Increased riboflavin production from activated bleaching earth by a mutant strain of Ashbya gossypii

Abstract

The production of riboflavin from vegetable oil was increased using a mutant strain of Ashbya gossypii. This mutant was generated by treating the wild-type strain with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Riboflavin production was 10-fold higher in the mutant compared to the wild-type strain. The specific intracellular catalase activity after 3 d of culture was 6-fold higher in the mutant than in the wild-type strain. For the mutant, riboflavin production in the presence of 40 mM hydrogen peroxide was 16% less than that in the absence of hydrogen peroxide, whereas it was 56% less for the wild-type strain. The isocitrate lyase (ICL) activity of the mutant was 0.26 mU/mg of protein during the active riboflavin production phase, which was 2.6-fold higher than the wild-type strain. These data indicate that the mutant utilizes the carbon flux from the TCA cycle to the glyoxylate cycle more efficiently than the wild-type strain, resulting in enhanced riboflavin production. This novel mutant has the potential to be of use for industrial-scale riboflavin production from waste-activated bleaching earth (ABE), thereby transforming a useless material into a valuable bioproduct.

Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers

“…RF overproduction occurs on different residues, including oily waste-activated bleaching earth (490). Selection for resistance to RF analogs was not reported, apparently due to the resistance of A. gossypii to these compounds (462).…”

Ashbya gossypii beyond industrial riboflavin production: A historical perspective and emerging biotechnological applications

“…The filamentous fungus A. gossypii presents several industrially attractive features, such as good performance in large-scale fermentation processes and ability to grow in inexpensive waste-derived substrates to high cell densities (Park et al, 2007;Tajima et al, 2009;Wei et al, 2013). Its capacity to secrete proteins and metabolites to the culture medium and to undergo autolysis during the late stationary phase of growth or at low temperature is also industrially appealing, as they facilitate downstream product recovery ( Together, these new tools are leveraging the A. gossypii potential as a cell factory organism.…”

“…Since the industrial establishment of this process, different mutagenic agents and screening procedures were adopted and combined in different manners (Table 1). UV radiation (Park et al 2007;Schmidt et al, 1996b;Wei et al, 2012), chemical mutagens (Lizama et al, 2007;Tajima et al, 2009) and disparity mutagenesis (Park et al, 2011) have been applied to create mutations. For the screening procedures, antimetabolites (mainly itaconate and oxalate) have been the most common way to isolate overproducing strains, essentially due to the correlation between the A. gossypii isocitrate lyase (Icl1p; glyoxylate cycle) activity and riboflavin production (Schmidt et al, 1996a(Schmidt et al, , 1996b.…”

“…Among the rational strategies tested (Table 2), the majority focused on increasing the flux of the riboflavin biosynthetic pathway and of the purine/glycine biosynthetic pathways by: (1) overexpressing the AgRIBs genes (Althöefer et al, 1999;Althöefer and Revuelta, 2003); (2) overexpressing the AgGLY1 gene, involved in the conversion of threonine to glycine (Monschau et al, 1998); (3) disrupting the AgSHM2 gene, which encodes a cytosolic serine hydroxymethyltransferase that converts glycine into serine (Schlüpen et al, 2003); (4) overexpressing the S. cerevisiae"s AGX1 gene, which is not present in A. gossypii and encodes an alanine:glyoxylate aminotransferase that converts glyoxylate into glycine (Kato and Park, 2006); (5) overexpressing several genes involved in the purine biosynthetic pathway such as AgADE4 (Jiménez et al, 2005), AgPRS2,4 and AgPRS3 (Jiménez et al, 2008); (6) deregulating the transcription factor AgBas1p that constitutively activates the purine and glycine biosynthetic pathways (Mateos et al, 2006); and (7) blocking the A. gossypii"s de novo pyrimidine biosynthetic pathway by deletion of the AgURA3 gene (Aguiar et al, 2014c) to increase the availability/accumulation of PRPP for the formation and/or accumulation of riboflavin precursors (GTP and ribulose-5P; Silva et al, 2015). Other strategies focused on improving riboflavin production from plant oils by overexpressing genes involved in the glyoxylate cycle, such as the AgICL1 (Käsler et al, 1997) and the AgMLS1 (encoding the malate synthase; Sugimoto et al, 2009), and on increasing the excretion of riboflavin into the medium by disrupting the gene encoding the vacuolar ATPase subunit A, AgVMA1 (Förster et al, 1999).…”

Improvement of medium components for high riboflavin production by Aspergillus terreus using response surface methodology

“…The instrument covers a wavelength range of 190-1100 nm. A 0.8 ml of centrifuged culture broth was mixed with 0.2 ml of 1 M NaOH and neutralized with 1 ml of 0.1 M potassium phosphate buffer (pH 6.0) as modified by Tajima et al (2009). The amount of riboflavin in the supernatant was measured quantitatively at 444 nm against substrate-free blank.…”