Abstract
BackgroundAspergillus niger is a filamentous fungus used for the majority of global citric acid production. Recent developments in genome editing now enable biotechnologists to engineer and optimize A. niger. Currently, however, genetic-leads for maximizing citric acid titers in industrial A. niger isolates is limited.ResultsIn this study, we try to engineer two citric acid A. niger production isolates, WT-D and D353, to serve as platform strains for future high-throughput genome engineering. Consequently, we used genome editing to simultaneously disrupt genes encoding the orotidine-5′-decarboxylase (pyrG) and non-homologous end-joining component (kusA) to enable use of the pyrG selection/counter selection system, and to elevate homologous recombination rates, respectively. During routine screening of these pyrG mutant strains, we unexpectedly observed a 2.17-fold increase in citric acid production when compared to the progenitor controls, indicating that inhibition of uridine/pyrimidine synthesis may increase citric acid titers. In order to further test this hypothesis, the pyrG gene was placed under the control of a tetracycline titratable cassette, which confirmed that reduced expression of this gene elevated citric acid titers in both shake flask and bioreactor fermentation. Subsequently, we conducted intracellular metabolomics analysis, which demonstrated that pyrG disruption enhanced the glycolysis flux and significantly improved abundance of citrate and its precursors.ConclusionsIn this study, we deliver two citric acid producing isolates which are amenable to high throughput genetic manipulation due to pyrG/kusA deletion. Strikingly, we demonstrate for the first time that A. niger pyrG is a promising genetic lead for generating citric acid hyper-producing strains. Our data support the hypothesis that uridine/pyrimidine biosynthetic pathway offer future avenues for strain engineering efforts.
Highlights
Aspergillus niger is a filamentous fungus used for the majority of global citric acid production
Gene disruption of pyrG and pyrG/kusA constructed by CRISPR/Cas9 system in A. niger In yeast and filamentous fungi, the pyrG gene has been widely used as nutritional/auxotrophic marker for fungal genetic manipulation [21, 22, 27,28,29,30]
With the advantage of counter selection using 5-fluoroorotic acid (5-FOA), pyrG could is utilized as the bidirectional selection marker for recyclable genome editing
Summary
Aspergillus niger is a filamentous fungus used for the majority of global citric acid production. Over the past 20 years, numerous technological advances have occurred in the A. niger field [5], including the development of recyclable selection markers, disruption of non-homologous end-joining systems for high throughput gene targeting, and, most recently, genome editing [6,7,8,9,10,11,12,13] Most of these tools and techniques have been developed and optimized in fully sequenced strain backgrounds. As sequencing and genome editing technologies are increasingly cheap and simple to develop, it is possible to expand the full A. niger toolkit to other industrially harnessed strains This is an attractive strategy, as optimization efforts in specific isolates can rapidly be applied at a commercial scale. The majority of genes with potential industrial applications to elevate citric acid production remain hypothetical and lack functional characterization in the laboratory [5, 16, 17]
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