Engineering Aspergillus niger for galactaric acid production: elimination of galactaric acid catabolism by using RNA sequencing and CRISPR/Cas9

Abstract

Background meso-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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0613-5) contains supplementary material, which is available to authorized users.

Sustainable Galactarate‐Based Polymers: Multi‐Enzymatic Production of Pectin‐Derived Polyesters

“…An alternative strategy to oxidize UA to AA is via mild biochemical route using enzymes. Examples of biotransformation of GalA into GA by engineered microorganisms based on GalA metabolism have been reported . The enzyme responsible for the oxidation in these studies is uronate dehydrogenase (UDH).…”

“…Examples of biotransformation of GalA into GA by engineered microorganisms based on GalA metabolism have been reported. [11][12][13] The enzyme responsible for the oxidation in these studies is uronate dehydrogenase (UDH). The industrial in vitro exploitation of UDH is not viable due to the requirement of expensive NADH as a cofactor.…”

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“…Further, to outcompete cohabitant bacteria and fungi in its natural environment, it is a naturally good secretor of organic acids including citric acid, oxalate and gluconate with applications in food, beverage, pharmaceutical and cosmetics industries (Andersen et al 2009). Also, it has been successfully redesigned to overproduce the building block itaconate used for the production of several synthetic polymers (Hossain et al 2016) and galactaric acid, the building block for nylon (Kuivanen et al 2016).…”

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“…Another high potential application area for the CRISPR technology is systematic genetic screening Proof-of-concept: Genome engineering for the production of galactaric acid from galacturonic acid [58] Both CRISPR and CRISPRi strategy for the production of various solvents [96] CRISPRi Corynebacterium glutamicum Lysine and glutamate…”