable imposed by cultural conditions for secretion of a particular protein is seldom reported. The filamentous form of A. niger was better for pectic enzyme synthesis, whereas the pellet form was optimum for citric acid production58. A deficiency of manganese leads to loss of growth polarity and formation of bulbous hyphae and increased citric acid production. Pellet form was also required for penicillin production by Penicillium chrysogenum. The mode of aeration can significantly affect morphology and the yield. In Aspergillus terreus, large fluffy pellet form obtained with supply of oxygen-enriched gas, but not with air, produced higher titers of lovastatin, a cholesterol lowering drug, than freely dispersed form59. A cross section of a 2 mm pellet of Penicillium chrysogenum showed differentiation of hyphae with cytoplasm-rich outer layer, partially lysed middle layers and disintegrated hyphae in the center60, indicating that the region of synthesis and release of product may be quite different. One of the more important parameters influencing culture morphology is aeration. For example, Trichophyton rubrum (a wooddegrading fungus) had different morphologies depending on its cultivation in baffled or unbaffled flasks. The pellet size in baffled flask was small and yield of MnP ligninase higher61. In Phanerochaete chrysosporium the ligninolytic activity (manganese peroxidase) was produced during differentiation of

spore62. These cells disappeared, coinciding with the time of enzyme secretion, suggesting that the differentiated cells acted as enzyme reservoir releasing their contents by autolysis process. Development of spores requires the presence of new enzymes for a limited period of time. Optimal productivity of secondary metabolites is dependent on a specific morphology, for example Penicillium urticae produced the antibiotics patulin and griseofulvin following conidiation63. Physical and chemical conditions of culture affect the expression of regulatory genes, affecting gross and microscopic morphology and the yield of product. Hyperbranching mutants Surprisingly, though filamentous fungi are known for their protein secreting abilities, the region of the hypha that secretes proteins is contentious. Wösten et al.64 found that in A. niger, protein synthesis occurs throughout growing hypha but glucoamylase is secreted only from the growing apical region of the hypha where nascent wall is laid down and is therefore porous. However, apical secretion may not be because the hyphal tip walls are relatively porous, but due to polarized nature of the hypha directing secretion vectorially at the tip. Nonetheless, this implies increased apical surface and continuous growth due to enhanced hyphal branching may increase productivity of strains. The mcb mutant of Neurospora crassa shows loss of growth polarity at 37 ºC with swollen hyphal tips, i.e. large increase in growing-surface area65. The effect is due to mutation in the gene encoding regulatory subunit of protein kinase. The mutant secreted 3-5-fold more extracellular proteins and a 20-fold increased level of carboxymethylcellulase relative to wild type. Another approach of obtaining higher protein yield may be through mutants with increased branching intensity. Hyperbranching mutants of Aspergillus oryzae produced higher amylase and protease on solid substrates66. The above results suggest that yields of extracellular protein by a filamentous fungus can be significantly increased by selecting strains with desirable morphologies for fermentation67. Modification of cell wall Why some proteins are secreted better than others, and why some strains secrete a protein better

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than others? Trevithick and Metzenberg68 found that if the quaternary structure of invertase of Neurospora is of a size that it can pass through the pores (40-70 Å) in the multi-layered network of cell wall, it was secreted externally as a light form. The large form of enzyme remained in the periplasm or was bound to cell wall. This observation is of unusual significance for it suggests that structural features of the protein and/or the cell wall are important determinants of level of secreted protein. Cellular regulatory mechanisms avoid wasteful synthesis of a protein that is unable to exit because of unfavourable pore size of the cell wall. Structural changes in mycelia with altered physical and chemical properties can occur in response to carbon sources69 affecting secretion. One example is α-glucosidase in the thermophilic, cellulolytic fungus Sporotrichum thermophile. Culture morphology of this fungus was strikingly different when grown with cellulose (filter paper) or its depolymerized form (cellobiose) as carbon source. In cellulose-medium the mycelium autolysed releasing the cell-wall-bound β-glucosidase whereas in cellobiose-medium the hyphae remained healthy in agglomerated state and β-glucosidase was not released into the medium70. That hyphal morphology is important in secretion is also emphasized by observations in a temperature-controlled morphological mutant of N. crassa. Increase in number of hyphal tips per hypha in secreted more cellulase relative to a wild-type strain65. The amount of protein secreted was seven-fold more when initial growth at 18 ºC was followed by growth at 37 ºC. In this mutant the site of secretion was not limited to the tip. Besides genetic factors, chemical environment of growth greatly affects thickness, chemical composition and structure of wall resulting in phenotypic change in the colony morphology71, 72. The results emphasize that structure of hyphal cell-wall is one of the important factors determining the amount of protein secreted. New expression hosts The fungi used currently do not fulfill all of the requirements, i.e., the production of enzymes stable at broad pH and temperature range, culture morphology giving non-viscous growth thereby reducing energy cost in operation in large-scale fermentation, free of undesirable pigment or protease enzymes. From the screening of more than 100 fungi,

Novozymes Inc. (www.novozymes.com) selected a filamentous fungus Fusarium venenatum as a new expression host which has the advantages of low secreted protease levels, low total spectrum of secreted protein, high level of heterologous expression, ‘favourable’ fermentation morphology and is ‘Generally Regarded As Safe’ (GRAS, i.e., no skin or respiratory allergy). Using F. venenatum the first microbe-produced recombinant alternative to animal trypsin was commercialized in November 2002. It has better stability than animal-derived trypsin. Dyadic International, Inc. has patented a novel-gene expression system based on a filamentous fungus Chrysosporium lucknowense that was isolated from alkaline soils in far-east Russia. This fungus grows at a broader range of pH from 4.5 to 9.0, compatible with stability of secreted proteins73. UV and N-methylN’-nitro-N-nitrosoguanidine treatment yielded protease-deficient mutants which formed dispersed fragmented mycelia and produced 200-fold more neutral cellulase with application in softening denim used in manufacture of jeans Molecular manipulations As proteins are significant constituents of all organic matter, fungi secrete proteases to utilize it. Not surprisingly, proteolytic destruction of enzyme is the single most important factor in low enzyme levels in the culture medium. By comparing the mRNA levels of a number of heterologous genes integrated in a single copy at a single site with the secreted levels of proteins, it was found that higher protein yields may be obtained through development of protease-deficient strains74, 75. In efforts to understand reasons of low yield, the authors compared protein levels of a number of heterologous genes integrated in a single copy at a defined locus, controlled by the expression signals of the host Aspergillus awamori endoxylanase gene. The results of mRNA analyses showed that mRNA stability is partly a reason for the low or undetectable protein levels. Other strategies are improvement of the mRNA stability by fusion with highly expressed genes 76; improvement in translation efficiency by construction of a synthetic gene with codon usage optimized for species; overproduction of protein disulphide isomerase77, 78, of chaperone79; and the use of protease-deficient strain so that the protein yield is not reduced 80, 81, 82.

318 Indian J Microbiol, December 2006

However, despite this knowledge, success has been limited. For example, lignin peroxidase of Phanerochaete chrysosporium could not be detected when fused to cellobiohydrolase-encoding cbh1 gene promoter of Trichoderma reesei 83; neither was ligninolytic activity (MnP) overproduced when cDNA encoding it was fused to the well-secreted glucoamylase in a protease-deficient strain of Aspergillus niger 44 . The results suggest that secretion of fusion protein is a complex process. Since the ability to secrete protein hydrolyzing an available substrate is essential for growth, use was made of the yeast temperature-sensitive mutants to identify steps in secretion. In these mutants a secretory step is normal at lower temperature but blocked only at a higher temperature. Using the temperature-sensitive mutants of yeast and invertase as a model secretory proteins the pathway (Fig. 5) indicated was84,85,: ER→ Berkeley bodies→Golgi →Secretory vesicles→Cell surface, i.e., after being translated in the ribosome, the protein is transported into the endoplasmic reticulum, where its subunits are assembled by correct disulphide bonds and the protein molecule correctly folded with the help of folding enzymes and chaperones85, and other modifications

such as disulphide bond formation and glycosylation86,87, take place catalyzed by an array of proteins. Following quality control checks the protein is transported into the Golgi and finally in carrier vesicles to the plasma membrane for fusion with plasma membrane and release into the periplasm between the plasma membrane and cell wall, the latter acting as a sieve allowing proteins of a particular size to pass through depending on its porosity. The low protein secretion can be due to defect in any of the steps. At least 23 genes with roles in secretory pathway have been identified in the yeast and a similar pathway is assumed in filamentous hypha. From the yeast genome sequence, about 20% of genes control functions that are related to cell wall biogenesis88, indicating that remodeling cell wall structure for enhancing protein secretion will be not be easy; it might even make the organism vulnerable to osmotic bursting due to lack or altered cross-linking of wall polymers89. Interacting factors Besides genetic factors, morphology of the fungus depends on the concentration of inoculum, composition of medium, the design of cultivation vessel (fermentor),

Fig. 5. Diagram of main events in the secretory path of protein. Abbreviations: N, nucleus; ER, endoplasmic reticulum; VAC, vacuole; G, Golgi ; SV, secretory vesicle. Fungi do not have stacked Golgi cisternae. Adapted from Conesa et al. (2001).

Fungal cell factory 319

although carried out by a different type of mycelium formed late in culture. A new finding is that of mycelial differentiation — of exploring hyphae penetrating into the substrate and of branching hyphae growing on surface — opening a new field of control of hyphal type in industrial fermentations92 (Vinck et al. 2005). When interacting factors in growth and differentiation are better understood, a system can be set up for best productivity, perhaps by slow feeding of nutrients, overlapping trophophase and idiophase. Human antibody production by fungi

Fig. 6. Growth forms of steroid- transforming mold Rhizopus nigricans. Fungus was grown in 100 ml medium/500 ml flask under different submerged cultivation conditions. Up, left: T=23 C, N=225 rpm, Inoculum= 103 spores/liter. Up, right; T= 19 C, N=150 rpm, Inoculum = 8 X 104 spores/liter; Bottom, left: T=23 C, N=100 rpm, Inoculum= 103 spores/liter; Bottom, right: T= 23 C, N=225 rpm, Inoculum = 107 spores/liter. Photo courtesy of Dr. Polona Znidarsic-Plazl (Food Technol. Biotechnol. 39: 237252, 2001)

addition of surfactants, mode of aeration (shearing forces), etc. In Rhizopus nigricans (Fig. 6) low inoculum resulted in small pellets which gave higher steroid transformation activity (progesterone 11α hydroxylation)57. In small pellets the ratio of growing hyphal length and inactive/dead hyphae is much higher. In concluding this section, we note that secretion of proteins is correlated with the period active growth of fungus (trophophase) or its release from autolysing cells (idiophase)57. Earlier, it had been thought that ligninolytic enzyme production occurs after primary growth has ceased in liquid-grown cultures due to nutrient limitation90 (Jeffries et al. 1981). However, Moukha et al. (1993) obtained different results with agar-surface-grown fungus sandwiched between two perforated membranes91. Though the radial growth of the fungal colony had stopped, new short branches were initiated at the colony centre which secreted Mn2+- dependent lignin peroxidase, suggesting that ligninase is produced by a specialized type of hyphae that develop after much of the assimilable carbon source has been consumed. In surface-grown cultures, this period coincided with accumulation of RNA transcripts and secretion of ligninase. Therefore lignin degradation is hyphal growth-associated process

Currently mammalian cell cultures are used for production of therapeutic proteins for inactivating or sequestering specific host proteins. These require expensive media of undefined composition for cultivation. Moreover, the average yield of antibody from hybridoma cells is about 100 milligram /liter. The major limitation in the therapeutic use of antibodies is producing a useful antibody in quantities required for clinical trials and use 93 (Nyssönen et al. 1993). Novozymes (USA), Genencor (USA) and DSM (The Netherlands) are using the yeast expression systems for producing large quantities of human-like antibody with addition of human-like N-glycan structures for therapeutic use is by a blending of fungal genetic techniques, recombinant DNA, and monoclonal antibody 94. Single chain antibody fragments have been produced in Aspergillus and Trichoderma as proteins fused to Trichoderma cellulase cbh1 promoter 95. Although fungi possess cell wall, hyphae of two related strains can fuse through short lateral branches to form a multinucleate mycelium containing a mixture of nuclei in a common cytoplasm (heterokaryon) without involving nuclear fusion. An exciting idea, based on fundamental knowledge being pursued by Neugenesis Corporation 96, 97 is to construct two separate vectors of light and heavy antibody chains as fusion proteins with a well-secreted protein (such as glucoamylase or cellobiohydrolase). A specific amino acid sequence is engineered between the secretory enzyme and the antibody chain in order that the secretory enzyme is clipped off by a host protease during the secretory process in the Golgi. These vectors are subsequently used to transform auxotrophic strains of N. crassa. The two transformed strains, i.e., one producing the light chain and the other producing the heavy chain (Fig 7) are fused to

320 Indian J Microbiol, December 2006

Heavy chain

Light chain

Heterokaryon formation

Auxotroph A

Auxotroph B

Fusion

Fermentation Heterokaryotic mycelium

Whole antibody

Fig. 7. A scheme for production of humanized antibody by heterokaryotic mycelium. Germinating spores of two auxotrophic strains (A and B) fuse to produce a heterokaryotic mycelium which can grow on a minimal medium lacking supplements because of complementation of non-allelic mutant genes. The mycelium secretes the heterologous whole antibody molecule. Based on a figure kindly provided by Dr. W. Dorsey Stuart (Novozyme-Neugenesis, Davis).

form a heterokaryon which produces both the antibody subunits and processes them into the intact monoclonal antibody molecules in synthetic medium of defined composition. As the information is classified, details are not available, Future researches “Each species has evolved to become a unique chemical factory producing substances in an unforgiving world.” This quote from E. O. Wilson98 is also true for fungi. However, the list of useful fungal products is small, and the list of exploited fungi even smaller - out of approximately 70,000 documented species of fungi, only a mere handful is exploited in industry despite the demonstration that fungi are sources of metabolites with antimicrobial, antidiabetic or anticancer properties99. One of the reasons is that isolation of fungi, their axenic culturing and investigations of their physiology and biochemistry is not fashionable science anymore. An alternative proposed in lieu of pure cultures of microorganisms is the direct extraction and cloning of DNA, obtained from unidentified mixtures (consortia) of microbial

communities taken from soil, seawater, insect guts, etc. 100, forming a metagenomic library, and finally its expression in E. coli 101. However, it is premature to infer general applicability of this approach to fungi. A coherent strategy of white biotechnology (i.e., the high-tech technology of using microorganisms by white-coated scientists and technicians) should combine traditional methods of new fungal hosts for microbial factories, and the development of suitable transformation protocols to accommodate the large gene clusters that are involved in secondary metabolite biosynthesis for a new species. Obtaining significant levels (greater than 10-15 percent of total protein) of heterologous protein requires fundamental researches — a comprehensive understanding of the signals for gene expression, strategies for adding, modifying or deleting regulatory genes; of the structure of gene product and its intracellular localization and interactions. Ultimately, since secretory proteins must exit through the cell wall, investigations of composition and three-dimensional structure of cell wall 102 and methods of manipulating its dynamic structure are important for improving the fungus’ secretion potential.

Fungal cell factory 321 Acknowledgments

15. van Brunt J (1986) Fungi: the perfect hosts? Biotechnology 4: 1057-1063.

I thank the following for granting permission to reproduce illustrations: John Gauntner (Sake World, Inc), W. Dorsey Stuart (Neugenesis Corporation), Polona Znidarsic-Plazl (University of Ljublanca), Bioengineering AG (Switzerland) and Novozymes Biotech Inc. (Denmark). Masayuki Machida (Ibaraki, Japan) advised on scientific nomenclature of koji mold, Manjuli Maheshwari edited the text and P. V. Balasubramanyam helped in graphics.

16. Foster JW (1947) Some introspections on mold metabolism. Bact Rev 11: 167-188.

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