Targeting enzymes to the right compartment: metabolic engineering for itaconic acid production by Aspergillus niger.

Itaconic acid is an unsaturated dicarboxylic acid which has a high potential as a biochemical building block. It can be microbially produced from some Aspergillus species, such as Aspergillus itaconicus and Aspergillus terreus. However, the achieved titers are significantly lower as compared to the citric acid production by A. niger. Heterologous expression of cis-aconitate decarboxylase in A. niger leads to the accumulation of small amounts of itaconic acid. Additional expression of aconitase, the second enzyme metabolically linking citric acid and itaconic acid improves productivity. However, proper organelle targeting of the enzymes appears to be an important point to consider. Here we compare the mitochondrial expression with the cytosolic expression of cis-aconitate decarboxylase or aconitase in A. niger. Heterologous expression of both enzymes in the mitochondria doubles the productivity compared to strains which express the enzymes in the cytosol. It is essential to target enzymes to the correct compartment in order to establish a proper flux through a compartmentalized pathway.

Six novel constitutive promoters for metabolic engineering of Aspergillus niger.

Genetic tools for the fine-tuning of gene expression levels are a prerequisite for rational strain optimization through metabolic engineering. While Aspergillus niger is an industrially important fungus, widely used for production of organic acids and heterologous proteins, the available genetic tool box for this organism is still rather limited. Here, we characterize six novel constitutive promoters of A. niger providing different expression levels. The selection of the promoters was based on published transcription data of A. niger. The promoter strength was determined with the ß-glucuronidase (gusA) reporter gene of Escherichia coli. The six promoters covered a GUS activity range of two to three orders of magnitude depending on the strain background. In order to demonstrate the power of the newly characterized promoters for metabolic engineering, they were used for heterologous expression of the cis-aconitate decarboxylase (cad1) gene of Aspergillus terreus, allowing the production of the building block chemical itaconic acid with A. niger. The CAD activity, dependent on the choice of promoter, showed a positive correlation with the specific productivity of itaconic acid. Product titers from the detection limit to up to 570 mg/L proved that the set of constitutive promoters is a powerful tool for the fine-tuning of metabolic pathways for the improvement of industrial production processes.

An efficient tool for metabolic pathway construction and gene integration for Aspergillus niger.

Metabolic engineering requires functional genetic tools for easy and quick generation of multiple pathway variants. A genetic engineering toolbox for A. niger is presented, which facilitates the generation of strains carrying heterologous expression cassettes at a defined genetic locus. The system is compatible with Golden Gate cloning, which facilitates the DNA construction process and provides high design flexibility. The integration process is mediated by a CRISPR/Cas9 strategy involving the cutting of both the genetic integration locus (pyrG) as well as the integrating plasmid. Only a transient expression of Cas9 is necessary and the carrying plasmid is readily lost using a size-reduced AMA1 variant. A high integration efficiency into the fungal genome of up to 100% can be achieved, thus reducing the screening process significantly. The feasibility of the approach was demonstrated by the integration of an expression cassette enabling the production of aconitic acid in A. niger.

Biochemistry of microbial itaconic acid production.

Itaconic acid is an unsaturated dicarbonic acid which has a high potential as a biochemical building block, because it can be used as a monomer for the production of a plethora of products including resins, plastics, paints, and synthetic fibers. Some Aspergillus species, like A. itaconicus and A. terreus, show the ability to synthesize this organic acid and A. terreus can secrete significant amounts to the media (>80 g/L). However, compared with the citric acid production process (titers >200 g/L) the achieved titers are still low and the overall process is expensive because purified substrates are required for optimal productivity. Itaconate is formed by the enzymatic activity of a cis-aconitate decarboxylase (CadA) encoded by the cadA gene in A. terreus. Cloning of the cadA gene into the citric acid producing fungus A. niger showed that it is possible to produce itaconic acid also in a different host organism. This review will describe the current status and recent advances in the understanding of the molecular processes leading to the biotechnological production of itaconic acid.

Community profiling and gene expression of fungal assimilatory nitrate reductases in agricultural soil.

Although fungi contribute significantly to the microbial biomass in terrestrial ecosystems, little is known about their contribution to biogeochemical nitrogen cycles. Agricultural soils usually contain comparably high amounts of inorganic nitrogen, mainly in the form of nitrate. Many studies focused on bacterial and archaeal turnover of nitrate by nitrification, denitrification and assimilation, whereas the fungal role remained largely neglected. To enable research on the fungal contribution to the biogeochemical nitrogen cycle tools for monitoring the presence and expression of fungal assimilatory nitrate reductase genes were developed. To the ~100 currently available fungal full-length gene sequences, another 109 partial sequences were added by amplification from individual culture isolates, representing all major orders occurring in agricultural soils. The extended database led to the discovery of new horizontal gene transfer events within the fungal kingdom. The newly developed PCR primers were used to study gene pools and gene expression of fungal nitrate reductases in agricultural soils. The availability of the extended database allowed affiliation of many sequences to known species, genera or families. Energy supply by a carbon source seems to be the major regulator of nitrate reductase gene expression for fungi in agricultural soils, which is in good agreement with the high energy demand of complete reduction of nitrate to ammonium.