“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-to-
batch 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/s00253-
018-8895-7
(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