Abstract
To construct a set of isogenic auxotrophic strains in Aspergillus niger suited for creating multiple gene deletion mutants and executing parasexual crossings, we have combined mutations in genes involved in colour pigmentation (fwnA and olvA) with well-selectable auxotrophic markers (pyrG, nicB, argB, and adeA). All markers, except for the pyrG marker, were introduced by targeted deletion, omitting UV mutagenesis of the strains. Aspergillus oryzae orthologous genes of the argB, nicB, and adeA markers were used as heterologous selection markers, and all markers were shown to complement to respective auxotrophic A. niger mutants. A quadruple auxotrophic marker was further constructed suitable for multiple gene deletions. Genome sequencing of two auxotrophic colour mutants JN3.2 (olvA::pyrG, argB::hygB) and JN6.2 (olvA::pyrG, nicB::hygB) revealed four SNPs between them in non-coding regions, indicating a high level of isogenicity between both strains. The availability of near-isogenic complementary auxotrophic colour mutants facilitates the selection of diploids and the isolation of haploid segregants from the diploid using the parasexual cycle.Electronic supplementary materialThe online version of this article (doi:10.1007/s00203-016-1240-6) contains supplementary material, which is available to authorized users.
Highlights
Aspergillus niger has attracted considerable interest as cell factories for the production of organic compounds or proteins (Andersen et al 2013; Meyer et al 2015; Pel et al 2007; Ward 2012)
A limiting factor for metabolic engineering in A. niger is the limited number of isogenic auxotrophic mutants with multiple auxotrophic markers, in which multiple gene deletion mutants can be made quickly without the need to recycle the selection markers
By targeted deletion of spore colour genes and auxotrophies, we constructed a set of near-isogenic strains suitable for parasexual crossings (Niu et al 2016)
Summary
Aspergillus niger has attracted considerable interest as cell factories for the production of organic compounds (citric acid and secondary metabolites) or (recombinant) proteins (Andersen et al 2013; Meyer et al 2015; Pel et al 2007; Ward 2012). A limiting factor for metabolic engineering in A. niger is the limited number of isogenic auxotrophic mutants with multiple auxotrophic markers, in which multiple gene deletion mutants can be made quickly without the need to recycle the selection markers. Selection markers such as the pyrG marker or the amdS marker are counter-selectable, but when multiple deletions need to be made, these markers need to be recycled, which is time-consuming. By targeted deletion of spore colour genes and auxotrophies, we constructed a set of near-isogenic strains suitable for parasexual crossings (Niu et al 2016). We performed genome sequencing of two auxotrophic colour mutants and confirmed the near-isogenicity between these auxotrophic mutants
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
