Optimization of Citric Acid Production from Glucose by Yarrowia lipolytica

Citric acid (CA) is mainly produced in a biotechnological process using Aspergillus niger. In this process, large amounts of wastes have to be removed. Since the use of Yarrowia lipolytica for CA production is an environmental compatible alternative method, the CA production was optimized in regard to growth temperature and pH as well as substrate and product inhibition. The highest value of the maximum specific growth rate at pH 6.5 was found to be μ_max = 0.192 h^–1, whereas the largest amount of CA of 24.91 g/L as well as the highest selectivity of the bioprocess (89.9 % CA) and the maximum yield (0.22 g_CA/g_Glucose) were obtained at pH 6.0. During the growth phase, the temperature optimum was found to be in the range of 30–34 °C (μ_max = 0.132 h^–1). Nevertheless, the highest concentration of CA during the production phase was obtained at 30 °C (41 g/L CA, 93.1 % CA, 0.55 g_CA/g_glucose). In studying the substrate inhibition of the process, a clear tendency of decrease in the maximum specific growth rate was detected when the initial glucose concentration was increased from 50 g/L (μ_max = 0.17 h^–1) to 200 g/L (μ_max = 0.055 h^–1). The addition of 120 g/L CA to the culture broth at the start of the production phase reduced the production of CA from 32.1 g/L to 7.4 g/L.     

Production of 1-decanol by metabolically engineered Yarrowia lipolytica

Medium-chain alcohols are used to produce solvents, surfactants, lubricants, waxes, creams, and cosmetics. In this study, we engineered the oleaginous yeast Yarrowia lipolytica to produce 1-decanol from glucose. Expression of a fatty acyl-CoA reductase from Arabidopsis thaliana in strains of Y. lipolytica previously engineered to produce medium-chain fatty acids resulted in the production of 1-decanol. However, the resulting titers were very low (<10 mg/mL), most likely due to product catabolism. In addition, these strains produced small quantities of 1-hexadecanol and 1-octadecanol. Deleting the major peroxisome assembly factor Pex10 was found to significantly increase 1-decanol production, resulting in titers exceeding 500 mg/L. It also increased 1-hexadecanoland and 1-octadecanol titers, though the resulting increases were less than those for 1-decanol. These results demonstrate that Y. lipolytica can potentially be used for the industrial production of 1-decanol and other fatty alcohols from simple sugars.     

Different effectors of dimorphism in Yarrowia lipolytica

Yarrowia lipolytica is an ascomycete with biotechnological potential. In common media, the fungus grows as a mixture of yeast-like and short mycelial cells. The environmental factors that affect dimorphism in the wild-type strain, W29, and its auxotrophic derivative, PO1a, were analyzed. In both strains, pH was the most important factor regulating the dimorphic transition. Mycelium formation was maximal at pH near neutrality and decreased as pH was lowered to become almost null at pH 3. Carbon and nitrogen sources, namely glucose and ammonium, were also important for mycelium formation; and their effect was antagonized by some alternative carbon and nitrogen sources. Citrate was an important positive effector of mycelium growth. Anaerobic stress induced formation of mycelial cells. The importance of the protein kinase A pathway was suggested by the inhibition of mycelium growth by cAMP. We propose that the interplay of these factors regulates the adaptation of the fungus, to better exploit its natural ecological niches.     

解脂耶罗维亚酵母产油脂的研究进展

单细胞油脂是生产生物柴油最理想的原料,随着化石能源的日益枯竭,单细胞油脂的生产受到广泛关注。解脂耶罗维亚酵母是生产单细胞油脂的最佳菌株,它能够利用诸多廉价底物作为碳源,在工业上有极大的应用前景。其遗传背景清晰,全基因组测序已完成,基因表达系统已构建。在此基础上对油脂累积途径进行了深入研究,多株油脂含量更高的菌株被构建。深入了解解脂耶罗维亚酵母的基因表达及油脂代谢系统,对日后对其进一步的代谢改造具有重要意义。     

Production of brown tyrosine pigments by the yeast Yarrowia lipolytica

AIMS: To study the mechanism of production of brown pigments from tyrosine in the yeast Yarrowia lipolytica. METHODS AND RESULTS: Pigment formation was followed during growth in tyrosine medium, and the presence of the pigment precursor in the medium was assessed by evaluating pigment formation after removing the cells at different times of incubation. It was observed that the pigment precursor accumulated outside the cells during the exponential phase of growth, but pigment formation only occurred during the stationary phase of growth and resulted from the oxidation of the precursor. Pigment formation was repressed by glucose and L-glutamine, and promoted by lactic acid, L-asparagine and glycine. Spectra of 1H and 13C-NMR revealed that the brown pigment was derived from tyrosine and was a polymer composed of a core of aromatic residues. CONCLUSION: The results indicate that pigments result from the extracellular accumulation and auto-oxidation of an intermediate of tyrosine catabolism. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the mechanism of pigment production from tyrosine in a yeast species.     

Production of citric acid with immobilized Yarrowia lipolytica

Summary Citric acid was produced with immobilized Yarrowia lipolytica yeast in repeated batch-shake-flask and air-lift fermentations. In active and passive immobilization methods calcium alginate, -carrageenan, polyurethane gel, nylon web and polyurethane foams were tested as carriers in repeated-batch fermentations. The highest citric acid productivity of 155 mg l^–1 h^–1 was reached with alginate-bead-immobilized cells in the first batch. A decrease in bead diameter from 5–6 mm to 2–3 mm increased the volumetric citric acid productivity threefold. In an air-lift bioreactor the highest citric acid productivity of 120 mg l^–1 h^–1 with a product concentration of 16.4 g l^–1 was obtained with cells immobilized in -carrageenan beads. Offprint requests to: H. Kautola     

代谢工程构建重组耶氏解脂酵母生产中长链聚羟基脂肪酸酯

中长链聚羟基脂肪酸酯(mcl-PHA)是一大类由微生物合成的天然生物聚酯,因具有可再生性和生物降解性越来越受到人们的关注。Mcl-PHA可由一些假单胞菌类利用自身的脂肪酸合成途径或β-氧化途径来合成。耶氏解脂酵母具有很好的脂/脂肪酸分解代谢能力,但是它体内缺乏PHA合成酶不能合成mcl-PHA。采用代谢工程策略构建重组解脂酵母,外源表达来自铜绿假单胞菌PAO1(Pseudomonas aeruginosa PAO1)的PHA合成酶。在PHA合成酶的C端添加PTS1过氧化物酶体定位信号序列,使其在过氧化物酶体内发挥功能,并对其编码基因PhaC1进行密码子优化得到oPhaC1。利用pINA1312载体构建表达框,借助载体上的zeta序列元件将oPhaC1基因表达框整合至酵母基因组,完成基因的稳定表达。重组菌PSOC在葡萄糖为唯一碳源的培养基中几乎不产PHA,添加0.5%的油酸时可合成占细胞干重0.67%的mcl-PHA。在含三油酸甘油酯的培养基中发酵72h产生1.51% mcl-PHA(wt%)。实验结果充分证明重组解脂酵母作为有潜力的微生物细胞工厂可以用于生产mcl-PHA,也为将来利用富含油脂和其他营养的餐厨垃圾水解液等廉价资源生产mcl-PHA打下基础。     

2,4,6-Trinitrotoluene Reduction by Carbon Monoxide Dehydrogenase from Clostridium thermoaceticum

Purified CO dehydrogenase (CODH) from Clostridium thermoaceticum catalyzed the transformation of 2,4,6-trinitrotoluene (TNT). The intermediates and reduced products of TNT transformation were separated and appear to be identical to the compounds formed by C. acetobutylicum, namely, 2-hydroxylamino-4,6-dinitrotoluene (2HA46DNT), 4-hydroxylamino-2,6-dinitrotoluene (4HA26DNT), 2,4-dihydroxylamino-6-nitrotoluene (24DHANT), and the Bamberger rearrangement product of 2,4-dihydroxylamino-6-nitrotoluene. In the presence of saturating CO, CODH catalyzed the conversion of TNT to two monohydroxylamino derivatives (2HA46DNT and 4HA26DNT), with 4HA26DNT as the dominant isomer. These derivatives were then converted to 24DHANT, which slowly converted to the Bamberger rearrangement product. Apparent K_m and k_cat values of TNT reduction were 165 ± 43 μM for TNT and 400 ± 94 s⁻¹, respectively. Cyanide, an inhibitor for the CO/CO₂ oxidation/reduction activity of CODH, inhibited the TNT degradation activity of CODH.     

Biotransformation Patterns of 2,4,6-Trinitrotoluene by Aerobic Bacteria

2,4,6-Trinitrotoluene (TNT), a toxic nitroaromatic explosive, accumulates in the environment, making necessary the remediation of contaminated areas and unused materials. Although bioremediation has been utilized to detoxify TNT, the metabolic processes involved in the metabolism of TNT have proven to be complex. The three aerobic bacterial strains reported here (Pseudomonas aeruginosa, Bacillus sp., and Staphylococcus sp.) differ in their ability to biotransform TNT and in their growth characteristics in the presence of TNT. In addition, enzymatic activities have been identified that differ in the reduction of nitro groups, cofactor preferences, and the ability to eliminate-NO₂ from the ring. The Bacillus sp. has the most diverse bioremediation potential owing to its growth in the presence of TNT, high level of reductive ability, and capability of removing-NO₂ from the nitroaromatic ring. Received: 16 May 1997 / Accepted: 19 July 1997     

Production of Lipase by Yarrowia lipolytica,I. Lipases from Yeasts (Review)

In this paper a review is given about the most important lipase-producing yeasts as well as the yield of lipase production. In order to find out the optimal conditions for lipase production by yeasts, it is necessary to investigate the influence of the main factors affecting lipase synthesis such as carbon and nitrogen sources, activators and inhibitors, interface-affecting agents, temperature, pH and inoculum. Besides the yield of lipase as an efficiency factor of fermentation and the substrate specificity of lipase, playing an important role in applications, are described.     

Production of erythritol and mannitol by Yarrowia lipolytica yeast in media containing glycerol

Glycerol is a by-product generated in large amounts during the production of biofuels. This study presents an alternative means of crude glycerol valorization through the production of erythritol and mannitol. In a shake-flasks experiment in a buffered medium, nine Yarrowia lipolytica strains were examined for polyols production. Three strains (A UV'1, A-15 and Wratislavia K1) were selected as promising producers of erythritol or/and mannitol and used in bioreactor batch cultures and fed-batch mode. Pure and biodiesel-derived crude glycerol media both supplemented (to 2.5 and 3.25?%) and not-supplemented with NaCl were applied. The best results for erythritol biosynthesis were achieved in medium with crude glycerol supplemented with 2.5?% NaCl. Wratislavia K1 strain produced up to 80.0?g?l(-1) erythritol with 0.49?g?g(-1) yield and productivity of 1.0?g?l(-1)?h(-1). Erythritol biosynthesis by A UV'1 and A-15 strains was accompanied by the simultaneous production of mannitol (up to 27.6?g?l(-1)). Extracellular as well as intracellular erythritol and mannitol ratios depended on the glycerol used and the presence of NaCl in the medium. The results from this study indicate that NaCl addition to the medium improves erythritol biosynthesis, and simultaneously inhibits mannitol formation.     

Production of Lycopene in the Non-Carotenoid-Producing Yeast Yarrowia lipolytica

The codon-optimized genes crtB and crtI of Pantoea ananatis were expressed in Yarrowia lipolytica under the control of the TEF1 promoter of Y. lipolytica. Additionally, the rate-limiting genes for isoprenoid biosynthesis in Y. lipolytica, GGS1 and HMG1, were overexpressed to increase the production of lycopene. All of the genes were also expressed in a Y. lipolytica strain with POX1 to POX6 and GUT2 deleted, which led to an increase in the size of lipid bodies and a further increase in lycopene production. Lycopene is located mainly within lipid bodies, and increased lipid body formation leads to an increase in the lycopene storage capacity of Y. lipolytica. Growth-limiting conditions increase the specific lycopene content. Finally, a yield of 16 mg g⁻¹ (dry cell weight) was reached in fed-batch cultures, which is the highest value reported so far for a eukaryotic host.     

Initial Stages of 2,4,6-Trinitrotoluene Transformation by Microorganisms

Screening of a wide range of microorganisms (32 strains) isolated from various anthropogenic and natural environments and of a number of collection strains showed that the early stages of 2,4,6-trinitrotoluene (TNT) transformation by the majority of the strains studied resulted in the formation of hydroxylaminodinitrotoluenes (HADNTs). The levels of HADNTs were in a number of cases comparable to the initial TNT level. The alternative reductive attack on TNT through the reduction of the aromatic ring was not characteristic of most of the prokaryotes studied. The susceptibility to the toxic effect of TNT was different for gram-positive and gram-negative bacteria.     

Improvement of lipase production from Yarrowia lipolytica

Extracellular lipase production by Yarrowia lipolytica was increased by mutant selection from 28 U/ml to 1000 U/ml. This activity was also reached in a 500 l bioreactor. The properties of the mutant lipase were the same of those of the wild type: M 38 kDa, optimum pH 7 and optimum temperature 37¡C.     

Biosynthesis of biotin vitamers by Yarrowia lipolytica

The hydrocarbon utilizing yeast Yarrowia lipolyyica NCYC 1421 produces biotin and its vitamers when grown on glucose in biotin-free media. Levels of production can be influenced by the medium composition. Growth in the presence of longchained fatty acids greatly increases biotin vitamer production. The biotin vitamers produced are normally dethiobiotin and 7-keto, 8-aminopelargonic acid. The addition of succinic acid at 0.5 g per litre causes the vitamer 7, 8-diaminopelargonic acid to be produced at high levels. The biotin antagonist α-dehydrobiotin inhibits the growth of Yarrowia lipolytica . Mutants can be readily isolated which show resistance to α-dehydrobiotin, but these do not produce greater amounts of biotin or its vitamers.     

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.     

Biotransformation of 2,4,6-Trinitrotoluene by Pure Culture Ruminal Bacteria

Twenty-one ruminal bacteria species were tested for their ability to degrade 2,4,6-trinitrotoluene (TNT) within 24 h. Butyrivibrio fibrisolvens, Fibrobacter succinogenes, Lactobacillus vitulinus, Selenomonas ruminantium, Streptococcus caprinus, and Succinivibrio dextrinosolvens were able to completely degrade 100 mg/L TNT, with <5% of the original TNT recovered as diaminonitrotoluene metabolites. Eubacterium ruminantium, Lactobacillus ruminis, Ruminobacter amylophilus, Streptococcus bovis, and Wolinella succinogenes were able to completely degrade 100 mg/L TNT, with 23–60% of the TNT recovered as aminodinitrotoluene and/or diaminonitrotoluene metabolites. Clostridium polysaccharolyticum, Megasphaera elsdenii, Prevotella bryantii, Prevotella ruminicola, Ruminococcus albus, and Ruminococcus flavefaciens were able to degrade 80–90% of 100 mg/L TNT. Desulfovibrio desulfuricans subsp. desulfuricans, Prevotella albensis, and Treponema bryantii degraded 50–80% of the TNT. Anaerovibrio lipolytica was completely inhibited by 100 mg/L TNT. These results indicate that a variety of rumen bacteria is capable of transforming TNT.     

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.     

Transformation of 2,4,6-Trinitrotoluene by Pseudomonas pseudoalcaligenes JS52

Pseudomonas pseudoalcaligenes JS52 grows on nitrobenzene via partial reduction of the nitro group and enzymatic rearrangement of the resultant hydroxylamine. Cells and cell extracts of nitrobenzene-grown JS52 catalyzed the transient formation of 4-hydroxylamino-2,6-dinitrotoluene (4HADNT), 4-amino-2,6-dinitrotoluene (4ADNT), and four previously unidentified metabolites from 2,4,6-trinitrotoluene (TNT). Two of the novel metabolites were identified by liquid chromatography/mass spectrometry and (sup1)H-nuclear magnetic resonance spectroscopy as 2,4-dihydroxylamino-6-nitrotoluene (DHANT) and 2-hydroxylamino-4-amino-6-nitrotoluene (2HA4ANT). A polar yellow metabolite also accumulated during transformation of TNT by cells and cell extracts. Under anaerobic conditions, extracts of strain JS52 did not catalyze the production of the yellow metabolite or release nitrite from TNT; moreover, DHANT and 2HA4ANT accumulated under anaerobic conditions, which indicated that their further metabolism was oxygen dependent. Small amounts of nitrite were released during transformation of TNT by strain JS52. Sustained transformation of TNT by cells required nitrobenzene, which indicated that TNT transformation does not provide energy. Transformation of TNT catalyzed by enzymes in cell extracts required NADPH. Transformation experiments with (sup14)C-TNT indicated that TNT was not mineralized; however, carbon derived from TNT became associated with cells. Nitrobenzene nitroreductase purified from strain JS52 transformed TNT to DHANT via 4HADNT, which indicated that the nitroreductase could catalyze the first two steps in the transformation of TNT. The unusual ability of the nitrobenzene nitroreductase to catalyze the stoichiometric reduction of aromatic nitro compounds to the corresponding hydroxylamine provides the basis for the novel pathway for metabolism of TNT.