Maximizing Protein Expression in Filamentous Fungi - Filamentous fungi are efficient protein producers that hold great promise for shortening product development cycles. - BioPharm International


Maximizing Protein Expression in Filamentous Fungi
Filamentous fungi are efficient protein producers that hold great promise for shortening product development cycles.

BioPharm International
Volume 19, Issue 5

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.
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.

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.

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