Engineering Aspergillus niger for galactaric acid production: elimination of galactaric acid catabolism by using RNA sequencing and CRISPR/Cas9
Backgroundmeso Galactaric acid is a dicarboxylic acid that can be produced by the oxidation of d galacturonic acid, the main constituent of pectin. Mould strains can be engineered to perform this oxidation by expressing the bacterial enzyme uronate dehydrogenase. In addition, the endogenous pathway for d galacturonic acid catabolism has to be inactivated. The filamentous fungus Aspergillus niger would be a suitable strain for galactaric acid production since it is efficient in pectin hydrolysis, however, it is catabolizing the resulting galactaric acid via an unknown catabolic pathway.ResultsIn this study, a transcriptomics approach was used to identify genes involved in galactaric acid catabolism. Several genes were deleted using CRISPR/Cas9 together with in vitro synthesized sgRNA. As a result, galactaric acid catabolism was disrupted. An engineered A. niger strain combining the disrupted galactaric and d galacturonic acid catabolism with an expression of a heterologous uronate dehydrogenase produced galactaric acid from d galacturonic acid. The resulting strain was also converting pectin rich biomass to galactaric acid in a consolidated bioprocess.ConclusionsIn the present study, we demonstrated the use of CRISPR/Cas9 mediated gene deletion technology in A. niger in an metabolic engineering application. As a result, a strain for the efficient production of galactaric acid from d galacturonic acid was generated. The present study highlights the usefulness of CRISPR/Cas9 technology in the metabolic engineering of filamentous fungi.
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