Industrial Biotechnology.docx

“Fungal industrial biotechnology: process, products and applications” As in any other industry, the biotechnological also seeks to achieve the highest yield and lowest production cost possible. Especially with products with a biological origin, minimizing the batch-tobatch variation, increasing the shelf-life and constantly developing new products and processes is also key. As explained by Prof.dr. Peter J. Punt, Dutch DNA is a company functioning at Utrecht Science Park that delivers fungal production hosts for the production of enzymes, proteins and organic acids, giving R&D solutions to companies in metabolic engineering using fungi there are many reasons for selecting these organisms for industrial purposes. Filamentous fungi are cell factories to produce primary metabolites (e.g. fatty acids, extracellular polysaccharides, organic acids), secondary metabolites (e.g. penicillin, cephalosporins), enzymes (e.g. amylases, celluloses, peroxidases) and other products. Other properties such as the ease of large-scale cultivation, low cost production, straightforward genetics and long history of safe use make them a great choice for industrial production. Typical examples of host strains include Ascomycetes (Aspergillus, Trichoderma, Penicillium, Myceliophthora), Basidiomycetes (Trametes) and Zygomycetes (Rhizopus). Dutch DNA conducted a bench mark study to compare 6 organisms to measure their potential as industrially relevant hosts: Escherichia coli, Corynebacterium glutamicum, Saccharomyces cerevisiae, Pichia stipitis, Aspergillus niger and Trichoderma reesei. In the overall performance results, A. niger and P. stipitis obtained the best results. The general conclusion from the company is that Aspergillus fungi are interesting industrial hosts. Examples of research conducted at Dutch DNA using Aspergillus are: strain development and organic acids production. The development of strains, for instance, includes the design of protease deficient and fermenter adapted mutants. The production of organic acids from fungi is basically the exploitation of the natural biosynthetic pathways they use to produce primary metabolites. It is believed that fungi produce organic acids as a strategy to fight competitors by decreasing the surrounding pH. Organic acids have many applications in the food, pharmaceutical and cosmetic industries. Aspergillus fungi are well known producers of this kind of substances, mainly citric, gluconic, malic and itaconic acids. The last one, for instance, is of great interest because of its application as co-monomer in the manufacture of polymers such as resins and synthetic fibres and it is produced by organisms like A. terreus and A. itaconicus. For the formation of itaconic acid, sugars such as glucose, xylose or arabinose are used by A. terreus to be converted to pyruvate in the cytosol via the metabolic pathway glycolysis and the pentose phosphate pathway. The produced pyruvate is transported into the mitochondria and then catalysed over the citrate cycle to citrate and cis-aconitate. Via a citrate malate antiporter or mitochondrial tricarboxylate transporter, cis-aconitate is imported into the cytosol to be converted into itaconate by cis-aconitate decarboxylase (CAD) and then, said itaconate enters using a transporter. In the reaction of 1 mol glucose, xylose, or arabinose, 1 mol of itaconic acid can be formed (1). Dutch DNA tested different batch conditions in order to enhance the production of itaconic acid. After selecting the most appropriate condition, the researchers performed a transcriptomics study in order to identify the genes in A. terreus that are involved in the production of the substance. An itaconic acid gene cluster was identified, including genes for the production of CAD and important transporters such as the Mitochondrial tricarboxylate transporter (MTT) and for a Major Facilitator Superfamily Transporter (MFS). The company selected the genes that show the largest difference in expression between non-producing and producing cells. This was expressed as two different ratios: the sample with highest and lowest itaconate titer and the sample with the highest and lowest itaconate productivity. In general, there are two basic goals in the industrial production of itaconic acid: the

genetic modification and the process optimization. For the genetic modification, it is necessary to enable the building block production via pathway construction and enhancing the production via gene insertion and deletion. For the process optimization, the ideal is to select the best cultivation and production medium and the best fermentation conditions (including temperature, agitation, pH, sugar mixture and concentration and many other factors). Other fungal industrial exploitation is the production of enzymes. The Aspergillus genus is one of the favourite expression systems in the production of industrial enzymes to companies such as Dutch DNA, in particular A. niger and A. oryzae due to their high titers of hydrolytic enzymes such as amylases and proteases. Glucoamylase (AMG), as an example, is a homologous protein of A. niger used for the conversion of starch to sweeteners and the production of ethanol. Amylases are added to detergents to enhance the removal of stains. In the sustainability field, enzymes are of great interest in the biofuel industry as they are used in the saccharification of lignocellulosic materials which are converted to bioethanol. Other enzymes of interest are glucose oxidases, pectinases, cellulases, lipases and xylanases (2). References (1) Kuenz, A., & Krull, S. (2018). Biotechnological production of itaconic acid—things you have to know. Applied Microbiology And Biotechnology, 102(9), 3901-3914. doi: 10.1007/s00253018-8895-7 (2) Quintanilla, D., Hagemann, T., Hansen, K., & Gernaey, K. (2015). Fungal Morphology in Industrial Enzyme Production—Modelling and Monitoring. Advances In Biochemical Engineering/Biotechnology, 29-54. doi: 10.1007/10_2015_309