Threonine Aldolase Overexpression plus Threonine Supplementation Enhanced Riboflavin Production in Ashbya gossypii

Riboflavin production in the filamentous fungus Ashbya gossypii is limited by glycine, an early precursor required for purine synthesis. We report an improvement of riboflavin production in this fungus by overexpression of the glycine biosynthetic enzyme threonine aldolase. The GLY1 gene encoding the threonine aldolase of A. gossypii was isolated by heterologous complementation of the glycine-auxotrophic Saccharomyces cerevisiae strain YM13 with a genomic library from A. gossypii. The deduced amino acid sequence of GLY1 showed 88% similarity to threonine aldolase from S. cerevisiae. In the presence of the GLY1 gene, 25 mU of threonine aldolase specific activity mg⁻¹ was detectable in crude extracts of S. cerevisiae YM13. Disruption of GLY1 led to a complete loss of threonine aldolase activity in A. gossypii crude extracts, but growth of and riboflavin production by the knockout mutant were not affected. This indicated a minor role of the enzyme in glycine biosynthesis of A. gossypii. However, overexpression of GLY1 under the control of the constitutive TEF promoter and terminator led to a 10-fold increase of threonine aldolase specific activity in crude extracts along with a 9-fold increase of riboflavin production when the medium was supplemented with threonine. This strong enhancement, which could not be achieved by supplementation with glycine alone, was attributed to an almost quantitative uptake of threonine and its intracellular conversion into glycine. This became evident by a subsequent partial efflux of the glycine formed.     

The improvement of riboflavin production in <Emphasis Type="Italic">Ashbya gossypii</Emphasis> via disparity mutagenesis and DNA microarray analysis

We generated a high riboflavin-producing mutant strain of Ashbya gossypii by disparity mutagenesis using mutation of DNA polymerase δ in the lagging strand, resulting in loss of DNA repair function by the polymerase. Among 1,353 colonies generated in the first screen, 26 mutants produced more than 3 g/L of riboflavin. By the second screen and single-colony isolation, nine strains that produced more than 5.2 g/L of riboflavin were selected as high riboflavin-producing strains. These mutants were resistant to oxalic acid and hydrogen peroxide as antimetabolites. One strain (W122032) produced 13.7 g/L of riboflavin in a 3-L fermentor using an optimized medium. This represents a ninefold improvement on the production of the wild-type strain. Proteomic analysis revealed that ADE1, RIB1, and RIB5 proteins were expressed at twofold higher levels in this strain than in the wild type. DNA microarray analysis showed that purine and riboflavin biosynthetic pathways were upregulated, while pathways related to carbon source assimilation, energy generation, and glycolysis were downregulated. Genes in the riboflavin biosynthetic pathway were significantly overexpressed during both riboflavin production and stationary phases, for example, RIB1 and RIB3 were expressed at greater than sixfold higher levels in this strain compared to the wild type. These results indicate that the improved riboflavin production in this strain is related to a shift in carbon flux from β-oxidation to the riboflavin biosynthetic pathway.     

Three biotechnical processes using Ashbya gossypii, Candida famata, or Bacillus subtilis compete with chemical riboflavin production

Chemical riboflavin production, successfully used for decades, is in the course of being replaced by microbial processes. These promise to save half the costs, reduce waste and energy requirements, and use renewable resources like sugar or plant oil. Three microorganisms are currently in use for industrial riboflavin production. The hemiascomycetes Ashbya gossypii, a filamentous fungus, and Candida famata, a yeast, are naturally occurring overproducers of this vitamin. To obtain riboflavin production with the Gram-positive bacterium Bacillus subtilis requires at least the deregulation of purine synthesis and a mutation in a flavokinase/FAD-synthetase. It is common to all three organisms that riboflavin production is recognizable by the yellow color of the colonies. This is an important tool for the screening of improved mutants. Antimetabolites like itaconate, which inhibits the isocitrate lyase in A. gossypii, tubercidin, which inhibits purine biosynthesis in C. famata, or roseoflavin, a structural analog of riboflavin used for B. subtilis, have been applied successfully for mutant selections. The production of riboflavin by the two fungi seems to be limited by precursor supply, as was concluded from feeding and gene-overexpression experiments. Although flux studies in B. subtilis revealed an increase both in maintenance metabolism and in the oxidative part of the pentose phosphate pathway, the major limitation there seems to be the riboflavin pathway. Multiple copies of the rib genes and promoter replacements are necessary to achieve competitive productivity. Received: 19 November 1999 / Accepted: 21 December 1999     

Importance of malate synthase in the glyoxylate cycle of <Emphasis Type="Italic">Ashbya gossypii</Emphasis> for the efficient production of riboflavin

The glyoxylate cycle is an anabolic pathway that is necessary for growth on nonfermentable carbon sources such as vegetable oils and is important for riboflavin production by the filamentous fungus Ashbya gossypii. The aim of this study was to identify malate synthase in the glyoxylate cycle of A. gossypii and to investigate its importance in riboflavin production from rapeseed oil. The ACR268C gene was identified as the malate synthase gene that encoded functional malate synthase in the glyoxylate cycle. The ACR268C gene knockout mutant lost malate synthase activity, and its riboflavin production and oil consumption were 10- and 2-fold lower, respectively, than the values of the wild-type strain. In contrast, the ACR268C gene-overexpressing strain showed a 1.6-fold increase in the malate synthase activity and 1.7-fold higher riboflavin production than the control strain. These results demonstrate that the malate synthase in the glyoxylate cycle has an important role not only in riboflavin production but also in oil consumption.     

Effect of vitamin E and menadione supplementation on riboflavin production and stress parameters in Ashbya gossypii

Ashbya gossypii is a filamentous fungus which overproduces riboflavin as a pseudo-secondary metabolite. Vitamin E supplemented at 1, 2.5 and 5 μM levels in the growth medium of A. gossypii increased the extracellular secretion of riboflavin and at 50, 100 and 240 μM levels reduced the biomass and riboflavin yield. With 2.5 μM vitamin E total riboflavin production and extracellular riboflavin secretion on day 2 was higher than non-supplemented control. By day 3 the production in supplemented was nearly the same as in non-supplemented, but the intracellular riboflavin levels were lower and extracellular levels higher. Supplemented cells showed increased levels of catalase, glutathione peroxidase, lipid peroxides and membrane lipid peroxides, and decreased glutathione indicating that vitamin E, a well-known antioxidant, had acted as a pro-oxidant at low levels of 2.5 μM and had increased the oxidative stress. Menadione, a well known oxidant also increased riboflavin production and secretion at 1.0, 2.5 and 5.0 μM level. This is the first report were vitamin E and menadione effects support the concept that overproduction of riboflavin is a stress induced phenomenon. These findings are not only of scientific interest but also useful for improving the industrial production of riboflavin.     

A developmental stage of hyphal cells shows riboflavin overproduction instead of sporulation in Ashbya gossypii

The hemiascomycete Ashbya gossypii develops a mycelium. Nutritional stress leads to its differentiation into sporangia. These generate spores. In parallel, the yellow pigment riboflavin is produced. Intracellularly accumulated riboflavin, made visible as a bright green fluorescence, was observed in only 60 % of the hyphal cells. For the remaining 40 %, it was unclear whether these cells simply export riboflavin or its biosynthesis remains down-regulated in contrast to the accumulating cells. The approach followed in this work was to convert the hyphae into protoplasts by enzymatic degradation of the cell wall. Afterwards, the protoplasts were sorted by fluorescence-activated cell sorting on the basis of riboflavin accumulation. When a reporter strain expressing lacZ under the control of the most important riboflavin biosynthesis promoter, RIB3, was used, green protoplasts were found to have more than tenfold greater reporter activity than hyaline protoplasts. This was true on the basis of total protein as well as on the basis of hexokinase specific activity, a marker for constitutive expression. These results allow the conclusion that hyphal cells of A. gossypii differ in phenotype regarding riboflavin overproduction and accumulation.     

Isolation of an oxalate-resistant Ashbya gossypii strain and its improved riboflavin production

An oxalate-resistant strain of Ashbya gossypii was naturally isolated from spores grown on an oxalate-containing medium, and its medium was optimized to improve riboflavin production. Riboflavin production by the resistant strain was three-fold higher than that by the wild-type organism when grown in flask cultures. Medium optimization increased the riboflavin production by the resistant strain to 5 g l⁻¹, which was five-fold higher than that obtained by the wild-type strain. The productivity was reproduced in a 3-l bioreactor. During the early growth phase, the specific activity of isocitrate lyase in the oxalate-resistant strain was slightly higher than that in the wild-type strain. Proteomic analysis of the oxalate-resistant strain revealed that the expression of aldose reductase and cobalamin-independent methionine synthase decreased significantly. This is the first report that describes the natural isolation of a riboflavin producer using an antimetabolite-containing medium to enhance the riboflavin production level. This method should also be useful for improving the productivity of other bioproducts since it does not require any mutations or genetic modifications of the microorganism.     

Effects of a riboflavin source suitable for use in organic broiler diets on performance traits and health indicators

Riboflavin (vitamin B₂) is essential for monogastric animals. It is mainly produced by recombinant microorganisms (Candida famata, Bacillus subtilis and Ashbya gossypii). The availability of genetically modified organism (GMO)-free riboflavin, obligatory in European organic agriculture, is a major issue. Besides, requirements for organic livestock might differ from conventional production because other genotypes and feed formulations are used. The effects of a fermentation suspension with a high native content of riboflavin produced with unmodified A. gossypii by fermentation were investigated at graded dosages as an alternative to conventional (GMO-based) riboflavin in slow-growing broilers on performance traits and health and welfare indicators. In 2 runs with 800 animals each, Ranger Gold™ broilers were fed with 4 dietary treatments. For starter diets (day 1 to 18), treatments included a basal diet (1) without any riboflavin supplementation (negative control, N-C), (2) with conventional riboflavin supplementation (Cuxavit B2 80% riboflavin) at 9.6 mg/kg (positive control, P-C), (3) with riboflavin supplementation from the alternative source at 3.5 mg/kg (A-low) and (4) with riboflavin supplementation from the alternative source at 9.6 mg/kg (A-high). For the finisher diet (day 29 until slaughtering), P-C and A-high were supplemented with 8.0 mg/kg and A-low with 3.5 mg/kg. Diets were formulated according to organic regulations. Animals were kept in floor pens with 20 chickens per pen. Weekly, BW, feed and water consumption were recorded. Every second week, animal-based health and welfare indicators (feather score and footpad dermatitis) were scored. Slaughter traits were assessed for five males and females per pen at 62/63 days of age. Final body weight of A-high differed from N-C and A-low, but not from P-C. From week 2 until six years of age, A-high had a higher daily weight gain when compared to all other groups. With 74.4%, dressing percentage was higher in A-high compared with all other groups (73.3%). Breast percentage of A-low was lower than that of both control groups but did not differ from A-high. The highest frequency of liver scores indicating fatty liver syndrome was found in P-C, followed by N-C and A-low. Feather scores did not respond to treatment; the highest frequency of mild footpad dermatitis was observed in A-high, however at a low prevalence. In conclusion, the tested fermentation suspension with a high native content of riboflavin derived from fermentation of A. gossypii can be used at levels of commercial recommendations as alternative to riboflavin produced from GMO in broiler feeding. Further studies must verify whether riboflavin can be reduced without inducing riboflavin deficiency in slow-growing broilers.     

Bioproduction of riboflavin: a bright yellow history

Riboflavin (vitamin B₂) is an essential nutrient for humans and animals that must be obtained from the diet. To ensure an optimal supply, riboflavin is used on a large scale as additive in the food and feed industries. Here, we describe a historical overview of the industrial process of riboflavin production starting from its discovery and the need to produce the vitamin in bulk at prices that would allow for their use in human and animal nutrition. Riboflavin was produced industrially by chemical synthesis for many decades. At present, the development of economical and eco-efficient fermentation processes, which are mainly based on Bacillus subtilis and Ashbya gossypii strains, has replaced the synthetic process at industrial scale. A detailed account is given of the development of the riboflavin overproducer strains as well as future prospects for its improvement.     

Phosphoribosyl pyrophosphate synthetase activity affects growth and riboflavin production in <Emphasis Type="Italic">Ashbya gossypii</Emphasis>

Background  

Phosphoribosyl pyrophosphate (PRPP) is a central compound for cellular metabolism and may be considered as a link between carbon and nitrogen metabolism. PRPP is directly involved in the de novo and salvage biosynthesis of GTP, which is the immediate precursor of riboflavin. The industrial production of this vitamin using the fungus Ashbya gossypii is an important biotechnological process that is strongly influenced by substrate availability.     

The Small GTP-Binding Proteins AgRho2 and AgRho5 Regulate Tip-Branching,Maintenance of the Growth Axis and Actin-Ring-Integrity in the Filamentous Fungus Ashbya gossypii

GTPases of the Rho family are important molecular switches that regulate many basic cellular processes. The function of the Rho2 and Rho5 proteins from Saccharomyces cerevisiae and of their homologs in other species is poorly understood. Here, we report on the analysis of the AgRho2 and AgRho5 proteins of the filamentous fungus Ashbya gossypii. In contrast to S. cerevisiae mutants of both encoding genes displayed a strong morphological phenotype. The Agrho2 mutants showed defects in tip-branching, while Agrho5 mutants had a significantly decreased growth rate and failed to maintain their growth axis. In addition, the Agrho5 mutants had highly defective actin rings at septation sites. We also found that a deletion mutant of a putative GDP-GTP-exchange factor (GEF) that was homologous to a Rac-GEF from other species phenocopied the Agrho5 mutant, suggesting that both proteins act in the same pathway, but the AgRho5 protein has acquired functions that are fulfilled by Rac-proteins in other species.     

Strain Design of Ashbya gossypii for Single-Cell Oil Production

Single-cell oil (SCO) represents a sustainable alternative for the oil industry. Accordingly, the identification of microorganisms with either higher lipidogenic ability or novel capacities for the transformation of raw materials constitutes a major challenge for the field of oil biotechnology. With this in mind, here, we were prompted to address the lipidogenic profile of the filamentous hemiascomycete Ashbya gossypii, which is currently used for the microbial production of vitamins. We found that A. gossypii mostly accumulates unsaturated fatty acids (FAs), with more than 50% of the total FA content corresponding to oleic acid. In addition, we engineered A. gossypii strains both lacking the beta-oxidation pathway and also providing ATP-citrate lyase (ACL) activity to block the degradation of FA and to increase the cytosolic acetyl-coenzyme A (CoA) content, respectively. The lipidogenic profile of the newly developed strains demonstrates that the mere elimination of the beta-oxidation pathway in A. gossypii triggers a significant increase in lipid accumulation that can reach 70% of cell dry weight. The use of A. gossypii as a novel and robust tool for the production of added-value oils is further discussed.