Forward Genetics by Genome Sequencing Uncovers the Central Role of the Aspergillus niger goxB Locus in Hydrogen Peroxide Induced Glucose Oxidase Expression

Abstract

Aspergillus niger is an industrially important source for gluconic acid and glucose oxidase (GOx), a secreted commercially important flavoprotein which catalyses the oxidation of β-D-glucose by molecular oxygen to D-glucolactone and hydrogen peroxide. Expression of goxC, the GOx encoding gene and the concomitant two step conversion of glucose to gluconic acid requires oxygen and the presence of significant amounts of glucose in the medium and is optimally induced at pH 5.5. The molecular mechanisms underlying regulation of goxC expression are, however, still enigmatic. Genetic studies aimed at understanding GOx induction have indicated the involvement of at least seven complementation groups, for none of which the molecular basis has been resolved. In this study, a mapping-by-sequencing forward genetics approach was used to uncover the molecular role of the goxB locus in goxC expression. Using the Illumina and PacBio sequencing platforms a hybrid high quality draft genome assembly of laboratory strain N402 was obtained and used as a reference for mapping of genomic reads obtained from the derivative NW103:goxB mutant strain. The goxB locus encodes a thioredoxin reductase. A deletion of the encoding gene in the N402 parent strain led to a high constitutive expression level of the GOx and the lactonase encoding genes required for the two-step conversion of glucose in gluconic acid and of the catR gene encoding catalase R. This high constitutive level of expression was observed to be irrespective of the carbon source and oxidative stress applied. A model clarifying the role of GoxB in the regulation of the expression of goxC involving hydrogen peroxide as second messenger is presented.

Genetic Characterization of Mutations Related to Conidiophore Stalk Length Development in Aspergillus niger Laboratory Strain N402

“…Within this 9kb region, three open reading frames (ORFs) (NRRL3_03857, NRRL3_03858, and NRRL3_03859) are located. As noted in a previous report (Laothanachareon et al, 2018), NRRL3_03857…”

“…Strains N401 and N402 are derivatives of wild-type A. niger strain N400 and were obtained by two consecutive rounds of UV mutagenesis [Laothanachareon et al, 2018; F. Debets (Wageningen University), personal communication]. Stalk lengths of N400, N401, and N402 were determined as described in the "Materials and Methods" section.…”

“…Therefore, Bos and co-workers isolated a morphological mutant from the gluconic acid producing A. niger wild-type strain CBS120.49 (also known as N400, NRRL3 or ATCC9092). The morphological mutant was generated in two independent mutagenesis steps and resulted in the laboratory strain N402 (also known as ATCC64974 or FGSCA733) [Laothanachareon et al, 2018;F. Debets (Wageningen University), personal communication].…”

DNA sequencing, genomes and genetic markers of microbes on fruits and vegetables

“…Genomic comparison revealed approximately 98% similarity between the six A. flavus species and 81% similarity between A. flavus and A. parasiticus species (Faustinelli et al , 2016). Fourteen strains of A. niger have been registered, the majority of which have been related to citrate production or sugar metabolism (Aguilar‐Pontes et al , 2018; Laothanachareon et al , 2018). A. carbonarius is another ochratoxin A‐producing member of this genus, for which one strain has been sequenced (NCBI genome ID 947).…”

An inducible and reversible genetics platform for novel drug target discovery

“…Second, identify the causative mutation by whole exome sequencing or whole genome sequencing [21][22][23] . This method is relatively quick and avoids the need for positional cloning.…”