Identification of Genes Associated with Morphology in Aspergillus niger by Using Suppression Subtractive Hybridization

Abstract

The morphology of citric acid production strains of Aspergillus niger is sensitive to a variety of factors, including the concentration of manganese (Mn 2؉ ). Upon increasing the Mn 2؉ concentration in A. niger (ATCC 11414) cultures to 14 ppb or higher, the morphology switches from pelleted to filamentous, accompanied by a rapid decline in citric acid production. The molecular mechanisms through which Mn 2؉ exerts effects on morphology and citric acid production in A. niger cultures have not been well defined, but our use of suppression subtractive hybridization has identified 22 genes responsive to Mn 2؉ . Fifteen genes were differentially expressed when A. niger was grown in media containing 1,000 ppb of Mn 2؉ (filamentous form), and seven genes were expressed in 10 ppb of Mn 2؉ (pelleted form). Of the 15 filament-associated genes, seven are novel and eight share 47 to 100% identity with genes from other organisms. Five of the pellet-associated genes are novel, and the other two genes encode a pepsin-type protease and polyubiquitin. All 10 genes with deduced functions are either involved in amino acid metabolism-protein catabolism or cell regulatory processes. Northern blot analysis showed that the transcripts of all 22 genes were rapidly enhanced or suppressed by Mn 2؉ . Steady-state mRNA levels of six selected filament-associated genes remained high during 5 days of culture in a filamentous state and remained low under pelleted growth conditions. The opposite behavior was observed for four selected pellet-associated genes. The full-length cDNA of the filament-associated clone, Brsa-25, was isolated. Antisense expression of Brsa-25 permitted pelleted growth and increased citrate production at concentrations of Mn 2؉ that were higher than the parent strain could tolerate. These results suggest the involvement of the newly isolated genes in the regulation of A. niger morphology.The morphology of filamentous fungi in fermentation processes is critical to maximum product output. The optimal morphology for the production of organic acids, enzymes, and secondary metabolites differs among fungi, but growth as small pellets is usually correlated with highly efficient fungal processes. For example, pelleted morphology is necessary for maximum production of citric acid by Aspergillus niger (9), itaconic acid by Aspergillus terreus (30), pravastatin precursor by Penicillium citrinum (17,47), and certain heterologous proteins by A. niger (57). It has been reported that filamentous growth is preferable for penicillin production by Penicillium chrysogenum (49) and fumaric acid production by Rhizopus arrhizus (6). The ability to obtain and maintain a particular morphology is one of the key parameters in the development of productive fungal fermentations. Empirically determined process conditions, such as agitation, dissolved oxygen concentration, substrate (carbon) concentration, nitrogen, phosphorous, and micronutrient concentrations, pH, ionic strength, and inoculum concentration have all been demonstrated to have effects...

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