Disruption of the SHM2 gene, encoding one of two serine hydroxymethyltransferase isoenzymes, reduces the flux from glycine to serine in Ashbya gossypii

Abstract

Riboflavin overproduction in the ascomycete Ashbya gossypii is limited by glycine, a precursor of purine biosynthesis, and therefore an indicator of glycine metabolism. Disruption of the SHM 2 gene, encoding a serine hydroxymethyltransferase, resulted in a significant increase in riboflavin productivity. Determination of the enzyme's specific activity revealed a reduction from 3 m-units/mg of protein to 0.5 m-unit/mg protein. The remaining activity was due to an isoenzyme encoded by SHM 1, which is probably mitochondrial. A hypothesis proposed to account for the enhanced riboflavin overproduction of SHM 2-disrupted mutants was that the flux from glycine to serine was reduced, thus leading to an elevated supply with the riboflavin precursor glycine. Evidence for the correctness of that hypothesis was obtained from (13)C-labelling experiments. When 500 microM 99% [1-(13)C]threonine was fed, more than 50% of the label was detected in C-1 of glycine resulting from threonine aldolase activity. More than 30% labelling determined in C-1 of serine can be explained by serine synthesis via serine hydroxymethyltransferase. Knockout of SHM 1 had no detectable effect on serine labelling, but disruption of SHM 2 led to a decrease in serine (2-5%) and an increase in glycine (59-67%) labelling, indicating a changed carbon flux.

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

“…Overexpression of GLY1, encoding the glycine biosynthetic enzyme threonine aldolase, strongly enhanced RF production, apparently due to an improved supply of glycine, the purine precursor (312). An increase in RF production was also achieved by disruption of the SHM2 gene, encoding one of two isoenzymes of serine hydroxymethyltransferase, due to a defect in the conversion of glycine to serine, which again improved the glycine supply (398). Conversion of glyoxylate into glycine was improved by heterologous expression of the alanine:glyoxylate aminotransferase gene from S. cerevisiae (217).…”

Purine Biosynthesis, Riboflavin Production, and Trophic-Phase Span Are Controlled by a Myb-Related Transcription Factor in the Fungus Ashbya gossypii

“…For A. gossypii, it has been shown that riboflavin production is correlated with the activity of the Rib3 protein (34). However, enhanced riboflavin overproduction has been reported only when GTP precursors are added to the medium or when the purine or glycine pathways are engineered to provide high concentrations of GTP precursors (16,21,25,35,38). This indicates that GTP availability is a limiting factor for riboflavin production in A. gossypii.…”

“…Liquid cultures were inoculated with 1 ϫ 10 6 spores per liter of medium and were performed on a rotary shaker at 120 rpm. The determination of riboflavin production was performed by high-performance liquid chromatography as described previously (35).…”

“…The productive phase is associated with a characteristic intense yellow color of the mycelia, due to the accumulation of the vitamin in the vacuolar compartment (9). During the past few years, different studies have been carried out with a view to improving the excretion of the vitamin (8) and also to increasing the metabolic flux for riboflavin biosynthesis (16,17,25,35).…”

Metabolic Engineering of the Purine Pathway for Riboflavin Production in Ashbya gossypii

“…The A. gossypii ATCC 10895 strain was used and was considered a wild-type strain. A. gossypii was cultured at 28°C using MA2 rich medium (8), synthetic complete medium (27), or synthetic minimal medium (26). A. gossypii transformation, genomic DNA and RNA isolation, Southern blot and Northern blot analyses, spores isolation, cell protein extraction, and determination of total riboflavin contents were carried out as previously described (24,26).…”