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     

Cheese yeasts

Numerous traditionally aged cheeses are surface ripened and develop a biofilm, known as the cheese rind, on their surfaces. The rind of such cheeses comprises a complex community of bacterial and fungal species that are jointly responsible for the typical characteristics of the various cheese varieties. Surface ripening starts directly after brining with the rapid colonization of the cheese surface by yeasts. The initially dominant yeasts are acid and salt-tolerant and are capable of metabolizing the lactate produced by the starter lactic acid bacteria and of producing NH₃ from amino acids. Both processes cause the pH of the cheese surface to rise dramatically. This so-called deacidification process enables the establishment of a salt-tolerant, Gram-positive bacterial community that is less acid-tolerant. Over the past decade, knowledge of yeast diversity in cheeses has increased considerably. The yeast species with the highest prevalence on surface-ripened cheeses are Debaryomyces hansenii and Geotrichum candidum, but up to 30 species can be found. In the cheese core, only lactose-fermenting yeasts, such as Kluyveromyces marxianus, are expected to grow. Yeasts are recognized as having an indispensable impact on the development of cheese flavour and texture because of their deacidifying, proteolytic, and/or lipolytic activity. Yeasts are used not only in the production of surface-ripened cheeses but also as adjunct cultures in the vat milk in order to modify ripening behaviour and flavour of the cheese. However, yeasts may also be responsible for spoilage of cheese, causing early blowing, off-flavour, brown discolouration, and other visible alterations of cheese.     

Magnetically responsive yeast cells: methods of preparation and applications

Magnetically modified yeast cells represent an interesting type of biocomposite material, applicable in various areas of bioanalysis, biotechnology and environmental technology. In this review, typical examples of magnetic modifications of yeast cells of the genera Saccharomyces, Kluyveromyces, Rhodotorula and Yarrowia are presented, as well as their possible applications as biocatalysts, active part of biosensors and biosorbents for the separation of organic xenobiotics, heavy metal ions and radionuclides. Copyright © 2014 John Wiley & Sons, Ltd.     

Tyrosine metabolism in pigment-forming Yarrowia lipolytica strains isolated from English and European speciality mould-ripened cheese exhibiting a brown discolouration defect

Yarrowia lipolytica s4fd was isolated from a UK-manufactured speciality mould-ripened cheese as the causative micro-organism of a brown surface discolouration spoilage defect. Comparative studies utilizing Y. lipolytica isolates indicated that inter-strain variations in pigment accumulation were attributable to differences in tyrosine uptake and metabolism to homogentisic acid. The range and activity profiles of proteolytic enzymes involved in tyrosine release and turnover in isolates that differed in pigment formation were, however, similar. Homogentisic acid formation was affected by physiological parameters, and approaches that may be developed by the cheesemaker to prevent spoilage losses associated with the development of the brown discolouration defect are discussed.     

Cloning of an epoxide hydrolase-encoding gene from Rhodotorula mucilaginosa and functional expression in Yarrowia lipolytica

Epoxide hydrolases (EHs), especially those of fungal origin, have the ability to catalyse the enantioselective hydrolysis of epoxides to their corresponding diols. Recombinant DNA technology has been used extensively to overproduce these catalysts for the efficient hydrolytic kinetic resolution of epoxides, which serve as high-value intermediates in the fine chemicals and pharmaceutical industries. Degenerate primers, based on data from available EH-encoding gene sequences, in conjunction with inverse PCR, were used to amplify the genomic EH-encoding gene from Rhodotorula mucilaginosa. The 2347 bp genomic sequence revealed a 1979 bp ORF containing nine introns. The cDNA sequence revealed an 1185 bp EH-encoding gene that translates into a 394 amino acid protein exhibiting low sequence homology towards the known EH proteins. The EH gene from R. mucilaginosa was functionally expressed in Yarrowia lipolytica using a constitutive integrative expression cassette. Whole-cell biotransformation of (2,3-epoxypropyl)benzene, using the recombinant EH, revealed activity and selectivity far superior to any other activity and selectivity reported in literature using wild-type organisms. The GenBank Accession No. for the R. mucilaginosa EH gene is AY627310.     

Glycerol valorization: New biotechnological routes

Biodiesel have drawn attention in the last decade as a renewable, biodegradable, and non-toxic fuel. Raw glycerol can become an important feedstock when biodiesel is applied on a large commercial scale. With the production of 10 kg of biodiesel from rapeseed oil, 1 kg of glycerol becomes available. Few microorganisms can be used for direct glycerol biovalorization. Yarrowia lipolytica is one of the most extensively studied “non-conventional” yeasts which is used as a model in the degradation study of hydrophobic substrates and in several other fields. Its affinity for hydrophobic compounds occurs because of the production of surface-active compounds and its differentiated cell wall. From glycerol, a hydrophobic compound easily assimilated by Y. lipolytica, it is possible to produce several substances of biotechnological importance, including biosurfactants and citric acid. Biosurfactants are potential candidates for many commercial applications in the petroleum, pharmaceutical, biomedical and food industrial processes. Citric acid has a broad use in the preparation of numerous industrial products and in many industrial areas, especially in food industry, which creates a large and ever-increasing demand for this chemical. Therefore, glycerol transformation by Y. lipolytica points to highly potential processes.     

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%.     

Citric acid,biomass and cellular lipid production by Yarrowia lipolytica strains cultivated on olive mill wastewater‐based media

BACKGROUND: Olive mill wastewaters (OMWs) are an important residue and several physico‐chemical and/or biotechnological methods have been proposed for their treatment. RESULTS: The ability of three Yarrowia lipolytica strains to grow on and convert glucose‐enriched OMWs into added‐value compounds in carbon‐ and nitrogen‐limited shake‐flask cultures was assessed. Remarkable decolorization (up to 63%) and non‐negligible removal of phenolic compounds (up to 34%, w/w) occurred. In nitrogen‐limited cultures, the accumulation of cellular lipids was favored by OMW addition into the medium. In contrast, although remarkable quantities of citric acid (Cit) were produced in control experiments (cultures without OMW addition), in which Cit up to 18.9 g L^?1 was produced with yield of Cit synthesized per sugar consumed ～0.73 g g^?1), adaptation of cultures to media supplemented with OMWs reduced the final Cit quantity and conversion yield values achieved. In OMW‐based media, the highest concentration of citric acid produced was 18.1 g L^?1, with conversion yield ～0.51 g g^?1. In carbon‐limited cultures, despite the presence of inhibitory compounds in the medium (e.g. phenols), biomass production was enhanced with the addition of OMWs. The highest biomass concentration achieved was 12.7 g L^?1, with biomass conversion yield per sugar consumed ～0.45 g g^?1. Fatty acid analysis of cellular lipid produced demonstrated that adaptation of all strains in OMW‐based media favored the synthesis of cellular lipids that contained increased concentrations of cellular oleic acid. CONCLUSIONS: The Y. lipolytica strains tested can be regarded as possible candidates for simultaneous OMWs remediation and production of added‐value compounds. Copyright © 2011 Society of Chemical Industry     

田口设计优化耶氏酵母生产γ-癸内酯

通过田口设计对产γ-癸内酯的培养基成分进行优化。根据单因素实验结果,利用Minitab软件设计正交实验,采用田口设计方法分析正交实验中各因素水平的均值情况和信噪比情况。实验结果表明,复合氮源即酵母膏和(NH4)2HPO4对γ-癸内酯产量影响极显著,MgSO.47H2O对γ-癸内酯产量影响显著。软件预测培养基的最优组合为麸皮20g/L、酵母膏7.5g/L、(NH4)2HPO416.5g/L、KH2PO46g/L、MgSO4.7H2O0.5g/L,预测产量为2.49g/L,信噪比为9.04dB,经验证γ-癸内酯产量为2.65g/L,信噪比为9.68dB,优化方案达到了预期效果,具有较好的稳定性。