Selective uptake of fatty acids by the yeast Yarrowia lipolytica

Yarrowia lipolytica, when cultivated on mixtures of free fatty acid substrates was found to remove C12:0, C14:0, ^Δ9C18:1, ^{Δ9, 12}C18:2 and ^{Δ9, 12, 15}C18:3 at significantly higher rates than C16:0 and C18:0, regardless of fatty acid composition of the initial substrate. C12:0, C14:0 and ^{Δ9, 12, 15}C18:3 were specifically and completely removed from the substrate, while ^Δ9C18:1 and ^{Δ9, 12}C18:2 concentrations decreased by 55‐80 wt‐% in comparison with initial concentrations. In contrast, concentration of C18:0 increased 2.1—3.5 fold in the substrate. Although C18:0 was removed slowly from the substrate, this fatty acid was selectively accumulated in the storage lipid. Inversely, only low quantities of ^Δ9C18:1 and ^{Δ9, 12}C18:2 and traces of C12:0, C14:0 and ^{Δ9, 12, 15}C18:3 were accumulated in the storage lipid. During storage lipid breakdown, cellular C16:0 and ^Δ9C18:1 were taken up more rapidly than C18:0. We concluded that the capability of Yarrowia lipolytica to selectively remove several fatty acids from the substrate and accumulate others could be used in modification of the composition of selected mixtures of fatty acids and probably of common fats, to produce “new” fats with a predetermined composition.     

The use of olive mill wastewater by wild type Yarrowia lipolytica strains: medium supplementation and surfactant presence effect

BACKGROUND: The aim of this work was to study the ability of two different wild type strains of the yeast Yarrowia lipolytica to grow on olive mill wastewater (OMW) and their potential to produce high‐value products such as lipases. Factors that affect cellular growth and OMW degradation were studied, such as nitrogen supplementation, cells concentration and surfactant addition. RESULTS: Both strains, W29 and IMUFRJ 50682, were able to grow in OMW with 19 g L^?1 of COD and approximately 800 mg L^?1 of total phenols concentration. The strain W29 presented the highest potential for extracellular lipase production in OMW medium. Lipase productivity was improved by the medium supplementation with ammonium sulphate up to 6 g L^?1, leading to 80% of COD degradation and 70% of total phenols reduction. The surfactant Tween 80 enhanced cell growth and COD degradation, but had a negative effect on lipase activity. CONCLUSIONS Y. lipolytica has a great potential for OMW valorisation by its use as culture medium for biomass and enzymes production. The operating conditions that favoured lipase production differ from the conditions that improve COD reduction. Copyright © 2008 Society of Chemical Industry     

Validated Growth Rate-Dependent Regulation of Lipid Metabolism in Yarrowia lipolytica

Given the strong potential of Yarrowia lipolytica to produce lipids for use as renewable fuels and oleochemicals, it is important to gain in-depth understanding of the molecular mechanism underlying its lipid accumulation. As cellular growth rate affects biomass lipid content, we performed a comparative proteomic analysis of Y. lipolytica grown in nitrogen-limited chemostat cultures at different dilution rates. After confirming the correlation between growth rate and lipid accumulation, we were able to identify various cellular functions and biological mechanisms involved in oleaginousness. Inspection of significantly up- and downregulated proteins revealed nonintuitive processes associated with lipid accumulation in this yeast. This included proteins related to endoplasmic reticulum (ER) stress, ER–plasma membrane tether proteins, and arginase. Genetic engineering of selected targets validated that some genes indeed affected lipid accumulation. They were able to increase lipid content and were complementary to other genetic engineering strategies to optimize lipid yield.     

Biosynthesis of lipids and organic acids by Yarrowia lipolytica strains cultivated on glucose

The biochemical behavior of wild‐type or genetically modified (presenting decreased expression of intracellular acyl‐CoA oxidases) Yarrowia lipolytica strains cultivated on commercial glucose was studied. Flask nitrogen‐limited cultures were performed favoring the production of organic acids (and potentially the accumulation of lipid). Nitrogen depletion induced secretion of citric acid, while intracellular lipid was not produced in high quantities. Maximum total citric acid up to 49 g/L (yield 0.85 g/g glucose) was produced. In some of the wild‐type strains, an increase of glucose in the medium also induced noticeable production of acetic acid. Increasing the amount of added glucose led to an increase in the total lipid quantity (%) produced, although in the stationary growth phase the concentration of lipid declined, indicating lipid degradation even for the genetically modified strains. Total lipid amount did not exceed the value of 14%, while neutral fractions increased with increase in glucose concentration. In all cases, the total microbial lipids and major lipid fractions were composed of C₁₆ and C₁₈ (principally unsaturated) fatty acids. Finally, in several of the strains cultured in media containing a low glucose concentration, unicellular morphology was observed, while at high glucose concentrations mycelia were predominant.     

Effect of lipids and surfactants on extracellular lipase production by Yarrowia lipolytica

The production of extracellular lipase in submerged cultures of Yarrowia lipolytica CECT 1240 has been investigated. Several compounds have been added to the culture medium, in order to assess their efficiency as inducers of lipase production. First, the effect of triglycerides (olive oil, sunflower oil, tributyrin) and fatty acids (oleic acid) has been studied. The highest activity level was obtained with sunflower oil (58 U cm^?3), followed by olive oil (49 U cm^?3). The cultures with tributyrin and oleic acid attained similar activities (33 U cm^?3). Then, several surfactants (Tween 80, Triton X‐100, gum arabic, polyethylene glycol 200) were added to the cultures with sunflower oil, in an attempt to increase the levels of extracellular lipase activity. The obtained activities were slightly lower than those achieved without surfactants. The assay of a wide range of surfactant concentrations in the case of PEG‐200 (with which the highest activity levels had been attained) did not improve the results. This strain secreted lipase concentrations two‐fold higher and showed significantly different behaviour towards the presence of surfactants in the culture medium, compared with other wild‐type Yarrowia lipolytica strains. Copyright © 2003 Society of Chemical Industry     

Biodegradation and utilization of waste cooking oil by Yarrowia lipolytica CECT 1240

Biodegradation of waste cooking oil and its application as lipase production inducer in cultures of Yarrowia lipolytica CECT 1240 have been investigated, both in shake flasks and a bench‐scale bioreactor. The ability of this strain to degrade the spent oil was evaluated by monitoring COD throughout the cultures, and a remarkable decrease was recorded (almost 90% decrease in oil COD after 3 days in bioreactor). Moreover, the addition of waste cooking oil to the medium led to a significant augmentation in extracellular lipase production by the yeast, compared to oil‐free cultures. This confirms the suitability of the studied residue as an inducer of lipase biosynthesis, which is a very interesting fact, from an economic standpoint. These results were confirmed when a fed‐batch strategy was proposed. Finally, some properties of the crude enzyme were studied, and compared to the enzymes obtained when non‐used oil was added to the medium. Practical application : New strategies to valorize wastes from the food and agro industries are attracting a great scientific interest due to the important advantages offered from an economic and environmental point of view. For this reason, the yeast Yarrowia lipolytica CECT 1240 is proposed for degrading waste cooking oils. This approach entails also another benefit in terms of lipolytic enzyme synthesis, since the addition of used up oils has a lipase inducer effect. The enormous interest in lipases is reflected in the number of applications that they present. The process was successfully carried out both in shake flasks and a bench‐scale bioreactor, allowing producing high levels of lipolytic activity at the same time that the COD was diminished up to nearly 90%.     

代谢工程改造解脂耶氏酵母生产脂肪酸及其衍生物

微生物来源的脂肪酸及其衍生物广泛应用于能源、材料和营养化学品等领域,可用于生产航空燃油、聚合物、增塑剂、润滑剂和食品添加剂等。解脂耶氏酵母是一种研究最为透彻的产油脂酵母,具有高产各种脂肪酸及其衍生物的潜力。本文综述了近年来解脂耶氏酵母遗传操作工具的发展,并介绍了通过代谢工程技术改造解脂耶氏酵母生产脂肪酸及其衍生物的进展,在此基础上,展望了通过构建解脂耶氏酵母细胞工厂合成特定脂肪酸及其衍生物的未来发展方向。     

Improvement of Yarrowia lipolytica Lipase Enantioselectivity by Using Mutagenesis Targeted to the Substrate Binding Site

Enhanced enantioselectivity : The resolution of 2‐bromo‐arylacetic acid esters by Lip2p lipase from Yarrowia lipolytica was improved through mutagenesis of the substrate binding site. Position 232 was identified as crucial for the discrimination. Saturation of this position led to the identification of variant V232S, which has a tremendously increased activity and E value as compared to the parental enzyme.

     

Evaluation of a Yarrowia lipolytica Strain Collection for Its Lipid and Carotenoid Production Capabilities

The oleaginous yeast Yarrowia lipolytica is capable of accumulating large amounts of fatty acids in the form of lipids which can serve as a platform polymer for a variety of applications. Additionally, through heterologous gene expression, Y. lipolytica is capable of producing carotenoids. Due to the observation that Y. lipolytica exhibits a high phenotypic inter‐strain variability, robotics and microwell scale cultivations are applied to examine 12 wild type strains of a Y. lipolytica collection. The strains are characterized with respect to their metabolic capabilities for fatty acid production as well as genetically manipulated to produce beta‐carotene. Furthermore, growth and production behavior of the strain collection at different temperatures as well as initial pH are assessed. A molecular discrimination between the strains is achieved by internal transcribed spacer (ITS)‐sequencing and polymerase chain reaction (PCR)‐based methods. The best performing strain with respect to lipid production produces ≈2% lipids per dry cell weight (DCW) and 8 mg g^?1 beta‐carotenoids in yeast nitrogen base (YNB) media. All strains show robust growth from 28 to 34 °C. Practical Applications: Unlocking nature's potential by screening natural isolates shows that even on inter‐strain level sufficient phenotypic diversity may arise. Automated growth‐based characterization of beta‐carotene‐producing strains in terms of media composition, effect of initial pH, and temperature tolerance shows that with modern cultivation techniques, rapid characterization of strain collections is feasible. Combining results of beta‐carotene and lipid formation could help to balance both pathways to improve the storage of hydrophobic compounds in the lipid droplets. The generalized findings could be further transferred to improve the production of any valuable compound, derived from the mevalonate pathway.     

Improving Lipid Production of Yarrowia lipolytica by the Aldehyde Dehydrogenase-Mediated Furfural Detoxification

Yarrowia lipolytica, the non-conventional yeast capable of high lipogenesis, is a microbial chassis for producing lipid-based biofuels and chemicals from renewable resources such as lignocellulosic biomass. However, the low tolerance of Y. lipolytica against furfural, a major inhibitory furan aldehyde derived from the pretreatment processes of lignocellulosic biomass, has restricted the efficient conversion of lignocellulosic hydrolysates. In this study, the furfural tolerance of Y. lipolytica has been improved by supporting its endogenous detoxification mechanism. Specifically, the endogenous genes encoding the aldehyde dehydrogenase family proteins were overexpressed in Y. lipolytica to support the conversion of furfural to furoic acid. Among them, YALI0E15400p (FALDH2) has shown the highest conversion rate of furfural to furoic acid and resulted in two-fold increased cell growth and lipid production in the presence of 0.4 g/L of furfural. To our knowledge, this is the first report to identify the native furfural detoxification mechanism and increase furfural resistance through rational engineering in Y. lipolytica. Overall, these results will improve the potential of Y. lipolytica to produce lipids and other value-added chemicals from a carbon-neutral feedstock of lignocellulosic biomass.     

Effect of Citrus essential oil addition upon growth and cellular lipids of Yarrowia lipolytica yeast

The chemical composition of the essential oil from fruits of the Greek citrus hybrid Citrus sinensis cv New Hall – Citrus aurantium was investigated by GC and GC-MS. Forty-four compounds were identified, representing 92.83% of the total oil. Limonene, myrcene, β-pinene, α-pinene and α-terpineol constituted the major components of the oil. The effect of the addition of this oil upon the behavior of Yarrowia lipolytica yeast was also investigated. The microorganism was aerobically grown on glucose and oil was added to the culture medium in various initial quantities (from 0.0 to 1.5 mL/L). The strain underwent significant inhibition exerted by the added essential oil; a decrease of the highest achieved biomass concentration, even if the oil was added to the culture medium at small concentrations (e.g. 0.3 mL/L), was observed. The more oil was added, the more the lag phase of the growth increased, while the biomass yield on glucose consumed decreased with the addition of the oil. When the essential oil was added to the medium, even at small concentrations, a significant increase of cellular medium-chain saturated fatty acids (12:0 and 14:0) resulting in an increase of saturated fatty acid content in the cellular lipids, was observed.     

The Role of Hexokinase and Hexose Transporters in Preferential Use of Glucose over Fructose and Downstream Metabolic Pathways in the Yeast Yarrowia lipolytica

The development of efficient bioprocesses requires inexpensive and renewable substrates. Molasses, a by-product of the sugar industry, contains mostly sucrose, a disaccharide composed of glucose and fructose, both easily absorbed by microorganisms. Yarrowia lipolytica, a platform for the production of various chemicals, can be engineered for sucrose utilization by heterologous invertase expression, yet the problem of preferential use of glucose over fructose remains, as fructose consumption begins only after glucose depletion what significantly extends the bioprocesses. We investigated the role of hexose transporters and hexokinase (native and fructophilic) in this preference. Analysis of growth profiles and kinetics of monosaccharide utilization has proven that the glucose preference in Y. lipolytica depends primarily on the affinity of native hexokinase for glucose. Interestingly, combined overexpression of either hexokinase with hexose transporters significantly accelerated citric acid biosynthesis and enhanced pentose phosphate pathway leading to secretion of polyols (31.5 g/L vs. no polyols in the control strain). So far, polyol biosynthesis was efficient in glycerol-containing media. Moreover, overexpression of fructophilic hexokinase in combination with hexose transporters not only shortened this process to 48 h (84 h for the medium with glycerol) but also allowed to obtain 23% more polyols (40 g/L) compared to the glycerol medium (32.5 g/L).     

A comprehensive study of lipase production by Yarrowia lipolytica CECT 1240 (ATCC 18942): from shake flask to continuous bioreactor

BACKGROUND: Lipases are commercially important enzymes, and the development and optimization of their production processes are of great interest. The diversity of behaviours between strains stresses the need for research on this topic, especially when bioreactor culture is considered. The study of a continuous operating mode is especially attractive, since very scarce information is available on its application to microbial lipases production. RESULTS: Lipase production in submerged cultures of Yarrowia lipolytica CECT 1240 (ATCC 18 942) has been investigated. Significant lipolytic activity (over 700 U dm^?3), mostly extracellular and membrane‐bound, was obtained in shake flasks using medium supplemented with olive oil. The culture was carried out in air‐lift and stirred tank bench‐scale bioreactors and the latter was selected. The influence of aeration and agitation rates was assessed in batch cultures, and agitation from 400–700 rpm and low aeration rates (i.e. 0.2 vvm) are recommended. Batch, fed‐batch and continuous operation were investigated, and regular enzyme production (up to 600 U dm^?3) was achieved with the latter. CONCLUSION: Lipase production by the selected strain was successfully carried out in shake flasks and bench‐scale bioreactors. After studying batch, fed‐batch and continuous processes, continuous culture in a stirred tank bioreactor was found best in terms of regular enzyme production, exceptionally good operational stability and good fitting of the results to mathematical models. Copyright © 2009 Society of Chemical Industry     

Production of Yarrowia lipolytica lipase LIP2 in Pichia pastoris using the nitrogen source‐regulated FLD1 promoter

BACKGROUND: Yarrowia lipolytica lipase LIP2 (YlLIP2) is an important industrial enzyme that has many potential applications. Although it has been successfully expressed in Pichia pastoris under the control of the AOX1 promoter (pAOX1), there have been many efforts to develop new alternative promoters to pAOX1 in order to avoid using methanol in the fermentation. Investigation of YlLIP2 production in P. pastoris using the formaldehyde dehydrogenase 1 promoter (pFLD1) is especially attractive, since little is known about its application in methanol‐free culture strategies. RESULTS: Three fed‐batch cultivations were performed to investigate the production of YlLIP2 in a pFLD1‐based system. When methanol was used as the fed‐batch feeding substrate, the maximum YlLIP2 activity obtained in a 10‐L bioreactor was 30 000 U mL^?1 after 143 h of culture, whereas the maximum YlLIP2 activity was further increased to 35 000 U mL^?1 by adopting a co‐induction strategy with methanol and methylamine as a mixed fed‐batch substrate. Furthermore, the maximum YlLIP2 activity reached 13 000 U mL^?1 after 80 h of cultivation in a methanol‐free culture. CONCLUSION: The expression levels of YlLIP2 in the pFLD1‐based system were comparable with those in a pAOX1‐based system. The results suggest that pFLD1 is an attractive alternative to pAOX1, and may make it feasible to induce high yields of protein expression. Copyright © 2011 Society of Chemical Industry     

A Novel Cre/lox-Based Genetic Tool for Repeated,Targeted and Markerless Gene Integration in Yarrowia lipolytica

The unconventional yeast Yarrowia lipolytica is extensively applied in bioproduction fields owing to its excellent metabolite and protein production ability. Nonetheless, utilization of this promising host is still restricted by the limited availability of precise and effective gene integration tools. In this study, a novel and efficient genetic tool was developed for targeted, repeated, and markerless gene integration based on Cre/lox site-specific recombination system. The developed tool required only a single selection marker and could completely excise the unnecessary sequences. A total of three plasmids were created and seven rounds of marker-free gene integration were examined in Y. lipolytica. All the integration efficiencies remained above 90%, and analysis of the protein production and growth characteristics of the engineered strains confirmed that genome modification via the novel genetic tool was feasible. Further work also confirmed that the genetic tool was effective for the integration of other genes, loci, and strains. Thus, this study significantly promotes the application of the Cre/lox system and presents a powerful tool for genome engineering in Y. lipolytica.     

On‐line prediction of crystallinity spatial distribution across polymer films using NIR spectral imaging and chemometrics methods

A rapid and non‐intrusive on‐line NIR imaging sensor is developed for monitoring spatio‐temporal crystallinity variations across the surface of polymer films. A multivariate image analysis and regression (MIA/MIR) approach is proposed and compared with standard NIR calibration techniques using averaged spectra or second order derivatives combined with PLS regression. Predictions of both the local and global crystallinity variations of HDPE, LDPE, and PP polymer samples were obtained with each approach. Our results show that small variations in crystallinity introduced by changes in cooling rates can be predicted within experimental errors. Crystallinity spatial distributions were also validated and found consistent with processing conditions.     

Preparation and characterization of organosilicon thin films for selective adhesion of Yarrowia lipolytica yeast cells

The adhesion of Yarrowia lipolytica and Saccharomyces cerevisiae on organosilicon thin films deposited on polycarbonate substrates was investigated through a series of adhesion tests in order to obtain a selective substrate for Y. lipolytica cell adhesion. Organosilicon thin films were prepared using atmospheric pressure surface barrier discharge. The surface was characterized by its total surface energy with its components calculated using the acid‐base theory. Assessment of adhesion via cell surface coverage was obtained by standard tests and also through image analysis of yeast cells on the organosilicon layers. The results show that it is possible to create organosilicon thin films for a selective adhesion of Y. lipolytica face to S. cerevisiae behaviour, for potential use in fixed bed biofilm reactors for industrial purposes involving enzyme‐based biotransformations. Copyright © 2007 Society of Chemical Industry