Carbon and nitrogen sources modulate lipase production in the yeast Yarrowia lipolytica

AIMS: To analyse the influence of nitrogen and carbon sources on extracellular lipase production by Yarrowia lipolytica-overproducing mutant in order to optimize its production in large-scale bioreactors. METHODS AND RESULTS: The level of lipase production and LIP2 induction, measured using an LIP2-LacZ reporter gene, were compared for different carbon and nitrogen sources and for different concentrations. The localization of the enzyme during growth was also determined by Western blotting analysis using a six-histidine-tagged lipase. SIGNIFICANCE AND IMPACT OF THE STUDY: Tryptone N1 and oleic acid are the most suitable nitrogen and carbon sources for the production of the extracellular lipase by the Y. lipolytica mutant. Higher levels of lipase production were obtained as the tryptone concentration increased in the culture medium. Such a positive correlation was not observed with oleic acid media where the highest lipolytic productivities were obtained in the presence of low concentration. We also demonstrate that in the presence of oleic acid, lipase is cell-bound during the growth phase before being released in the media. CONCLUSIONS: This work provides a better understanding of the mechanism controlling LIP2 expression and, thus, extracellular lipase production in the yeast Y. lipolytica.     

Selection of new over-producing derivatives for the improvement of extracellular lipase production by the non-conventional yeast Yarrowia lipolytica

The non-conventional yeast Yarrowia lipolytica produces an extracellular lipase encoded by the LIP2 gene. Mutant strains with enhanced productivity were previously obtained either by chemical mutagenesis or genetic engineering. In this work, we used one of these mutants, named LgX64.81 to select new overproducing strains following by amplification of the LIP2 gene. We also developed a process for lipase production in bioreactors and compared lipase production levels in batch and fed-batch cultures. Batch culture led to a lipase production of 26450 U ml(-1) in a media containing olive oil and tryptone as carbon and nitrogen sources. Feeding of a combination of tryptone and olive oil at the end of the exponential growth phase yielded to lipase activity of 158246 U ml(-1) after 80 h of cultivation. In addition this production system developed for the extracellular lipase could also be applied for other heterologous protein production since we have demonstrated that LgX64.81 is an interesting alternative host strain.     

Characterization of an extracellular lipase encoded by LIP2 in Yarrowia lipolytica

We isolated the LIP2 gene from the lipolytic yeast Yarrowia lipolytica. It was found to encode a 334-amino-acid precursor protein. The secreted lipase is a 301-amino-acid glycosylated polypeptide which is a member of the triacylglycerol hydrolase family (EC 3.1.1.3). The Lip2p precursor protein is processed by the KEX2-like endoprotease encoded by XPR6. Deletion of the XPR6 gene resulted in the secretion of an active but less stable proenzyme. Thus, the pro region does not inhibit lipase secretion and activity. However, it does play an essential role in the production of a stable enzyme. Processing was found to be correct in LIP2(A) (multiple LIP2 copy integrant)-overexpressing strains, which secreted 100 times more activity than the wild type, demonstrating that XPR6 maturation was not limiting. No extracellular lipase activity was detected with the lip2 knockout (KO) strain, strongly suggesting that extracellular lipase activity results from expression of the LIP2 gene. Nevertheless, the lip2 KO strain is still able to grow on triglycerides, suggesting an alternative pathway for triglyceride utilization in Y. lipolytica.     

Identification and characterisation of LIP7 and LIP8 genes encoding two extracellular triacylglycerol lipases in the yeast Yarrowia lipolytica

In the lipolytic yeast Yarrowia lipolytica, the LIP2 gene was previously reported to encode an extracellular lipase. The growth of a Deltalip2 strain on triglycerides as sole carbon source suggest an alternative pathway for triglycerides utilisation in this yeast. Here, we describe the isolation and the characterisation of the LIP7 and LIP8 genes which were found to encode a 366 and a 371-amino acid precursor protein, respectively. These proteins which belong to the triacylglycerol hydrolase family (EC 3.1.1.3) presented a high homology with the extracellular lipase CdLIP2 and CdLIP3 from Candida deformans. The physiological function of the lipase isoenzymes was investigated by creating single and multi-disrupted strains. Lip7p and Lip8p were found to correspond to active secreted lipases. The lack of lipase production in a Deltalip2 Deltalip7 Deltalip8 strain suggest that no additional extracellular lipase remains to be discovered in Y. lipolytica. The substrate specificity towards synthetic ester molecules indicates that Lip7p presented a maximum activity centred on caproate (C6) while that of Lip8p is in caprate (C10).     

Overproduction of lipase by<Emphasis Type="Italic"> Yarrowia lipolytica</Emphasis> mutants

Non-genetically modified mutants with increased capacities of extracellular lipase production were obtained from Yarrowia lipolytica strain CBS6303 by chemical mutagenesis. Of the 400 mutants isolated, LgX64.81 had the highest potential for the development of an industrial lipase production process. This mutant exhibits lipase production uncoupled from catabolite repression by glucose, and a 10-fold increased productivity upon addition of oleic acid. Using a LIP2- LacZ reporter gene, we demonstrate that the mutant phenotype originates from a trans-acting mutation. The glucose uptake capacity of LgX64.81 is reduced 2.5-fold compared to the wild-type-strain, and it exhibits high lipase production on glucose medium. A trans-acting mutation in a gene involved in glucose transport could thus explain this mutant phenotype.     

New finding and optimal production of a novel extracellular alkaline lipase from Yarrowia lipolytica NRRL Y-2178

Lipases are industrially useful versatile enzymes that catalyze numerous different reactions including hydrolysis of triglycerides, transesterification, and chiral synthesis of esters under natural conditions. Although lipases from various sources have been widely used in industrial applications, such as in food, chemical, pharmaceutical, and detergent industries, there are still substantial current interests in developing new microbial lipases, specifically those functioning in abnormal conditions. We screened 17 lipase-producing yeast strains, which were prescreened for substrate specificity of lipase from more than 500 yeast strains from the Agricultural Research Service Culture Collection (Peoria, IL, U.S.A.), and selected Yarrowia lipolytica NRRL Y-2178 as a best lipase producer. This report presents new finding and optimal production of a novel extracellular alkaline lipase from Y. lipolytica NRRL Y-2178. Optimal c ulture conditions f orlipase production by Y. lipolytica NRRL Y-2178 were 72 h incubation time, 27.5 degrees C, pH 9.0. Glycerol and glucose were efficiently used as the most efficient carbon sources, and a combination of yeast extract and peptone was a good nitrogen source for lipase production by Y. lipolytica NRRL Y-2178. These results suggested that Y. lipolytica NRRL Y-2178 showsgood industrial potential as a new alkaline lipase producer.     

Autocloning and amplification of LIP2 in Yarrowia lipolytica

We synthesized a Yarrowia lipolytica strain overproducing lipase for industrial applications by using long terminal repeat (zeta) of the Y. lipolytica retrotransposon Ylt1 and an allele of URA3 with a promoter deletion to construct JMP3. JMP3 is a derivative of plasmid pHSS6 carrying a NotI-NotI cassette which contains a defective URA3 allele, a polylinker sequence, and the zeta region for targeting to multiple sites in the genome of the recipient. We inserted the LIP2 gene (encoding extracellular lipase) under the control of the strong POX2 promoter into JMP3 to generate JMP6. The pHSS6 region was removed by NotI digestion prior to transformation. Two Y. lipolytica strains transformed with the JMP6 LIP2 cassette had a mean of 10 integrated copies devoid of the Escherichia coli region, corresponding to an autocloning event. The copy number in the transformants was stable even after 120 generations in nonselective and lipase-inducing conditions. The resulting strains could produce 0.5 g of active lipase per liter in the supernatant, 40 times more than the single-copy strain with the LIP2 promoter. This work provides a new expression system in Y. lipolytica that results in strains devoid of bacterial DNA and in strains producing a high level of lipase for industrial uses, waste treatment, and pancreatic insufficiency therapy.     

Investigation of the effect of different extracellular factors on the lipase production by <Emphasis Type="Italic">Yarrowia lipolityca</Emphasis> on the basis of a scale-down approach

The influence of three extracellular factors (namely, the methyl oleate dispersion in the broth, the dissolved oxygen variations, and the pH fluctuation) on the lipase production by Y. lipolytica in batch bioreactor has been investigated in different scale-down apparatus. These systems allow to reproduce the hydrodynamic phenomena encountered in large-scale equipments for the three specified factors. The effects of the extracellular factors have been observed at three distinct levels: the microbial growth, the extracellular lipase production, and the induction of the gene LIP2 encoding for the main lipase of Y. lipolytica. Among the set of environmental factors investigated, the dissolved oxygen fluctuations generated in a controlled scale-down reactor (C-SDR) have led to the more pronounced physiological effect by decreasing the LIP2 gene expression level. The other environmental factors observed in a partitioned scale-down reactor, i.e., the methyl oleate dispersion and the pH fluctuations, have led to a less severe stress traduced only by a decrease of the microbial yield and thus of the extracellular lipase specific production rate.     

SOA genes encode proteins controlling lipase expression in response to triacylglycerol utilization in the yeast Yarrowia lipolytica

The oleaginous yeast Yarrowia lipolytica efficiently metabolizes hydrophobic substrates such as alkanes, fatty acids or triacylglycerol. This yeast has been identified in oil-polluted water and in lipid-rich food. The enzymes involved in lipid breakdown, for use as a carbon source, are known, but the molecular mechanisms controlling the expression of the genes encoding these enzymes are still poorly understood. The study of mRNAs obtained from cells grown on oleic acid identified a new group of genes called SOA genes (specific for oleic acid). SOA1 and SOA2 are two small genes coding for proteins with no known homologs. Single- and double-disrupted strains were constructed. Wild-type and mutant strains were grown on dextrose, oleic acid and triacylglycerols. The double mutant presents a clear phenotype consisting of a growth defect on tributyrin and triolein, but not on dextrose or oleic acid media. Lipase activity was 50-fold lower in this mutant than in the wild-type strain. The impact of SOA deletion on the expression of the main extracellular lipase gene ( LIP2 ) was monitored using a LIP2 -β-galactosidase promoter fusion protein. These data suggest that Soa proteins are components of a molecular mechanism controlling lipase gene expression in response to extracellular triacylglycerol.     

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.     

Beta-fructofuranosidase production by 2-deoxyglucose resistant mutants of aspergillus niger in submerged and solid-state fermentation

Aspergillus niger produces extracellular beta-fructofuranosidase under submerged (SmF) and solid state fermentation (SSF) conditions. After UV mutagenesis of conidiospores of A. niger, 2-deoxyglucose (10 g/l) resistant mutants were isolated on Czapek's minimal medium containing glycerol as a carbon source and the mutants were examined for improved production of beta-fructofuranosidase in SmF and SSF conditions. One of such mutant DGRA-1 overproduced beta-fructofuranosidase in both SmF and SSF conditions. In SmF, the mutant DGRA-1 showed higher beta-fructofuranosidase productivity (110.8 U/l/hr) than the wild type (48.3 U/l/hr). While in SSF the same strain produced 322 U/l/hr of beta-fructofuranosidase, 2 times higher than that of wild type (154.2 U/l/hr). In SmF, both wild type and mutants produced relatively low level of beta-fructofuranosidase in medium containing sucrose with glucose than from the sucrose medium. However in SSF, the DGRA-1 mutant grown in sucrose and sucrose+ glucose did not show any difference with respect to beta-fructofuranosidase production. These results indicate that the catabolite repression of beta-fructofuranosidase synthesis is observed in SmF whereas in SSF such regulation was not prominent.     

解脂亚罗酵母P12单倍体的制备

【背景】目前解脂亚罗酵母在实验研究和工业生产方面的应用越来越广泛,但相较于常规酵母而言,解脂亚罗酵母缺乏简便有效的遗传转化体系,致使其在基因表达调控方面存在较大困难。同时,酵母的染色体倍性也会对基因敲除效果产生影响,选择单倍体细胞作为功能基因改造的受体可以避免等位基因之间相互作用的影响,解决多倍体细胞基因敲除不完全的问题。【目的】以解脂亚罗酵母诱变菌株P12为研究对象,以不同方法分离得到单倍体菌株,建立解脂亚罗酵母单倍体的制备方法。【方法】分别采用固体和液体McClary产孢培养基诱导解脂亚罗酵母菌株产生子囊孢子,培养条件为30℃,固体7-14 d;液体200 r/min,2-4 d。以2%浓度的蜗牛酶33℃水浴裂解子囊孢子细胞壁3 h,通过染色镜检和PCR鉴定筛选单倍体细胞。【结果】镜检结果表明,解脂亚罗酵母在液体产孢培养基中产孢速度较快,相同视野下孢子数约为固体产孢培养基的3.7倍,在固体产孢培养基中产孢质量较好。初步探索并筛选得到6株解脂亚罗酵母P12 B型单倍体菌株。【结论】解脂亚罗酵母P12 B型单倍体菌株的获得可为后续继续开展基因工程操作奠定基础。     

Hydrophobic substrate utilisation by the yeast Yarrowia lipolytica, and its potential applications

The alkane-assimilating yeast Yarrowia lipolytica degrades very efficiently hydrophobic substrates such as n-alkanes, fatty acids, fats and oils for which it has specific metabolic pathways. An overview of the oxidative degradation pathways for alkanes and triglycerides in Y. lipolytica is given, with new insights arising from the recent genome sequencing of this yeast. This includes the interaction of hydrophobic substrates with yeast cells, their uptake and transport, the primary alkane oxidation to the corresponding fatty alcohols and then by different enzymes to fatty acids, and the subsequent degradation in peroxisomal beta-oxidation or storage into lipid bodies. Several enzymes involved in hydrophobic substrate utilisation belong to multigene families, such as lipases/esterases (LIP genes), cytochromes P450 (ALK genes) and peroxisomal acyl-CoA oxidases (POX genes). Examples are presented demonstrating that wild-type and genetically engineered strains of Y. lipolytica can be used for alkane and fatty-acid bioconversion, such as aroma production, for production of SCP and SCO, for citric acid production, in bioremediation, in fine chemistry, for steroid biotransformation, and in food industry. These examples demonstrate distinct advantages of Y. lipolytica for their use in bioconversion reactions of biotechnologically interesting hydrophobic substrates.     

Yarrowia lipolytica mutants devoid of pyruvate carboxylase activity show an unusual growth phenotype

We have cloned and characterized the gene PYC1, encoding the unique pyruvate carboxylase in the dimorphic yeast Yarrowia lipolytica. The protein putatively encoded by the cDNA has a length of 1,192 amino acids and shows around 70% identity with pyruvate carboxylases from other organisms. The corresponding genomic DNA possesses an intron of 269 bp located 133 bp downstream of the starting ATG. In the branch motif of the intron, the sequence CCCTAAC, not previously found at this place in spliceosomal introns of Y. lipolytica, was uncovered. Disruption of the PYC1 gene from Y. lipolytica did not abolish growth in glucose-ammonium medium, as is the case in other eukaryotic microorganisms. This unusual growth phenotype was due to an incomplete glucose repression of the function of the glyoxylate cycle, as shown by the lack of growth in that medium of double pyc1 icl1 mutants lacking both pyruvate carboxylase and isocitrate lyase activity. These mutants grew when glutamate, aspartate, or Casamino Acids were added to the glucose-ammonium medium. The cDNA from the Y. lipolytica PYC1 gene complemented the growth defect of a Saccharomyces cerevisiae pyc1 pyc2 mutant, but introduction of either the S. cerevisiae PYC1 or PYC2 gene into Y. lipolytica did not result in detectable pyruvate carboxylase activity or in growth on glucose-ammonium of a Y. lipolytica pyc1 icl1 double mutant.     

Strain improvement of Aspergillus niger for enhanced lipase production

The enhancement of lipase production from Aspergillus niger was attempted by ultraviolet (UV) and nitrous acid mutagenesis, and the mutants were selected on media containing bile salts. Nitrous acid mutants exhibited increased efficiency for lipase production when compared with UV mutants in submerged fermentation. The hyperproducing UV and nitrous acid mutants were further subjected to a second step of mutagenesis to devise an economical and ecofriendly technique for lipase production by the effective use of hydrocarbons. One percent kerosene was found to be optimal for lipase production, and one of the mutant strains NAII exhibited 2.53 times more increased lipase activity than the parental strain did. This investigation indicates a possible role for the A. niger mutant strains in the biodegradation of oil-polluted environments for the development of ecofriendly technologies.     

Strong hybrid promoters and integrative expression/secretion vectors for quasi-constitutive expression of heterologous proteins in the yeast Yarrowia lipolytica

The industrial yeast Yarrowia lipolytica secretes high amounts of an alkaline extracellular protease encoded by the XPR2 gene. The industrial use of the XPR2 promoter was however hindered by its complex regulation. We designed hybrid promoters, based on tandem copies of the XPR2 promoter UAS1 region. In contrast to native XPR2 promoter, these hybrid promoters were not repressed by the preferred carbon and nitrogen sources, nor by acidic conditions, and they did not require the presence of peptones in the culture medium. They exhibited a strong quasi-constitutive activity, similar when carried on either integrative or replicative plasmids. We used these hybrid promoters to direct the production of bovine prochymosin, using XPR2 secretion signals. The production of active chymosin was several fold higher than with previously available Y. lipolytica promoters (up to 160 mg/l). Integrative vectors based on the hybrid promoters, allowing the easy insertion of a heterologous gene and its expression or expression/secretion in Y. lipolytica, were designed. We also designed new Y. lipolytica recipient strains with good secreting abilities, able to grow on sucrose, and devoid of extracellular proteases. These new tools will add to the interest of Y. lipolytica as a host for heterologous protein production.     

Genetic studies on the yeast Saccharomycopsis lipolytica. Inactivation and mutagenesis

Spontaneous mutants of Saccharomycopsis lipolytica were selected and partially characterized. Several antibiotics and antimetabolites were used for selection of spontaneous resistant mutants from Saccharomycopsis lipolytica. The frequencies of such mutants were mainly arranged between 1 X 10(-7) and 5 X 10(-6) mutants per cell. But one class of glucosamine resistant mutants (GAMRA) occurred more frequently. Among the resistant mutants different types of dominant and recessive resistant mutants could be observed. UV light was used for inactivation of cells and induction of mutants from S. lipolytica. Comparing four haploid strains only small differences were detected in sensitivity to UV light. UV light at a dosage of 135 J/m2 was applied to increase the mutant frequencies in three haploid strains. Besides auxotrophic, temperature sensitive and colony morphology mutants, some new mutant types like small colony forming mutants, red-brown coloured mutants, some new mutant types like small colony forming mutants, red-brown coloured mutants, allylalcohol, glucosamine, 2-deoxyglucose or nystatin resistant mutants, hitherto not described for S. lipolytica, were isolated and partially characterized.     

Synthesis citric and isocitric acids by Yarrowia lipolytica during yeasts growth on rapeseed oils

The native strain Yarrowia lipolytica VKM Y-2373 grown in a complete medium exhibited the maximum lipase activity at the concentration of rapeseed oil of at least 5.0 g/l. In the course of yeast growth, no considerable changes were observed in the glycerol concentration, the proportions of the major free fatty acids formed via oil hydrolysis, or the fatty acid composition of oil. Under nitrogen limitation of cell growth, the accumulation of citric acids reached 77.1 g/l with predominance of isocitric acid at pH 6.0, whereas at pH 4.5, almost equal amounts of citric and isocitric acids were produced. Cultivation of the mutant strain Y. lipolytica N 1 at pH 4.5 resulted in the predominant accumulation of citric acid (66.6 g/l) with an insignificant amount of isocitric acid. In the period of intense acid synthesis, high production of lipase was observed.     

Survival and liquid holding recovery in UV-sensitive strains of Saccharomyces cerevisiae after treatment with chemical mutagens and radiation.

Two UV-sensitive mutants of Saccharomyces cerevisiae rad 3 and rad 6 were tested for sensitivity to X-rays, MMS, EMS, HNO2 and DEB. Rad 3 mutant is more sensitive than the wild type strain only to HNO2 and DEB, while rad 6 is cross sensitive both to X-rays and all chemicals tested. Liquid holding recovery (LHR) was studied by comparison of cell survival immediately after mutagen treatment and after 5 days of storage in phosphate buffer. LH greatly increases cell survival of rad 3 mutant after DEB and slightly after EMS, MMS and HNO2, while after UV treatment LH significantly decreases survival of this mutant. LH increases survival of rad 6 mutant after exposure to UV, MMS and HNO2, but decreases survival of DEB-treated cells. Exposure of wild type strain to LH results in an increase of survival after UV, and DEB but not after MMS and HNO2. The results suggest that LHR is a strain- and mutagen-specific phenomenon and cannot be explained within the present knowledge of repair processes in yeast.