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

Abstract

Ashbya gossypii is a natural riboflavin overproducer used in the industrial production of the vitamin. We have isolated an insertional mutant exhibiting higher levels of riboflavin production than the wild type. DNA analysis of the targeted locus in the mutant strain revealed that a syntenic homolog of the Saccharomyces cerevisiae BAS1 gene, a member of the Myb family of transcription factors, was inactivated. Directed gene disruption of AgBAS1 confirmed the phenotype observed for the insertional mutant, and the ⌬bas1 mutant also showed auxotrophy for adenine and several growth defects, such as a delay in the germination of the spores and an abnormally prolonged trophic phase. Additionally, we demonstrate that the DNA-binding domain of AgBas1p is able to bind to the Bas1-binding motifs in the AgADE4 promoter; we also show a clear nuclear localization of a green fluorescent protein-Bas1 fusion protein. Real-time quantitative PCR analyses comparing the wild type and the ⌬bas1 mutant revealed that AgBAS1 was responsible for the adenine-mediated regulation of the purine and glycine pathways, since the transcription of the ADE4 and SHM2 genes was virtually abolished in the ⌬bas1 mutant. Furthermore, the transcription of ADE4 and SHM2 in the ⌬bas1 mutant did not diminish during the transition from the trophic to the productive phase did not diminish, in contrast to what occurred in the wild-type strain. A C-terminal deletion in the AgBAS1 gene, comprising a hypothetical regulatory domain, caused constitutive activation of the purine and glycine pathways, enhanced riboflavin overproduction, and prolonged the trophic phase. Taking these results together, we propose that in A. gossypii, AgBAS1 is an important transcription factor that is involved in the regulation of different physiological processes, such as purine and glycine biosynthesis, riboflavin overproduction, and growth.

Strain Design of Ashbya gossypii for Single-Cell Oil Production

“…A. gossypii has been considered a paradigm of sustainable white biotechnology through its use in the industrial overproduction of riboflavin and other vitamins (16). Indeed, we have previously described metabolically engineered strains of A. gossypii that are able to produce 10-fold more riboflavin than a wild-type (WT) strain (17)(18)(19), and similar results have been achieved for the production of folic acid (J. L. Revuelta, unpublished results). The fermentation processes using A. gossypii have several advantages, such as the mycelial autolysis that occurs in late stationary phase, which avoids costly recovery steps, and its ability to grow in different oil sources and oil-containing wastes (16,20).…”

“…A. gossypii is considered to be a model organism for the biotechnological production of vitamins, nucleotides, and proteins (19,29,30). Furthermore, it has been reported that A. gossypii is able to accumulate a significant amount of lipid bodies in its multinucleated hyphae (21) (Fig.…”

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

“…In E. ashbyii the shift from growth to the production phase was accompanied by derepression of GTP cyclohydrolase II and FAD synthetase, whereas the activity of RF synthase was only slightly changed (239). In A. gossypii, the RF production phase was characterized by a strong increase in transcription of several RF biosynthesis genes (RIB3, RIB4, and RIB5 but not RIB2 and RIB7), as well as many other genes involved in transcription, translation, membrane transport, cell architecture, and metabolism (209,292,396,397). The mechanisms involved in the shift from growth to production phase and in the induction of RF synthesis and the nature of the messenger(s) that induces RF oversynthesis remain unknown.…”

“…A protocol for insertion mutagenesis of A. gossypii has been developed (391), and a disruption mutant defective in gene BAS1 (encoding a member of the Myb family of transcription factors) has been tagged. Corresponding disruption and knockout strains overexpressed RF (292). BAS1 proved to be involved in adeninemediated repression of the de novo biosynthesis of purine nucleotides.…”

“…Furthermore, it participates in the regulation of the glycine pathway, spore germination, and the duration of the trophic phase. Deletion of the C-terminal part of BAS1 resulted in constitutive activation of the adenine and glycine pathways, thereby enhancing RF overproduction (292).…”