Fermentation of n-Paraffins by Yeast

Nutritional requirement of Candida lipolytica AJ 5004 and its productivity of α-ketoglutarate were further studied.

It became clear that this yeast required only thiamine as grown factor, and even if the yeast was cultured in chemically defined medium containing adequate amount of thiamine, it was able to produce as high yield of α-ketoglutarate as in the medium containing 0.02% of corn steep liquor.

It was also shown that the rate of convertion of n-paraffin to α-ketoglutarate gradually increased as the concentration of n-paraffins was decreased or as the incubation time was prolonged. A very high rate of conversion, 71%, was obtained after prolonged culture, for 5 days, with a culture medium containing 8% of n-paraffins.

The productivity of α-ketoglutarate from C₉- to C₂₀-alkanes by the yeast was maximum in the range from C₁₅ to C₁₉, especially from C₁₇ to C₁₉.     

Fermentation of n-Paraffins by Yeast

Screening test for obtaining microorganisms which produce l-amino acids or organic acids from n-paraffins were carried out. Fourteen strains of microorganisms which seemed to belong to the yeast showed strong ability to produce α-ketoglutaric acid. A representative strain of these microorganisms was identified as Candida lipolytica AJ 5004.

Optimal conditions for production of α-ketoglutarate using Candida lipolytica AJ 5004 were also studied. Under the condition thus obtained using a culture medium of 8 weight % of n-paraffins, the yeast accumulated 59% of α-ketoglutarate to the substrate added after three days culture.     

Mode of Metabolism of 1-Tetradecene by Candida Yeasts and Citrates Production

Metabolic pathways for the oxidation of 1-tetradecene by Candida lipolytica were investigated and compared with those for Candida tropicalis in order to elucidate the difference in the productivity of citrates reported in the previous paper. No difference was found in the pathways for the initial stage of oxidation of 1-tetradecene between the two strains, indicating that the difference in the productivity of citrates is not attributable to the metabolic pathways. The metabolic rate of 1-tetradecene with C. lipolytica was found to be much lower than that with C. tropicalis. The production of citrates was much enhanced in the presence of surfactants which were known to be stimulative for microbial metabolism of hydrocarbons and 11 ~ 15 g/liters was attained on the 6th day of cultivation.     

Microbiological Studies of Coli-aerogenes Bacteria

α-Ketoglutarate was obtained in a very small amount by the oxidative fermentation of acetate with either a growing culture or the washed cells of Escherichia coli. This microorganism was also observed to accumulate a considerable amount of α-ketoglutarate as the oxidation-product of C₄-dicarboxylic acids such as succinate, fumarate, malate and oxalacetate. The addition of acetate to the reaction mixtures containing either C₃- or C₄-acids brought about an increase in the yield of α-ketoglutarate. The bacteria of coli-aerogenes revealed an ability of oxidizing tricarboxylic acids under suitable conditions, but there was no noticeable production of α-ketoglutarate. The formation of glyoxylate was observed to occur during the degradation of citrate by the bacteria of coli-aerogenes. Finally, a cyclic mechanism of aerobic carbon-metabolism in the bacteria was propounded and discussed.     

Effects of pyruvate dehydrogenase subunits overexpression on the α-ketoglutarate production in Yarrowia lipolytica WSH-Z06

Yarrowia lipolytica WSH-Z06 harbours a promising capability to oversynthesize α-ketoglutarate (α-KG). Its wide utilization is hampered by the formation of high concentrations of pyruvate. In this study, a metabolic strategy for the overexpression of the α and β subunits of pyruvate dehydrogenase E1, E2 and E3 components was designed to reduce the accumulation of pyruvate. Elevated expression level of α subunit of E1 component improved the α-KG production and reduced the pyruvate accumulation. Due to a reduction in the acetyl-CoA supply, neither the growth of cells nor the synthesis of α-KG was restrained by the overexpression of β subunit of E1, E2 and E3 components. Furthermore, via the overexpression of these thiamine pyrophosphate (TPP)-binding subunits, the dependency of pyruvate dehydrogenase on thiamine was diminished in strains T1 and T2, in which α and β subunits of E1 component were separately overexpressed. In these two recombinant strains, the accumulation of pyruvate was insensitive to variations in exogenous thiamine. The results suggest that α-KG production can be enhanced by altering the dependence on TPP of pyruvate dehydrogenase and that the competition for the cofactor can be switched to ketoglutarate dehydrogenase via separate overexpression of the TPP-binding subunits of pyruvate dehydrogenase. The results presented here provided new clue to improve α-KG production.     

Propionate Enhances Synthesis and Secretion of Bile Acids in Primary Cultured Rat Hepatocytes via Succinyl CoA

To discover the role of propionate produced by colonic bacteria, this study examined the secretion of bile acids and cholesterol 7α-hydroxylase activity in the primary cultured hepatocytes. Addition of propionate (2 mM) to the medium for 48 h caused an increase in the bile acid secretion and enzyme activity, while acetate and butyrate had no significant influence. Bile acid secretion was increased by the addition of succinyl CoA and its precursor substances (α -ketoglutarate, valine, isoleucine, and methionine), but not malate and oxaloacetate, which are the metabolites of succinyl CoA. α -Ketoglutarate and valine also increased the activity of cholesterol 7α -hydroxylase. Since cholesterol 7α -hydroxylase is a microsomal cytochrome P-450 enzyme and the formation of δ-aminolevulinate from succinyl CoA in the mitochondria is the rate-controlling step for the subsequent synthesis of heme proteins, propionate may affect bile acid synthesis via elevation of mitochondrial succinyl CoA.     

Induction and Citric Acid Productivity of Fluoroacetate-sensitive Mutant Strains of Candida lipolytica

To establish a novel process for the economical production of citric acid from n-paraffins by yeast, attempts were made to obtain some mutant strains capable of producing citric acid in higher yield without (+)-isocitric acid.

From among the mutant strains derived from Candida lipolytica ATCC 20114, which produced citric acid and (+)-isocitric acid in the ratio of about 60:40 from n-paraffins, a citrate non-utilizing mutant strain, K-20, and a fluoroacetate-sensitive mutant strain , S-22, were selected on the basis of high citric acid and low (+)-isocitric acid productivity.

The mutant strain S-22 showed extremely poor growth in a medium containing sodium citrate as the sole carbon source and extremely high sensitivity to fluoroacetate. The production ratio of citric acid and (+)-isocitric acid by the mutant strain was changed to 97:3, and the yield of the citric acid from n-paraffins, charged to the fermentation medium, reached 145%(w/w).     

α-KETOGLUTARATE UPTAKE IN HUMAN FIBROBLASTS

Glutamine requirements are increased during injury, in particular to sustain the needs of rapidly growing cells. This includes fibroblasts involved in wound healing. α-Ketoglutarate (α-KG) has been proved to be a potent precursor of glutamine. However, little is known about the process of its cell uptake. Since this first step could be crucial in α-KG metabolism, we have characterized α-ketoglutarate uptake in fibroblasts. Total uptake of α-ketoglutarate was linear up to 1mmol and temperature independent. Rate of uptake was independent of the presence of Na⁺in the medium. Competition studies with another ketoacid demonstrated the non-specificity of α-ketoglutarate uptake. In addition, 4-hydroxy-α-cyanocinnamate, a known inhibitor of anion transport, was ineffective on α-ketoglutarate uptake. Taken as a whole, these data provide evidence that α-ketoglutarate uptake in fibroblast occurs by an unmediated diffusion process. This suggests that α-ketoglutarate uptake is not the controlling step in fibroblasts, i.e. only the availability of extracellular α-ketoglutarate. This could be an advantage since during injury, cell membrane depolarization and dissipation of Na⁺gradient may limit cellular glutamine uptake.     

Relationship between Aconitate Hydratase Activity and Citric Acid Productivity in Fluoroacetate-sensitive Mutant Strain of Candida lipolytica

Fluoroacetate-sensitive mutant strains, K–20 and S–22, of Candida lipolytica could not grow or could only slightly grow on agar media containing di- or tricarboxylic acid involved in the TCA-cycle as the sole source of carbon. Relative activities of aconitate hydratase in the cells of the mutant strains, K-20 and S-22, were approximately 1/10 and 1/100, against that of the parent strain, respectively. This facts support the statement that the mutant strains were extremely sensitive to monofiuoroacetate.

The aconitate hydratase activities of these mutant strains and the parent strain corresponded well to the citric to (+)-isocitric acid ratio in the final fermented broths.     

The substrate specificity of two yeast strains utilizing hydrocarbons

StrainsCandida lipolytica 4-1 andCandida lipolytica K were compared in their growth and dawaxing capacities during batch growth on model gas oil. The model gas oil was composed of a mixture of even-numbered puren-alkanes (n-decane ton-dotriacontane) dissolved in dewaxed gas oil. The results show that both strains differ in their substrate specificity and in the sequence of utilization of individualn-alkanes. Strain K, previously used for dewaxing of mineral oil, has its substrate specificity shifted toward the highern-alkanes.     

Isolation and Determination of Yeasts Utilizing Kerosene as a Sole Source of Carbon

In order to produce microbial cell substances from petroleum, 83 strains of kerosene-utilizing yeasts, as a sole source of carbon, were isolated from 37 materials in contact with petroleum in the petroleum refinery. They could be distributed in either of 15 cultural groups with their colony appearances. Fifteen representative strains in 15 cultural groups were served for determination and identified with the following species: Candida tropicalis, 9 strains; C. guilliermondii, 2 strains; C. intermedia, 2 strains; C. pulcherrima, 1 strains; Torulopsis pinus, 1 strain.

In order to clarify what the ability of hydrocarbon utilization means biologically, 46 standard strains were served for test, of which the following 5 strains could utilize kerosene as a sole source of carbon: Candida albicans IAM 4888; C. arborea IAM 4147; C. lipolytica IAM 4947; C. tropicalis IAM 4862 and IAM 4924. Considering the result, the ability of utilizing kerosene would seem to characterize the genus, but it was not evident that it would characterize the species.

C. tropicalis Pk-233 gave the best cell yield among the above strains when kerosene was employed as a sole source of carbon and moreover, in the production of the cells of Pk-233, employing kerosene as a carbon material was compared with employing glucose.     

Microbiological Studies of Coli-aerogenes Bacteria

α-Ketoglutarate was formed from the various carbohydrates including lactose, maltose, sucrose, d-glucose, d-fructose, d-galactose, d-mannose, d-mannitol, l-rhamnose, d-xylose, l-arabinose and glycerin. The influence of pH of the reaction mixture were tested, and inorganic phosphate was observed to be indispensable for α-ketoglutarate-fermentation. A cell of E. coli grown statically on glucose was found to reveal an ability of producing α-ketoglutarate under aerobic conditions. Optically dextro lactic acid was potent in the formation of a-ketoglutaric acid. The following reagents revealed the inhibiting effect on α-ketoglutarate-fermentation; CuSO₄, AgNO₃, iodoacetate, 2, 4-dinitrophenol, NaN₃, 3-sulfanilamido-6-methoxypyridazine and arsenite, while, kanamycin and 8-azaguanine has no inhibiting effect. When E. coli was grown in a glucose-medium, a small supply of air increased the yield of acetate against decreasing α-ketoglutarate.     

Competition between n-alkane-assimilating yeasts and bacteria during colonization of sandy soil microcosms

An n-alkane-assimilating strain of Candida tropicalis was selected in sandy soil inoculated with microorganisms from contaminated sites. Competition experiments with n-alkane utilizers from different strain collections confirmed that yeasts overgrow bacteria in sandy soil. Acidification of the soil is one of the colonization factors useful for the yeasts. It can be counteracted by addition of bentonite, a clay mineral with high ion exchange capacity, but not, however, by kaolin. Strains of different yeast species showed different levels of competitiveness. Strains of Arxula adeninivorans, Candida maltosa, and Yarrowia lipolytica overgrew strains of C. tropicalis, C. shehatae or Pichia stipitis. Two strains of C. maltosa and Y. lipolytica coexisted during several serial transfers under microcosm conditions. Received: 20 October 1999 / Received revision: 26 January 2000 / Accepted: 27 January 2000     

α-Ketoglutaric acid production from rapeseed oil by Yarrowia lipolytica yeast

The possibility of using rapeseed oil as a carbon source for microbiological production of α-ketoglutaric acid (KGA) has been studied. Acid formation on the selective media has been tested in 26 strains of Yarrowia lipolytica yeast, and the strain Y. lipolytica VKM Y-2412 was selected as a prospective producer of KGA from rapeseed oil. KGA production by the selected strain was studied in dependence on thiamine concentration, medium pH, temperature, aeration, and concentration of oil. Under optimal conditions (thiamine concentration of 0.063 μg?g cells^?1, pH?3.5, 30 °C, high dissolved oxygen concentration (pO₂) of 50 % (of air saturation), and oil concentration in a range from 20 to 60 g?l^?1), Y. lipolytica VKM Y-2412 produced up to 102.5 g?l^?1 of KGA with the mass yield coefficient of 0.95 g?g^?1 and the volumetric KGA productivity (Q _KGA) of 0.8 g?l^?1?h^?1.     

The teleomorph state of Candida deformans Langeron & Guerra and description of Yarrowia yakushimensis comb. nov.

Yarrowia lipolytica is the only known species in the teleomorph (i.e. sexual) genus Yarrowia and has its anamorph (i.e. asexual state) classified in the genus Candida Berkhout as Candida lipolytica. This species can be found readily in nature, has significant industrial value and is important to the food and medical fields. Candida deformans was first considered a variety of C. lipolytica and was later listed as a synonym of this species. More recent studies based on sequence variation in the nuclear rRNA gene sequences suggested C. deformans to be a separate species with no known teleomorph linked to it. In this study we show that C. deformans strains, obtained from South Africa, can mate with strains present in the CBS yeast collection and produce a Yarrowia teleomorph, described here as Yarrowia deformans. Strains of Candida yakushimensis nom. inval., a species also belonging to the Yarrowia phylogenetic clade, were also studied and described as a novel species of Yarrowia.     

Establishment of genomic library technology mediated by non-homologous end joining mechanism in Yarrowia lipolytica

Genomic variants libraries are conducive to obtain dominant strains with desirable phenotypic traits. The non-homologous end joining (NHEJ), which enables foreign DNA fragments to be randomly integrated into different chromosomal sites, shows prominent capability in genomic libraries construction. In this study, we established an efficient NHEJ-mediated genomic library technology in Yarrowia lipolytica through regulation of NHEJ repair process, employment of defective Ura marker and optimization of iterative transformations, which enhanced genes integration efficiency by 4.67, 22.74 and 1.87 times, respectively. We further applied this technology to create high lycopene producing strains by multi-integration of heterologous genes of CrtE, CrtB and CrtI, with 23.8 times higher production than rDNA integration through homologous recombination (HR). The NHEJ-mediated genomic library technology also achieved random and scattered integration of loxP and vox sites, with the copy number up to 65 and 53, respectively, creating potential for further application of recombinase mediated genome rearrangement in Y. lipolytica. This work provides a high-efficient NHEJ-mediated genomic library technology, which enables random and scattered genomic integration of multiple heterologous fragments and rapid generation of diverse strains with superior phenotypes within 96 h. This novel technology also lays an excellent foundation for the development of other genetic technologies in Y. lipolytica.

     

Heavy metal tolerance in marine strains of Yarrowia lipolytica

Heavy metal tolerance of two marine strains of Yarrowia lipolytica was tested on solid yeast extract peptone dextrose agar plates. Based on minimum inhibitory concentration esteems, it is inferred that the two strains of Y. lipolytica were tolerant to heavy metals such as Pb(II), Cr(III), Zn(II), Cu(II), As(V), and Ni(II) ions. The impact of various heavy metal concentrations on the growth kinetics of Y. lipolytica was likewise assessed. With increased heavy metal concentration, the specific growth rate was reduced with delayed doubling time. Furthermore, biofilm development of both yeasts on the glass surfaces and in microtitre plates was assessed in presence of different heavy metals. In microtitre plates, a short lag phase of biofilm formation was noticed without the addition of heavy metals in yeast nitrogen base liquid media. A lag phase was extended over increasing metal concentrations of media. Heavy metals like Cr(VI), Cd(II), and As(V) are contrastingly influenced on biofilms’ formation of microtitre plates. Other heavy metals did not much influence on biofilms development. Thus, biofilm formation is a strategy of Y. lipolytica under stress of heavy metals has significance in bioremediation process for recovery of heavy metals from contaminated environment.

     

Multiple gene integration to promote erythritol production on glycerol in Yarrowia lipolytica

Objective

Erythritol (1,2,3,4-butanetetrol) is a 4-carbon sugar alcohol that occurs in nature as a metabolite or storage compound. In this study, a multiple gene integration strategy was employed to enhance erythritol production in Y. lipolytica.

Results

The effects on the production of erythritol in Y. lipolytica of seven key genes involved in the erythritol synthesis pathway were evaluated individually, among which transketolase (TKL1) and transaldolase (TAL1) showed important roles in enhancing erythritol production. The combined overexpression of four genes (GUT1, TPI1, TKL1, TAL1) and disruption of the EYD1 gene (encoding erythritol dehydrogenase), resulted in produce approximately 40 g/L erythritol production from glycerol. Further enhanced erythritol synthesis was obtained by overexpressing the RKI1 gene (encoding ribose 5-phosphate isomerase) and the AMPD gene (encoding AMP deaminase), indicating for the first time that these two genes are also related to the enhancement of erythritol production in Y. lipolytica.

Conclusions

A combined gene overexpression strategy was developed to efficiently improve the production of erythritol in Y. lipolytica, suggesting a great capacity and promising potential of this non-conventional yeast in converting glycerol into erythritol.

     

Applying pathway engineering to enhance production of alpha-ketoglutarate in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

α-Ketoglutarate (α-KG), one of short-chain carboxylates of high commercial relevance, has been widely used in food, medicine, chemical, and cosmetic fields. Compared to other carboxylates, α-KG occupies key positions in the tricarboxylate cycle (TCA cycle) and amino acid metabolic pathway, the over-accumulation of α-KG is restricted both by tighter carbon and nitrogen regulation process. Biotechnology production of α-KG on large industrial level has been impeded by many obstacles. This review aims at highlighting and stating recent efforts toward improving the yield and titer of α-KG in the strains of Yarrowia lipolytica to reach industrial relevance. Fermentation process optimization concerning feedstock utilization, dissolved oxygen controlling, pH manipulation and establishment of fed-batch process, have been assessed and evaluated. Moreover, pathway engineering routes have been applied for enhancing carbon commitment to α-KG, blocking competing pathways, regenerating of co-factors and regulating of carboxylate transporters to facilitate production and accumulation of α-KG. Although no engineered strain can satisfy the requirements of industrial production relevance to date, these strategies provide many clues for accelerating strain development for α-KG production.     

The yeast genus Yarrowia gen. nov.

The ascigerous teleomorph of Candida lipolytica (Harrison) Diddens et Lodder, previously classified as Endomycopsis lipolytica Wickerham et al. and as Saccharomycopsis lipolytica (Wickerham et al.) Yarrow, has been assigned to the new genus Yarrowia. Yarrowia lipolytica (Wickerham et al.) comb. nov. is the type species for the genus.The remaining species of Saccharomycopsis are revised.