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

Abstract

Purine nucleotides are essential precursors for living organisms because they are involved in many important processes, such as nucleic acid synthesis, energy supply, and the biosynthesis of several amino acids and vitamins such as riboflavin. GTP is the immediate precursor for riboflavin biosynthesis, and its formation through the purine pathway is subject to several regulatory mechanisms in different steps. Extracellular purines repress the transcription of most genes required for de novo ATP and GTP synthesis. Additionally, three enzymes of the pathway, phosphoribosyl pyrophosphate (PRPP) amidotransferase, adenylosuccinate synthetase, and IMP dehydrogenase, are subject to feedback inhibition by their end products. Here we report the characterization and manipulation of the committed step in the purine pathway of the riboflavin overproducer Ashbya gossypii. We report that phosphoribosylamine biosynthesis in A. gossypii is negatively regulated at the transcriptional level by extracellular adenine. Furthermore, we show that ATP and GTP exert a strong inhibitory effect on the PRPP amidotransferase from A. gossypii. We constitutively overexpressed the AgADE4 gene encoding PRPP amidotransferase in A. gossypii, thereby abolishing the adenine-mediated transcriptional repression. In addition, we replaced the corresponding residues (aspartic acid 310 , lysine 333 , and alanine 417 ) that have been described to be important for PRPP amidotransferase feedback inhibition in other organisms by site-directed mutagenesis. With these manipulations, we managed to enhance metabolic flow through the purine pathway and to increase the production of riboflavin in the triple mutant strain 10-fold (228 mg/liter).

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(WT) strain (17)(18)(19), and similar results have been achieved for the production of folic acid (J. L. Revuelta, unpublished results).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).…”

“…The other A. gossypii strains used in the study are listed in Table S1 in the supplemental material. A. gossypii was cultured at 28°C using MA2 rich medium, synthetic complete (SC) medium, or synthetic minimal medium lacking leucine (SCϪleu) (17). For lipid accumulation analyses, the carbon source of the MA2 medium was either 8% glucose or 1% glucose plus 2% oleic acid (Sigma), previously emulsified by sonication in the presence of 0.02% Tween 40 (22).…”

Biosynthesis of Vitamins by Probiotic Bacteria

“…Currently, A. gossypii, Candida famata, and B. subtilis are exploited for riboflavin production, with riboflavin production levels reaching 15 g/L, 20 g/L, and 14 g/L, respectively [19][20][21]. In A. gossypii, metabolic engineering increased riboflavin production almost 10-fold [22]. A. gossypii has also been targeted as a microorganism to overproduce riboflavin using oil waste [23].…”

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

“…To alleviate feedback inhibition of this enzyme by ATP and GTP, site-specific mutagenesis of the ADE4 gene by replacement of three amino acid residues was carried out. Constitutive overexpression of this engineered enzyme resulted in a 10-fold increase in RF synthesis (195). A. gossypii contains two phosphoribosyl pyrophosphate synthetases, encoded by genes PRS2,4 and PRS3.…”

“…Conversion of glyoxylate into glycine was improved by heterologous expression of the alanine:glyoxylate aminotransferase gene from S. cerevisiae (217). To enhance the metabolic flux through the purine nucleotide biosynthesis pathway, the key regulatory gene ADE4, encoding phosphoribosyl pyrophosphate amidotransferase, was cloned and overexpressed under the control of the strong constitutive promoter of glyceraldehyde phosphate dehydrogenase to abolish repression caused by exogenous adenine (195). To alleviate feedback inhibition of this enzyme by ATP and GTP, site-specific mutagenesis of the ADE4 gene by replacement of three amino acid residues was carried out.…”