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

Thanaporn Laothanachareon,Bart Nijsse,Juan Antonio Tamayo-Ramos,Peter J. Schaap

doi:10.3389/fmicb.2018.02269

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.

Highlights

Aspergillus niger is a high performance microbial cell factory and used for cost effective production of food additives, pharmaceuticals and industrial enzymes
Since the N402 master strain was first selected for having the cspA1 phenotype, which could only be obtained after 2 consecutive rounds of UV mutagenesis and selection, we surmised that this trait may be polygenic, and that the N402 master strain could have acquired multiple unlinked mutations which are directly passed on to the NW102 and NW103 goxB strains
As this could hamper molecular identification of specific loci, we first obtained a highquality A. niger N402 draft genome sequence using a hybrid of Illumina and PacBio RS II sequencing, and subsequently mapped NW103 Illumina reads against the scaffolds, while focusing on linkage group II

Summary

Introduction

Aspergillus niger is a high performance microbial cell factory and used for cost effective production of food additives, pharmaceuticals and industrial enzymes. Having the abilities of a natural tolerance toward acid media as well as a high secretory capacity, A. niger is exploited as a cell factory to produce citric and gluconic acid (Meyer et al, 2011). N402 showed a phenotype different from its origin, notably the development of shorter conidiophores (cspA1), a property that makes this strain much more amendable to genetic manipulations in a laboratory setting than wild type strains

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