Maximizing Protein Expression in Filamentous Fungi

Filamentous fungi are widely used in industry to produce small-molecule metabolites. Examples include antibiotics like penicillin, cholesterol-lowering drugs like Lovastatin, and food ingredients like citric acid. Because of their biological niche as microbial scavengers, filamentous fungi are also uniquely adapted to produce and secrete proteins. In environments rich in biological polymers, such as forest floors, the fungi thrive by secreting enzymes that degrade the polymers to produce monomers that can be readily used as nutrients for growth. The natural ability of fungi to produce proteins has been widely exploited, particularly in the production of industrial enzymes. While the levels of protein production in natural isolates are generally not high enough for commercial exploitation, improved strains and processes can lead to enormous yield increases, making it possible to produce yields of tens of grams of protein per liter of fermentation culture.

Richard P. Burlingame, PhD

Traditionally, these yield increases have been achieved through mutagenesis and screening for increased production of proteins of interest. While effective, this approach is useful only for the overproduction of endogenous proteins in isolates containing the enzymes of interest. For each new protein product, a lengthy strain and process development program is required, often with an organism for which there is little if any biochemical, physiological, or genetic knowledge.

Jan C. Verdoes, PhD

Since the 1980s, modern molecular genetic techniques have been applied to a number of filamentous fungi.^1,2 These methods have led to the development of various fungal hosts, such as various species of Aspergillus, Trichoderma, Neurospora, Fusarium, and Chrysosporium. With modern technologies, improved production of both native and non-native proteins can be achieved, shortening product development cycles and leading to greater exploitation of the physiological attributes that make fungi efficient protein producers.

STRATEGIES FOR MAXIMIZING PROTEIN YIELDS

Molecular cloning for high-level expression and secretion. To express endogenous or heterologous genes, the promoter of the gene to be expressed is generally replaced by a promoter sequence from a highly expressed gene of the fungal host organism or a closely related organism. This replacement results in higher expression levels and the ability to work under well-established and optimized fermentation conditions. Constitutive promoters from the highly expressed genes of central metabolic pathways—such as glycolytic genes—are common. Alternatively, inducible promoters from highly expressed genes specific to the particular host are also used frequently. Examples include those from the Aspergillus niger glucoamylase encoding gene (glaA); the A. oryzae α-amylase encoding gene (amy); and the Trichoderma reesei or Chrysosporium lucknowense cellobiohydrolase I encoding genes (cbh1). For a new fungal host, these promoters can be validated by analyzing the expression levels of single-copy integrants of reporter gene constructs at a specific locus. Another validation option is identifying host-specific strong promoters by determining the major secreted proteins in the new fungal host grown under various culture conditions. The expression signals from those genes will often be useful for the expression of other genes, whether from the host organism itself or from different organisms.

Gene expression constructs must then be introduced into cells of the fungal host. In fungi there are three major gene-transfer strategies: treating fungal cells with lytic enzymes to create protoplasts; biolistic bombardment; and Agrobacterium-mediated transformation. Various transformation frequencies, some of them yielding up to several thousands transformants per microgram of DNA, have been reported.¹

Various approaches to improve transcription have been used in fungi. For the expression of heterologous genes, especially of nonfungal origin, codon-optimized, synthetic genes can improve the transcription rate. The gene is adapted to the preferred codon usage of the host strain, based on the codon usage data of a large number of well-expressed or secreted proteins. To obtain high- level expression of a particular gene, a well-established procedure is targeting multiple copies of the recombinant gene constructs to the locus of a highly expressed endogenous gene. An advantage of such a gene replacement is knowing that the integration occurs in a region of the genome that is actively transcribed. When the target locus is that of a highly-expressed secreted protein, the reduced load on the secretion pathway may facilitate expression and secretion of the recombinant gene product.

For the secretion of foreign proteins, fusion strategies are used to facilitate translocation in the secretion pathway and to protect the heterologous protein from degrading. An amino terminal fusion with an efficiently secreted protein is generally preferred. A truncated form of the A. niger glucoamylase (GII) gene has been used successfully in several fungal hosts.³ To separate the fusion partner from the target gene, one can use the endogenous secretion machinery of the fungal host or engineer cleavage sites between the fusion partner and target. During normal fungal protein secretion, passage of the protein through the cell membrane is accompanied by proteolytic cleavage of the signal sequence from the mature protein in a sequence-specific manner. When such a recognition sequence is engineered between the fusion partner and target protein, cleavage results, with the secretion of the protein of interest separated from the fusion partner. Sometimes, however, it is desirable to maintain intact fusion proteins, if they are more stable than the corresponding independent target protein. In those cases, protease recognition sites that are not recognized by the cellular secretion machinery can be engineered between the secretion partner and target protein, with subsequent in vitro cleavage and separation of the target from the fusion partner.

Reducing proteolytic degradation. One of the major challenges in fungal biotechnology, especially for the production of pharmaceutical proteins, is that the recombinant protein is often degraded by proteases of the fungal host. Reducing protease activities can be achieved by combining different approaches. One involves selecting or screening for fungal host strains with low protease activity. This can be accomplished either by direct selection, using "suicide" selection substrates, or by mutagenesis and screening for reduced protease activity on indicator plates. Other approaches are identifying and inactivating genes for specific proteases, identifying and modifying wide-domain protease regulators, and optimizing medium composition and fermentation protocols.⁴ The isolation of mutant strains that can produce higher levels of a specific heterologous protein with subsequent removal of the expression construct is another strategy used to produce host strains with reduced proteolytic activity.⁵

Post-translational modification. Protein glycosylation is an important consideration in the production of eukaryotic proteins and is especially important for therapeutics. Non-native glycosylation can influence serum half-life, function, and immunogenicity of therapeutic proteins. Considerable progress has been made in modifying the glycosylation patterns of fungi, especially in the methylotrophic yeast Pichia pastoris.⁶ While there has been considerable effort in understanding glycan biosynthesis,³ the progress toward glycan modification in filamentous fungi is not as advanced as in Pichia. Modification of native fungal glycan structures to make them human-identical or human-compatible, whether by in vivo methods or by in vitro remodeling⁷ will be important if filamentous fungi are going to be viable alternative systems for the production of proteins of pharmaceutical interest.

Figure 1. At left is a microscopic image of wild type Chrysosporium lucknowense. The filamentous nature of this organism results in highly viscous cultures, surface matting, and clumping. A nonviscous mutant "propagule" is shown at right. The nonfilamentous strain offers advantages for high-throughput screening, gene expression, and protein production.

Morphology control. An interesting strategy for improving protein production in filamentous fungi is morphology control. The filamentous nature of fungi leads to high viscosities in fermentors, resulting in difficulties with agitation, mixing, and aeration in submerged fermentation. These difficulties result in poor oxygen and nutrient transfer to the growing cultures, reducing their capacity for growth and protein production. One approach to reducing viscosity is isolating low-viscosity mutant strains. Figure 1 compares wild-type and low-viscosity strains of Chrysosporium lucknowense. The morphology of the low-viscosity strain is characterized by mycelial fragmentation and the formation of discrete elements known as "propagules." Cultures composed of these propagules exhibit lower viscosity, better nutrient and oxygen transfer, and higher protein production. The latter results from the inherent properties of the strain itself and an amenability to more nutrient and oxygen-rich culture conditions than would be otherwise unattainable. As illustrated in Figure 2, the dramatic decreases in culture viscosity are accompanied by increased protein production per unit of biomass. When compared with its parent strain, the low-viscosity mutant produces about twice the total protein yield when the two strains are grown under similar conditions. Moreover, when improved culture conditions afforded by the low viscosity are applied, an additional three-fold increase in protein production is achieved.

Figure 2. The low-viscosity strain UV18-25 exhibits approximately a 50-fold lower viscosity in fermentors than the high-viscosity strain NG7C-19 (left side). This results in a four-fold increase in the amount of protein produced per unit of biomass under similar fermentation conditions (center). On the right side of the graph: When the two strains were grown under identical fermentation conditions, the protein yield of the low-viscosity strain was about twice that of the high-viscosity strain (blue vs. green bars). Under culture conditions optimized for the low-viscosity strain, three-fold further increases were obtained (yellow bar). The latter culture conditions were not attainable with the high-viscosity strain due to mixing and aeration limitations.

A more recent approach to improved morphology control is identifying genes and proteins involved in morphology control, as well as genes that are differentially expressed under conditions where fungal cultures exhibit different morphological characteristics.⁸ In those studies, 15 such genes were identified, and it is anticipated that controlled expression of some of these genes will influence the morphology of fungi used to produce commercial products.

A third approach to viscosity reduction is altering feeding strategies in fermentors. By using a pulse-feed strategy of a limiting carbon source, reductions in viscosity and mean mycelial particle size were observed in fermentations with a strain of Aspergillus oryzae producing recombinant glucoamylase.⁹ These changes were accompanied by an increase in glucoamylase production in those strains.

Genomics and proteomics of fungi. Researchers increasingly are using high-throughput methods for genomic sequencing and proteomic analysis to investigate the fundamental biology of fungi. To date, about 80 fungal strains (including both yeasts and filamentous fungi) have been or are being sequenced, including fungi of medical, agricultural, and industrial importance.¹⁰ The sequencing and annotation of fungi facilitates, for example, identification of genes and proteins involved in protein synthesis, protein secretion, protein degradation, morphology control, and post-translational modifications. Furthermore, whole genome transcriptional analysis using microarrays will be possible when specific genomes are sequenced and annotated. Armed with this knowledge, investigators will be able to rapidly discover, characterize, and modify genes involved with the basic cellular processes of protein production. Differential analysis of gene transcription and protein production patterns under various conditions will enable researchers to identify the factors controlling these basic cellular processes. As more genomic and proteomic information becomes available, this knowledge-based improvement of fungal host strains and fermentation processes for the commercial production of proteins and metabolites is becoming more and more common.

INTEGRATING SCREENING, PRODUCTION, AND MANUFACTURING SYSTEMS

To satisfy the protein expression needs of the various stages of drug discovery and development, an ideal fungal expression system should have the ability to (i) express a wide variety of eukaryotic proteins; (ii) express proteins in a biologically active form; (iii) produce proteins in a rapid and inexpensive fashion; (iv) seamlessly interface with laboratory automation; (v) produce sufficient quantities of protein to support the confirmation of hits and the many other activities involved in transforming a hit into a drug; and (vi) scale up to commercial drug production. With these needs in mind, an integrated technology platform is currently under development using Chrysosporium lucknowense gene expression technology coupled with high-throughput screening.¹¹ The morphology of this fungal strain enables this technology platform. Its low viscosity in culture and formation of propagules allows the growth of this fungus in microtiter cultures and the manipulation of cultures in liquid handling systems utilizing robotics. These characteristics enable high-throughput screening of gene expression libraries, either for gene discovery or for screening variants created using molecular evolution.¹² The ability to grow in microtiter plates can also facilitate gene expression and strain development processes. This miniaturization allows large numbers of transformants to be screened for the presence of genes and gene products, using high-throughput hybridization and blotting methods. Finally, as described earlier, the low-viscosity phenotype allows more versatile and robust culture conditions to optimize protein manufacturing by fermentation.

Using a single organism for gene discovery, gene evolution, protein engineering, gene expression, and product manufacturing offers significant advantages in the product development area by increasing the probability of success and decreasing development time. Such an integrated system will be anticipated to eliminate bottlenecks in the drug development process that are inherent when different organisms are used for gene discovery, protein evolution, early expression for preclinical testing, and product manufacturing. Screening by expression will likely ensure that improved variants will also be well expressed, and successful expression at the laboratory scale will more than likely lead to successful expression in the final manufacturing process. The downside of integrating the technologies is that creating the platform requires the development and seamless integration of several complex technologies. Filamen-tous fungi are genetically and physiologically less tractable than bacteria or yeast. However, the physiological advantages of filamentous fungi—high level protein production and secretion, post-translational modification, and proper folding—suggest that once the technical obstacles are overcome, the technology platform will be especially valuable as the number of biopharmaceutical candidates continues to grow.

Richard P.Burlingame,PhD, is the director of R&D for Dyadic International, 140 Intracoastal Pointe Dr., Jupiter, FL 33477; tel 561.743.8333; rburlingame@dyadic-group.com

Jan C.Verdoes,PhD, Dyadic Nederland, BV, Utrechtseweg 48, 3704 HE Zeist, The Netherlands; tel. +31.30.6944015, jverdoes@dyadic-group.com

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