High-level production of extracellular lipase by Yarrowia lipolytica mutants from methyl oleate

The yeast Yarrowia lipolytica degrades efficiently low-cost hydrophobic substrates for the production of various added-value products such as lipases. To obtain yeast strains producing high levels of extracellular lipase, Y. lipolytica DSM3286 was subjected to mutation using ethyl methanesulfonate (EMS) and ultraviolet (UV) light. Twenty mutants were selected out of 1600 mutants of Y. lipolytica treated with EMS and UV based on lipase production ability on selective medium. A new industrial medium containing methyl oleate was optimized for lipase production. In the 20 L bioreactor containing new industrial medium, one UV mutant (U6) produced 356 U/mL of lipase after 24h, which is about 10.5-fold higher than that produced by the wild type strain. The properties of the mutant lipase were the same as those of the wild type: molecular weight 38 kDa, optimum temperature 37°C and optimum pH 7. Furthermore, the nucleotide sequences of extracellular lipase gene (LIP2) in wild type and mutant strains were determined. Only two silent substitutions at 362 and 385 positions were observed in the ORF region of LIP2. Two single substitutions and two duplications of the T nucleotide were also detected in the promoter region. LIP2 sequence comparison of the Y. lipolytica DSM3286 and U6 strains shows good targets to effective DNA recombinant for extracellular lipase of Y. lipolytica.     

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.     

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.     

Yarrowia lipolytica lipase production enhanced by increased air pressure

Aims: To study the cellular growth and morphology of Yarrowia lipolytica W29 and its lipase and protease production under increased air pressures. Methods and Results: Batch cultures of the yeast were conducted in a pressurized bioreactor at 4 and 8 bar of air pressure and the cellular behaviour was compared with cultures at atmospheric pressure. No inhibition of cellular growth was observed by the increase of pressure. Moreover, the improvement of the oxygen transfer rate (OTR) from the gas to the culture medium by pressurization enhanced the extracellular lipase activity from 96·6 U l^?1 at 1 bar to 533·5 U l^?1 at 8 bar. The extracellular protease activity was reduced by the air pressure increase, thereby eliciting further lipase productivity. Cell morphology was slightly affected by pressure, particularly at 8 bar, where cells kept the predominant oval form but decreased in size. Conclusions: OTR improvement by total air pressure rise up to 8 bar in a bioreactor can be applied to the enhancement of lipase production by Y. lipolytica. Significance and Impact of the Study: Hyperbaric bioreactors can be successfully applied for yeast cells cultivation, particularly in high‐density cultures used for enzymes production, preventing oxygen limitation and consequently increasing overall productivity.     

Evaluating aqueous two-phase systems for Yarrowia lipolytica extracellular lipase purification

In this study an aqueous two-phase system (ATPS) composed of polyethylene glycol (PEG) and potassium phosphate was tested for the purification of lipase from Yarrowia lipolytica IMUFRJ 50682. Ultrafiltration and precipitation with acetone and kaolin were also used as traditional comparison methods Ultrafiltration was a good method with a purification factor of 6.55, but protease was also purified in this extract. For the precipitation with acetone and kaolin lower values of lipase and protease activity were found in relation to the original crude enzyme extract. Under the best conditions of ATPS (pH 6 and 4 °C), the purification fold was greater than 40 and selectivity was almost 500. Lipase was recovered in the salty phase which makes it easier to purify it. The optimum pH and temperature ranges for purified lipase with this system was 6–7 and 35–40 °C, respectively. Lipase thermostability was increased in relation to crude extract after the purification with the PEG/phosphate buffer system for temperatures lower than 50 °C. All enzyme extracts showed good stability to a wide pH range. Y. lipolytca lipase was successfully purified by using ATPS in a single downstream processing step and presented good process characteristics after this treatment.     

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.     

Analysis of Yarrowia lipolytica extracellular lipase Lip2p glycosylation

Wild-type (WT) Yarrowia lipolytica strain secretes a major extracellular lipase Lip2p which is glycosylated. In silico sequence analysis reveals the presence of two potential N-glycosylation sites (N113IS and N134NT). Strains expressing glycosylation mutant forms were constructed. Esterase activities for the different forms were measured with three substrates: p-nitrophenol butyrate (p-NPB), tributyrin and triolein. Sodium dodecyl sulfate polacrylamide gel electrophoresis analysis of supernatant indicated that the suppression of the two sites of N-glycosylation did not affect secretion. S115V or N134Q mutations led to lipase with similar specific activity compared with WT lipase while a T136V mutation reduced specific activity toward p-NPB and tributyrin. Electrospray ionization MS of the WT entire protein led to an average mass of 36 950 Da, higher than the mass deduced from the amino acid sequence (33 385 Da) and to the observation of at least two different mannose structures: Man(8)GlcNAc(2) and Man(9)GlcNAc(2). LC-tandem MS analysis of the WT Lip2p after trypsin and endoproteinase Asp-N treatments led to high coverage (87%) of protein sequence but the peptides containing N113 and N134 were not identified. We confirmed that the presence of N-glycosylation occurred at both N113 and N134 by MS of digested proteins obtained after enzymatic deglycosylation or from mutant forms.     

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.     

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.     

Purification and characterization of the extracellular lipase Lip2 from Yarrowia lipolytica

An extracellular lipase from Yarrowia lipolytica (YlLip2) has been purified by ion exchange chromatography on Q sepharose FF, followed by hydrophobic interaction chromatography on butyl sepharose FF. SDS-PAGE showed that the molecular weight of this lipase is about 38 kDa. N-terminal amino acid sequencing and MALDI-TOF mass spectral analysis showed that this lipase is encoded by gene LIP2 (GenBank accession no. AJ012632). Enzymatic deglycosylation showed that this lipase is a glycosylated protein which contains about 12% sugar. The corresponding deglycosylated lipase remained 88% specific activity of untreated lipase. There was a high amino acid sequence identity (91%) between YlLip2 and Candida deformans lipase CdLip1 (GenBank accession no. AJ428393). The optima temperature and pH for the purified lipase was 40 °C and 8.0, respectively. The lipase showed a preference for long chain fatty acid methyl esters (C12–C16), with the highest activity toward methyl myristate (C14). Lipase activity was stimulated by Ca²⁺ and Mg²⁺ and inhibited by Zn²⁺, Ni²⁺ and Cu²⁺, whereas EDTA had no effect on its activity. A 0.1% of Tween 80 and Span 65 increased slightly the enzyme activity and SDS inhibited it.     

Genetic control of extracellular protease synthesis in the yeast Yarrowia lipolytica

Depending on the pH of the growth medium, the yeast Yarrowia lipolytica secretes an acidic protease or an alkaline protease, the synthesis of which is also controlled by carbon, nitrogen, and sulfur availability, as well as by the presence of extracellular proteins. Previous results have indicated that the alkaline protease response to pH was dependent on YlRim101p, YlRim8p/YlPalF, and YlRim21p/YlPalH, three components of a conserved pH signaling pathway initially described in Aspergillus nidulans. To identify other partners of this response pathway, as well as pH-independent regulators of proteases, we searched for mutants that affect the expression of either or both acidic and alkaline proteases, using a YlmTn1-transposed genomic library. Four mutations affected only alkaline protease expression and identified the homolog of Saccharomyces cerevisiae SIN3. Eighty-nine mutations affected the expression of both proteases and identified 10 genes. Five of them define a conserved Rim pathway, which acts, as in other ascomycetes, by activating alkaline genes and repressing acidic genes at alkaline pH. Our results further suggest that in Y. lipolytica this pathway is active at acidic pH and is required for the expression of the acidic AXP1 gene. The five other genes are homologous to S. cerevisiae OPT1, SSY5, VPS28, NUP85, and MED4. YlOPT1 and YlSSY5 are not involved in pH sensing but define at least a second protease regulatory pathway.     

Heterologous protein expression and secretion in the non-conventional yeast Yarrowia lipolytica: a review

The production of heterologous proteins is a research field of high interest, with both academic and commercial applications. Yeasts offer a number of advantages as host systems, and, among them, Yarrowia lipolytica appears as one of the most attractive. This non-conventional dimorphic yeast exhibits a remarkable regularity of performance in the efficient secretion of various heterologous proteins. This review presents the main characteristics of Y. lipolytica, and the genetic and molecular tools available in this yeast. A particular emphasis is given to newly developed tools such as efficient promoters, a non-homologous integration method, and an amplification system using defective selection markers. A table recapitulates the 42 heterologous proteins produced until now in Y. lipolytica. A few relevant examples are exposed in more detail, in order to illustrate some peculiar points of the Y. lipolytica physiology, and to offer a comparison with other production systems. This amount of data demonstrates the global reliability and versatility of Y. lipolytica as a host for heterologous production.     

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.     

Carbon source impact on Yarrowia lipolytica KKP 379 lipase production

The production of lipases by microorganisms is strongly influenced by the culture conditions. The optimum culture conditions for enzyme production are strain- and species-dependent. The aim of this study was to evaluate the impact of the carbon source used in the culture medium on the profile of lipases produced by Yarrowia lipolytica KKP 379. We observed a different pattern of extracellular and cell-bound lipase production, which was the highest in the early exponential phase. The extracellular lipase activity increased in the late exponential phase due to the lower accumulation of lipase molecules in cell walls. The best carbon source for extracellular lipase production by Y. lipolytica KKP 379 was olive oil. Glucose, dodecane and olive oil had a positive effect on biomass yield. Dodecane and/or glycerol utilization in microbiological lipase production was possible, but this process could not proceed without the addition of some activators such as olive oil in the cultivation medium.     

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.     

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.     

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.     

Production of recombinant Rhizopus oryzae lipase by the yeast Yarrowia lipolytica results in increased enzymatic thermostability

The gene encoding Rhizopus oryzae lipase (ROL) was expressed in the non-conventional yeast Yarrowia lipolytica under the control of the strong inducible XPR2 gene promoter. The effects of three different preprosequence variants were examined: a preprosequence of the Y. lipolytica alkaline extracellular protease (AEP) encoded by XPR2, the native preprosequence of ROL, and a hybrid variant of the presequence of AEP and the prosequence of ROL. Lipase production was highest (7.6 U/mL) with the hybrid prepropeptide. The recombinant protein was purified by ion-exchange chromatography. The ROL included 28 amino acids of the C-terminal region of the prosequence, indicating that proteolytic cleavage occurred below the KR site through the activity of the Kex2-like endoprotease. The optimum temperature for recombinant lipase activity was between 30 and 40 °C, and the optimum pH was 7.5. The enzyme was shown not to be glycosylated. Furthermore, recombinant ROL exhibited greater thermostability than previously reported, with the enzyme retaining 64% of its hydrolytic activity after 30 min of incubation at 55 °C.     

Genomic organization of the yeast Yarrowia lipolytica

We produced electrophoretic karyotypes of the reference strain E150 and of seven other isolates from different geographical origins to study the genomic organization of the dimorphic yeast Yarrowia lipolytica. These karyotypes differed in the number and size of the chromosomal bands. The karyotype of the reference stain E150 consisted of five bands of between 2.6 and 4.9 Mb in size. This strain contained at least five rDNA clusters, from 190 to 620 kb in size, which were scattered over most of the chromosomes. The assignment of 43 markers, including rRNA genes and three centromeres, to the E150 bands defined five linkage groups. Hybridization to the karyotypes of other isolates with pools of markers of each linkage group showed that linkage groups I, II, IV and V were conserved in the strains tested whereas group III was not and was split between at least two chromosomes in most strains. Use of a meganuclease I-SceI site targeted to one locus of E150 linkage group III showed that two chromosomes actually comigrated in band III of this strain. Our results are compatible with six chromosomes defining the haploid complement of strains of Y. lipolytica and that, despite an unprecedented chromosome length polymorphism, the overall structure of the genome is conserved in different isolates. Received: 27 March 1997; in revised form: 8 July 1997 / Accepted: 9 July 1997