Development of a homologous transformation system for Aspergillus niger based on the pyrG gene

Abstract

The development of a homologous transformation system for Aspergillus niger is described. The system is based on the use of an orotidine-5'-phosphate decarboxylase deficient mutant (pyrG) and a vector, pAB4-1, which contains the functional A. niger pyrG gene as a selection marker. Transformation of the A. niger pyrG mutant with pAB4-1 resulted in the appearance of stable Pyr+ transformants at a frequency of 40 transformants per microgram of DNA. In 90% of these transformants integration had occurred at the resident pyrG locus, resulting either in replacement of the mutant allele by the wild-type allele (60%) or in insertion of one or two copies of the vector (40%). The A. niger pyrG mutant could also be transformed with the vector pDJB2 containing the pyr4 gene of Neurospora crassa, at a frequency of 2 transformants per microgram of DNA. Integration at the resident pyrG locus was not found with this vector. The vector pAB4-1 is also capable of transforming an Aspergillus nidulans pyrG mutant to Pyr+. The pyrG transformation system was used for the introduction of a non-selectable gene into A. niger.

Database mining and transcriptional analysis of genes encoding inulin-modifying enzymes of Aspergillus niger

“…The A. niger strain used for the sequencing of the genome by DSM is CBS 513.88 (a natural derivative of strain NRRL 3122). Strain AB4.1 is a pyrG derivative of N402 (van Hartingsveldt et al, 1987) and was used to construct the creA deletion strain. A. niger strains were grown in minimal medium (MM) (Bennett & Lasure, 1991) spores ml 21 and incubated at 30 uC in a rotary shaker at 300 r.p.m.…”

Enhanced heterologous protein production in Aspergillus niger through pH control of extracellular protease activity

“…This strain produces two types of GLA, the plasmid encoded type that is part of the fusion protein, and a homologous type produced by the host. The strain AB4.1 is a pyrG1 derivative of N402 [17] and N402 is a cspA1 derivative of strain ATCC 9029 [4]. The medium for A. niger cultures was YM broth (Difco), which consisted of yeast extract 3.0 g/l, malt extract 3.0 g/l, peptone 5.0 g/l and dextrose 10 g/l.…”

The effect of environmental conditions on extracellular protease activity in controlled fermentations of Aspergillus niger

“…For decades they have been commonly exploited as commercial production organisms for a variety of enzymes. With the development of transformation systems for these industrially important members of the genus (Buxton et al, 1985;Kelly & Hynes, 1985;van Hartingsveldt et al, 1987;Iimura et al, 1987;Unkles et al, 1989), the expression of large quantities of heterologous proteins seemed within reach as well. And indeed, nowadays Aspergillus species dominate the list of host organisms for the commercial production of enzymes from fungal origin (according to the Association of Manufacturers and Formulators of Enzyme Products at www.amfep.org).…”