The peculiarities of succinic acid production from rapeseed oil by Yarrowia lipolytica yeast

The process of succinic acid (SA) production represents the combination of microbial synthesis of α-ketoglutaric acid from rapeseed oil by yeast Yarrowia lipolytica VKM Y-2412 and subsequent decarboxylation of α-ketoglutaric acid by hydrogen peroxide to SA that leads to the production of 69.0 g l^?1 of SA and 1.36 g l^?1 of acetic acid. SA was isolated from the culture broth filtrate in a crystalline form. The SA recovery from the culture filtrate has certain difficulties due to the presence of residual triglycerides of rapeseed oil. The effect of different methods of the culture filtrate treatment and various sorption materials on the coagulation of triglycerides was studied, and as a result, the precipitation of residual triglycerides by acetone was chosen. The subsequent isolation procedures involved the decomposition of H₂O₂ in the filtrate followed by filtrate bleaching and acidification with a mineral acid, evaporation of filtrate, and SA extraction with ethanol from the residue. The purity of crystalline SA isolated from the culture broth filtrate achieved 97.6–100 %. The product yield varied from 62.6 to 71.6 % depending on the acidity of the supernatant.     

Isocitric acid production from rapeseed oil by Yarrowia lipolytica yeast

Production of d _S-threo-isocitric acid (ICA) by yeast meets serious difficulties since it is accompanied by a simultaneous production of citric acid (CA) in significant amounts that reduces the yield of desired product. In order to develop an effective process of ICA production, 60 yeast strains of different genera (Candida, Pichia, Saccharomyces, Torulopsis, and Yarrowia) were tested for their ability to produce ICA from rapeseed oil; as a result, wild-type strain Yarrowia lipolytica VKM Y-2373 and its mutant Y. lipolytica 704-UV4-A/NG50 were selected as promising ICA producers. The effects of temperature, pH, aeration, and concentrations of rapeseed oil, iron, and itaconic acid on ICA production by selected strains were studied. Under optimal conditions (pH 6.0; aeration 50–55 %; rapeseed oil concentration of 20–60 gl^?1, iron ion concentration of 1.2 mg l^?1, and itaconic acid amount of 30 mM), selected strains of Y. lipolytica produced predominantly ICA with a low amount of a by-product, CA.     

Organic acid production by the yeast Yarrowia lipolytica (a review)

The review sums up the results of studies of (1) physiological growth characteristics of the yeast Yarrowia lipolytica, cultured in the presence of diverse carbon sources (n-alkanes, glucose, and glycerol), and (2) superhigh synthesis of organic acids, which were performed at the Skryabin Institute of Biochemistry and Physiology of Microorganisms of the Russian Academy of Sciences. Microbiological processes of obtaining alpha-ketoglutaric, pyruvic, isocitric, and citric acids are discussed.     

Engineering of unconventional yeast Yarrowia lipolytica for efficient succinic acid production from glycerol at low pH

Yarrowia lipolytica is considered as a potential candidate for succinic acid production because of its innate ability to accumulate citric acid cycle intermediates and its tolerance to acidic pH. Previously, a succinate-production strain was obtained through the deletion of succinate dehydrogenase subunit encoding gene Ylsdh5. However, the accumulation of by-product acetate limited further improvement of succinate production. Meanwhile, additional pH adjustment procedure increased the downstream cost in industrial application. In this study, we identified for the first time that acetic acid overflow is caused by CoA-transfer reaction from acetyl-CoA to succinate in mitochondria rather than pyruvate decarboxylation reaction in SDH negative Y. lipolytica. The deletion of CoA-transferase gene Ylach eliminated acetic acid formation and improved succinic acid production and the cell growth. We then analyzed the effect of overexpressing the key enzymes of oxidative TCA, reductive carboxylation and glyoxylate bypass on succinic acid yield and by-products formation. The best strain with phosphoenolpyruvate carboxykinase (ScPCK) from Saccharomyces cerevisiae and endogenous succinyl-CoA synthase beta subunit (YlSCS2) overexpression improved succinic acid titer by 4.3-fold. In fed-batch fermentation, this strain produced 110.7 g/L succinic acid with a yield of 0.53 g/g glycerol without pH control. This is the highest succinic acid titer achieved at low pH by yeast reported worldwide, to date, using defined media. This study not only revealed the mechanism of acetic acid overflow in SDH negative Y. lipolytica, but it also reported the development of an efficient succinic acid production strain with great industrial prospects.     

Production of succinic acid at low pH by a recombinant strain of the aerobic yeast Yarrowia lipolytica

Biotechnological production of weak organic acids such as succinic acid is most economically advantageous when carried out at low pH. Among naturally occurring microorganisms, several bacterial strains are known to produce considerable amounts of succinic acid under anaerobic conditions but they are inefficient in performing the low‐pH fermentation due to their physiological properties. We have proposed therefore a new strategy for construction of an aerobic eukaryotic producer on the basis of the yeast Yarrowia lipolytica with a deletion in the gene coding one of succinate dehydrogenase subunits. Firstly, an original in vitro mutagenesis‐based approach was proposed to construct strains with Ts mutations in the Y. lipolytica SDH1 gene. These mutants were used to optimize the composition of the media for selection of transformants with the deletion in the Y. lipolytica SDH2 gene. Surprisingly, the defects of each succinate dehydrogenase subunit prevented the growth on glucose but the mutant strains grew on glycerol and produced succinate in the presence of the buffering agent CaCO₃. Subsequent selection of the strain with deleted SDH2 gene for increased viability allowed us to obtain a strain capable of accumulating succinate at the level of more than 45 g L^?1 in shaking flasks with buffering and more than 17 g L^?1 without buffering. The possible effect of the mutations on the utilization of different substrates and perspectives of constructing an industrial producer is discussed. Biotechnol. Bioeng. 2010;107:673–682. © 2010 Wiley Periodicals, Inc.     

产琥珀酸重组解脂酵母发酵副产物乙酸的代谢控制

琥珀酸是一种高附加值的有机酸,广泛用于食品、化工和农药领域。解脂酵母Yarrowia lipolytica作为新型强健的非传统酵母,近年来逐渐吸引了研究者的注意。前期通过基因敲除琥珀酸脱氢酶基因构建了一株产琥珀酸的重组解脂酵母PGC01003。由于糖酵解和TCA循环流量不协调,PGC01003分泌大量副产物乙酸,限制了琥珀酸产量的进一步提高。为降低乙酸的溢出,实现自然低pH值发酵生产琥珀酸,首先干扰旁路代谢,异源表达来自鼠沙门氏菌的乙酰辅酶A合酶,乙酸的产量下降至4.6 g/L,比对照降低了24.6%。而基因敲除乙酰辅酶A水解酶基因得到的重组菌PGC11505,发酵96 h乙酸分泌量只有0.4 g/L,琥珀酸产量提高到7.0 g/L,琥珀酸的转化率为0.30 g/g,为进一步构建高产琥珀酸的细胞工厂奠定基础。     

The citric acid production from raw glycerol by Yarrowia lipolytica yeast and its regulation

The optimal cultivation conditions ensuring the maximal rate of citric acid (CA) biosynthesis by glycerol-grown mutant Yarrowia lipolytica NG40/UV7 were found to be as follows: growth limitation by inorganic nutrients (nitrogen, phosphorus, or sulfur), 28 °C, pH 5.0, dissolved oxygen concentration (pO₂) of 50 % (of air saturation), and pulsed addition of glycerol from 20 to 80 g L^?1 depending on the rate of medium titration. Under optimal conditions of fed-batch cultivation, in the medium with pure glycerol, strain Y. lipolytica NG40/UV7 produced 115 g L^?1 of CA with the mass yield coefficient of 0.64 g g^?1 and isocitric acid (ICA) amounted to 4.6 g L^?1; in the medium with raw glycerol, CA production was 112 g L^?1 with the mass yield coefficient of 0.90 g g^?1 and ICA amounted to 5.3 g L^?1. Based on the activities of enzymes involved in the initial stages of raw glycerol assimilation, the tricarboxylic acid cycle and the glyoxylate cycle, the mechanism of increased CA yield from glycerol-containing substrates in Y. lipolytica yeast was explained.     

Perspectives of nervonic acid production by Yarrowia lipolytica

Biotechnology Letters - Nervonic acid (cis-15-tetracosenoic acid, 24:1Δ15) is a long chain monounsaturated fatty acid, mainly exists in white matt er of the human brains. It plays an important...     

Selectivity in citric acid production by Yarrowia lipolytica

This experimental study reports about production selectivity in the fermentation of glucose to citric acid by Yarrowia lipolytica as a function of substrate concentration. Batch runs featuring biomass growth and one or two citric acid production phases were carried out in a 15-l stirred tank fermentor. The presented results demonstrate that working at high initial substrate concentration in the production phase is beneficial both in terms of a higher production rate of citric acid, the desired metabolite (reaching 0.077 h(-1)) and of a higher utilization degree of the employed carbon source (yield up to 0.384 g(c.a.)/g(glucose)). The production rate of isocitric acid, the major undesired metabolite, was found to be practically constant over the tested initial substrate concentration range.     

Citric acid production from sucrose using a recombinant strain of the yeast Yarrowia lipolytica

The yeast Yarrowia lipolytica is able to secrete high amounts of several organic acids under conditions of growth limitation and carbon source excess. Here we report the production of citric acid (CA) in a fed-batch cultivation process on sucrose using the recombinant Y. lipolytica strain H222-S4(p67ICL1) T5, harbouring the invertase encoding ScSUC2 gene of Saccharomyces cerevisiae under the inducible XPR2 promoter control and multiple ICL1 copies (10–15). The pH-dependent expression of invertase was low at pH 5.0 and was identified as limiting factor of the CA-production bioprocess. The invertase expression was sufficiently enhanced at pH 6.0–6.8 and resulted in production of 127–140 g l⁻¹ CA with a yield Y _CA of 0.75–0.82 g g⁻¹, whereas at pH 5.0, 87 g l ⁻¹ with a yield Y _CA of 0.51 gg⁻¹ were produced. The CA-productivity Q _CA increased from 0.40 g l ⁻¹ h⁻¹ at pH 5.0 up to 0.73 g l ⁻¹ h⁻¹ at pH 6.8. Accumulation of glucose and fructose at high invertase expression level at pH 6.8 indicated a limitation of CA production by sugar uptake. The strain H222-S4(p67ICL1) T5 also exhibited a gene–dose-dependent high isocitrate lyase expression resulting in strong reduction (<5%) of isocitric acid, a by-product during CA production.     

Metabolic engineering for ricinoleic acid production in the oleaginous yeast Yarrowia lipolytica

Although there are numerous oleochemical applications for ricinoleic acid (RA) and its derivatives, their production is limited and subject to various safety legislations. In an effort to produce RA from alternative sources, we constructed a genetically modified strain of the oleaginous yeast Yarrowia lipolytica. This strain is unable to perform β-oxidation and is invalidated for the native triacylglycerol (TAG) acyltransferases (Dga1p, Dga2p, and Lro1p) and the ?12 desaturase (Fad2p). We also expressed the Ricinus communis ?12 hydroxylase (RcFAH12) under the control of the TEF constitutive promoter in this strain. However, RA constituted only 7 % of the total lipids produced by this modified strain. By contrast, expression of the Claviceps purpurea hydroxylase CpFAH12 in this background resulted in a strain able to accumulate RA to 29 % of total lipids, and expression of an additional copy of CpFAH12 drove RA accumulation up to 35 % of total lipids. The co-expression of the C. purpurea or R. communis type II diacylglycerol acyltransferase (RcDGAT2 or CpDGAT2) had negative effects on RA accumulation in this yeast, with RA levels dropping to below 14 % of total lipids. Overexpression of the native Y. lipolytica PDAT acyltransferase (Lro1p) restored both TAG accumulation and RA levels. Thus, we describe the consequences of rerouting lipid metabolism in this yeast so as to develop a cell factory for RA production. The engineered strain is capable of accumulating RA to 43 % of its total lipids and over 60 mg/g of cell dry weight; this is the most efficient production of RA described to date.     

Engineering the oleaginous yeast Yarrowia lipolytica for high-level resveratrol production

Resveratrol is a plant secondary metabolite with multiple health-beneficial properties. Microbial production of resveratrol in model microorganisms requires extensive engineering to reach commercially viable levels. Here, we explored the potential of the non-conventional yeast Yarrowia lipolytica to produce resveratrol and several other shikimate pathway-derived metabolites (p-coumaric acid, cis,cis-muconic acid, and salicylic acid). The Y. lipolytica strain expressing a heterologous pathway produced 52.1 ± 1.2 mg/L resveratrol in a small-scale cultivation. The titer increased to 409.0 ± 1.2 mg/L when the strain was further engineered with feedback-insensitive alleles of the key genes in the shikimate pathway and with five additional copies of the heterologous biosynthetic genes. In controlled fed-batch bioreactor, the strain produced 12.4 ± 0.3 g/L resveratrol, the highest reported titer to date for de novo resveratrol production, with a yield on glucose of 54.4 ± 1.6 mg/g and a productivity of 0.14 ± 0.01 g/L/h. The study showed that Y. lipolytica is an attractive host organism for the production of resveratrol and possibly other shikimate-pathway derived metabolites.     

Chemostat study of citric acid production from glycerol by Yarrowia lipolytica

The aim of the study was to examine how the dilution rate and the chemical composition of the production medium impacts on the synthesis of citric acid by the Yarrowia lipolytica strain Wratislavia AWG7 from glycerol in a chemostat culture. The yeast Y. lipolytica Wratislavia AWG7, an acetate (acet(-)) and morphological (fil(-)) mutant, was cultured in a nitrogen- and phosphorus-limited medium at the dilution rate of 0.009-0.031h(-1) in the chemostat. Under steady-state conditions, the increase in the dilution rate was paralleled by the decrease in citric acid concentration (from 86.5 to 51.2gL(-1)), as well as by the increase in the volumetric rate (from 0.78 to 1.59gL(-1)h(-1)) and specific rate (from 0.05 to 0.18gg(-1)h(-1)) of citric acid production. The yield of the production process varied from 0.59 to 0.67gg(-1). In a 550-h continuous culture of the yeast test, at a dilution rate of 0.01h(-1), in a medium with enhanced concentrations of carbon, nitrogen and phosphorus sources, the concentration of citric acid, the concentration of biomass and the volumetric rate of citric acid production were 97.8gL(-1), 22.2gL(-1) and 0.98gL(-1)h(-1), respectively. The yield of the process decreased to 0.49gg(-1). The number of dead cells did not exceed 1% while that of the budding cells accounted for about 20%. Owing to the low content of isocitric acid and polyols, the fermentation process was characterized by a high purity. This study has produced the following finding: the double mutant Y. lipolytica AWG7 is an effective citric acid producer, with the ability to preserve its properties unchanged during the long run of the continuous chemostat process. This is a valued technological feature of such mutants.     

Correction to: Perspectives of nervonic acid production by Yarrowia lipolytica

Biotechnology Letters -     

Role of beta-oxidation enzymes in gamma-decalactone production by the yeast Yarrowia lipolytica.

Some microorganisms can transform methyl ricinoleate into gamma-decalactone, a valuable aroma compound, but yields of the bioconversion are low due to (i) incomplete conversion of ricinoleate (C(18)) to the C(10) precursor of gamma-decalactone, (ii) accumulation of other lactones (3-hydroxy-gamma-decalactone and 2- and 3-decen-4-olide), and (iii) gamma-decalactone reconsumption. We evaluated acyl coenzyme A (acyl-CoA) oxidase activity (encoded by the POX1 through POX5 genes) in Yarrowia lipolytica in lactone accumulation and gamma-decalactone reconsumption in POX mutants. Mutants with no acyl-CoA oxidase activity could not reconsume gamma-decalactone, and mutants with a disruption of pox3, which encodes the short-chain acyl-CoA oxidase, reconsumed it more slowly. 3-Hydroxy-gamma-decalactone accumulation during transformation of methyl ricinoleate suggests that, in wild-type strains, beta-oxidation is controlled by 3-hydroxyacyl-CoA dehydrogenase. In mutants with low acyl-CoA oxidase activity, however, the acyl-CoA oxidase controls the beta-oxidation flux. We also identified mutant strains that produced 26 times more gamma-decalactone than the wild-type parents.     

Citric acid production by Yarrowia lipolytica cultivated on olive-mill wastewater-based media

Yarrowia lipolytica ACA-DC 50109 cultivated on olive-mill wastewater (O.M.W.)-based media, enriched with commercial-industrial glucose, presented an efficient cell growth. Parameters of growth were unaffected by the presence of O.M.Ws in the growth medium. In diluted O.M.Ws enriched with high glucose amounts (initial sugar concentration, 65 g l(-1)), a notable quantity of total citric acid was produced (28.9 g l(-1)). O.M.W.-based media had a noteworthy stimulating effect on the production of citric acid, since both final citric acid concentration and conversion yield of citric acid produced per unit of sugar consumed were higher when compared with the respective parameters obtained from trials without added O.M.W. Adaptation of the strain in O.M.W.-based media favoured the biosynthesis of cellular unsaturated fatty acids (principally of oleic and palmitoleic acids). Additionally, a non-negligible decrease of the phenolic compounds in the growth medium [up to 15% (wt/wt)], a slight decrease of the phyto-toxicity, and a remarkable decolourisation of the O.M.W. were observed. All these results suggest the potentiality of O.M.Ws utilisation in the fermentation process of citric acid production.     

190 The peculiarities of regulation of Yarrowia lipolytica yeast and citric acid overproduction

The growth of Yarrowia lipolytica yeast as well the biosynthesis of citric acid on rapeseed oil were studied. It was indicated that the initial step of assimilation of rapeseed oil in the yeast Y. lipolytica is their hydrolysis by extracellular lipases with the formation of glycerol and fatty acids, which appear in the medium in the phase of active growth. The concentrations of these metabolites change insignificantly upon further cultivation. Lipase and the key enzymes of glycerol metabolism (glycerol kinase) and the glyoxylate cycle responsible for the metabolism of fatty acids (isocitrate lyase and malate synthase) are induced just at the beginning of the growth phase and remain active in the course of further cultivation. These results, taken together, suggest that glycerol and fatty acids according in the medium do not suppress the metabolism of each other. The fact that glycerol and fatty acids can be consumed simultaneously is of special importance for the development of the efficient regime of oil feeding, Y. lipolytica produced citric acid (175?g/L) with a yield of 150%. It should be noted that the simultaneous utilization of two different substrates is not typical of micro-organisms, which first assimilate one of the two available substrates (commonly, a carbohydrate), whereas the assimilation of the other substrate starts only after the first substrate is fully consumed from the medium. Indeed, upon the cultivation of Y. lipolytica on the mixture of glucose and oleic acid, the latter substrate began to be utilized only when the concentration of glucose decreased. The glycolytic enzyme pyruvate dehydrogenase was induced from the first hours of cultivation and remained at high levels until the exhaustion of glucose in the medium. At the same time, the activities of isocitrate lyase and malate synthase were very low during the metabolism of glucose, but were rapidly induced (approximately in 10 times) after the exhaustion of glucose in the medium. When Y. lipolytica was grown on the mixture of glucose and hexadecane, the dynamics of growth and substrate consumption was typical of the diauxie phenomenon: the utilization of hexadecane began only in several hours after the time when glucose was completely exhausted in the cultivation medium. In this case, the exhaustion of glucose arrested growth and the culture resumed growth only after a lag period. The assay of enzymes showed that the glycolytic enzyme pyruvate dehydrogenase was active during the phase of growth on glucose, whereas the enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase were active during the phase of growth on hexadecane. In recent years in the literature, there are data that the different sugars produce signals which modify the conformation of certain proteins that, in turn, directly or through a regulatory cascade affect the expression of the genes subject to catabolite repression. These genes are not all controlled by a single set of regulatory proteins (Cho et al. 2009), but there are different circuits of repression for different groups of genes (Gancedo 1990). We will discuss the possible metabolic regulation in the case of Y. lipolytica.     

Effect of copper on acid phosphatase activity in yeast Yarrowia lipolytica

Acid phosphatase (APase) activity of the yeast Yarrowia lipolytica increased with increasing Cu2+ concentrations in the medium. Furthermore, the enzyme in soluble form was stimulated in vitro by Cu2+, Co2+, Ni2+, Mn2+ and Mg2+ and inhibited by Ag+ and Cd2+. The most effective ion was Cu2+, especially for the enzyme from cultures in medium containing Cu2+, whereas APase activity in wall-bound fragments was only slightly activated by Cu2+. The content of cellular phosphate involving polyphosphate was decreased by adding Cu2+, regardless of whether or not the medium was rich in inorganic phosphate. Overproduction of the enzyme stimulated by Cu2+ might depend on derepression of the gene encoding the APase isozyme.