Production of carboxylic acids by metabolically engineered Yarrowia lipolytica: a review / 生物工程学报

Yarrowia lipolytica is a non-conventional yeast with unique physiological and metabolic characteristics. It is suitable for production of various products due to its natural ability to utilize a variety of inexpensive carbon sources, excellent tolerance to low pH, and strong ability to secrete metabolites. Currently, Y. lipolytica has been demonstrated to produce a wide range of carboxylic acids with high efficiency. This article summarized the progress in engineering Y. lipolytica to produce various carboxylic acids by using metabolic engineering and synthetic biology approaches. The current bottlenecks and solutions for high-level production of carboxylic acids by engineered Y. lipolytica were also discussed, with the aim to provide useful information for relevant studies in this field.

The recent advances in developing gene editing and expression tools and the synthesis of natural products in Yarrowia lipolytica / 生物工程学报

Yarrowia lipolytica, as an important oleaginous yeast, has been widely used in metabolic engineering. Y. lipolytica is considered as an ideal host for the production of natural products such as terpenes, polyketides and flavonoids, due to its ability to utilize a variety of hydrophobic substrates, high stress tolerance to acid and salt, high flux of tricarboxylic acid cycle and the ability in providing abundant the common precursor acetyl-CoA. Recently, more and more tools for genetic editing, gene expression and regulation has been developed in Y. lipolytica, which facilitate the metabolic engineering of Y. lipolytica for bio-manufacturing. In this review, we summarized the recent progresses in developing gene expression and natural product synthesis in Y. lipolytica, and also discussed the challenges and possible solutions in heterologous synthesis of natural products in this yeast.

Butter whey and corn steep liquor as sole raw materials to obtain a bioemulsifier from Yarrowia lipolytica for food oil-in-water emulsions / Soro de manteiga e milhocina como únicas matérias primas para obter bioemulsificante de Yarrowia lipolytica para emulsões óleo-em-água alimentícias

ABSTRACT: A synthetic medium containing glucose, glycerol, yeast extract (YE), and ammonium sulfate (AS) was compared to several low-cost media in their ability to produce high emulsification index (EI). The goal was to reduce the production costs of an emulsifier with application in food oil-in-water emulsions. To this end, agro-industrial by-products were screened for bioemulsifier production from Yarrowia lipolytica. The statistical analysis showed that the EIs of media containing residual frying oil from palm oil (RFO_palm) or soybean oil (RFO_soy), residual liquid from butter production (butter whey, BWhey) or cheese production (cheese whey, CWhey), supplemented with YE and AS were similar to the EI of the synthetic medium. The replacement of YE by corn steep liquor (CSL) also resulted in similar EI, except for RFO_soy. BWhey was tested with CSL without AS and similar EI (66.8%) was detected in comparison to that of the same medium with AS (66.3%). The cell-free broth obtained after Y. lipolytica growth in BWhey+CSL was successfully used to obtain vegetable oil-in-water emulsions indicating its potential application in food products.

RESUMO: O meio sintético contendo glicose, glicerol, extrato de levedura (YE) e sulfato de amônio (AS) foi comparado a meios de baixo custo para produzir alto índice de emulsificação (EI). Para este fim, os subprodutos agroindustriais foram rastreados quanto à produção de bioemulsificante por Yarrowia lipolytica. A análise estatística mostrou que EI de meios contendo óleo de fritura residual de óleo de palma (RFO_palm) ou óleo de soja (RFO_soy), líquido residual da produção de manteiga (soro de manteiga, BWhey) ou da produção de queijo (soro de queijo, CWhey), suplementado com YE e AS foram semelhantes ao EI do meio sintético. A substituição do YE por milhocina (CSL) também resultou em EI semelhante, exceto no RFO_soy. O BWhey foi testado com CSL sem AS e EI semelhante (66,8%) foi detectado em comparação com o mesmo meio com AS (66,3%). O meio isento de células obtido após o crescimento de Y. lipolytica no meio BWhey + CSL foi utilizado com sucesso para obter emulsões de óleo vegetal em água, indicando sua potencial aplicação em produtos alimentícios.

Enhanced pyruvic acid yield in an osmotic stress-resistant mutant of Yarrowia lipolytica

BACKGROUND: Pyruvic acid (PA), a vital α-oxocarboxylic acid, plays an important role in energy and carbon metabolism. The oleaginous yeast Yarrowia lipolytica (Y. lipolytica) has considerable potential for the production of PA. An increased NaCl concentration reportedly increases the biomass and PA yield of Y. lipolytica. RESULTS: To increase the yield of PA, the NaCl-tolerant Y. lipolytica A4 mutant was produced using the atmospheric and room temperature plasma method of mutation. The A4 mutant showed growth on medium containing 160 g/L NaCl. The PA yield of the A4 mutant reached 97.2 g/L at 120 h (0.795 g/g glycerol) in a 20-L fermenter with glycerol as the sole carbon source, which was 28.9% higher than that of the parental strain. CONCLUSION: The PA yield from Y. lipolytica can be improved by increasing its NaCl tolerance.

Green (Detox) juice physicochemical properties and stabilization effect of naturals emulsifiers / Propriedades físico-químicas do suco verde (detox) e efeito de estabilização de emulsificantes naturais

ABSTRACT: Green or "detox" juice is a mixture of fruit juice with vegetables, which has been used intensively by consumers seeking for healthy food. Physicochemical properties of Green juice were accessed in the present research, which brings new insights for the use of this beverage in human diet. A total phenolic content of 2833.60 mg GAE (Gallic acid equivalent)/ g of juice and a Total Antioxidant Capacity by FRAP of 323.62 µM Fe2SO4 / g of juice and by ABTS•+ of 333.11 µM Trolox/ g of juice, indicated good antioxidant properties. Low energy and reducing sugar content indicate its use for low calorie diet, but low carbohydrate and protein content prove that Green juice cannot be used as meal replacement. The addition of a microbial biosurfactant (YlBio) and chia gel as bioemulsifiers was tested in the Green juice formulation to reduce solid decantation and increase consistency. YlBio and chia gel were able to change the Newtonian behavior of the Green juice to a Pseudoplastic behavior due to stabilization properties and also increase consistency, without the need to add synthetic stabilizers.

RESUMO: O suco verde ou "detox" é uma mistura de suco de frutas com vegetais que tem sido intensamente utilizado por consumidores que buscam alimentos saudáveis. As propriedades físico-químicas do suco verde foram avaliadas no presente trabalho, o que traz novas perspectivas para o uso dessa bebida na dieta humana. Um conteúdo fenólico total de 2833,60 mg de EAG (equivalente em ácido gálico) / g de suco) e uma capacidade antioxidante total por FRAP de 323,62 µM de Fe2SO4 / g de suco e por ABTS•+ de 333,11 µM de Trolox / g de suco, indicam boas propriedades antioxidantes. Um baixo teor de energia e açúcar redutor indica seu uso em dietas de baixa caloria, mas o baixo teor de carboidratos e proteínas prova que o suco verde não pode ser usado como substituto de refeição. A adição de um biossurfactante microbiano (YlBio) e do gel de chia no suco foi testada na formulação do suco verde, para reduzir a decantação de sólidos e aumentar a consistência. YlBio e o gel de chia foram capazes de mudar o comportamento do suco de fluido Newtoniano para um fluido pseudoplástico devido às propriedades estabilizantes, e também aumentaram a consistência do suco, sem a necessidade de adição de estabilizantes sintéticos.

An efficient method of Yarrowia lipolytica immobilization using oil- and emulsion-modified bacterial cellulose carriers

Background: Yarrowia lipolytica is a nonconventional, dimorphic yeast with multiple biotechnological applications. Considering the size of Y. lipolytica cells and a plethora of its morphological forms (spherical cells or hyphae and pseudohyphae), it is highly difficult to select a suitable carrier for this useful microorganism. Bacterial cellulose (BC) is currently considered one of the most promising immobilization carriers. In the current study, the usefulness of oil- and emulsion-modified BCs as a carrier for Y. lipolytica immobilization was investigated. Static and agitated cultures were conducted in media supplemented with oil or emulsion to improve carrier porosity. Results: It was found that the application of oil- and emulsion-modified BCs correlated with significantly higher efficiency of Y. lipolytica immobilization and hence higher yield than the yield achieved with an unmodified carrier. Increased efficiency of immobilization correlated with BC porosity-related parameters, which, in turn, depended on the size of oil droplets introduced into the culture medium. Moreover, changes in porosity-related parameters caused by the addition of oil or emulsion to the medium were observed when the cultures were conducted only under static conditions and not under agitated conditions. Conclusion: The application of oil- and emulsion-modified BCs as carriers significantly increased the efficiency of Y. lipolytica immobilization as compared to unmodified BC. The addition of oil or emulsion to the culture medium can be a simple but effective method to modify the porosity of BC-based carriers.

One step production of isomalto-oligosaccharides by engineered Yarrowia lipolytica yeast co-displayed β-amylase and α-transglucosidase / 生物工程学报

Isomalto-oligosaccharides (IMO) have good physiochemical properties and excellent physiological functions to make it widely used in food, medicine, feed, cosmetics and other industries. However, the procedures for industrial production of IMO are complicated. Therefore, it is necessary to develop an economical and easy-to-operate method. The genes encoding for β-amylase and α-transglucosidase were fused and co-displayed on the yeast cell surface of Yarrowia lipolytica which can convert liquefied starch to IMO in one step. The highest IMO purity of 75.3% was obtained using the displayed fusion-enzyme at 50 °C. This method showed potential application in IMO production.

Control of acetic acid metabolism of recombinant Yarrowia lipolytica for efficient succinic acid production / 生物工程学报

Succinic acid is a high value-added organic acid widely used in food, chemical and pesticide industries. As a new robust non-conventional yeast, Yarrowia lipolytica attracts more and more attention due to its potential for industrial applications. Previously, we obtained a succinic acid-producing strain through gene deletion of succinic acid dehydrogenase subunit encoding gene Ylsdh5, resulting in the strain of PGC01003. However, the recombinant strain produced large amount of acetic acid due to imbalance between glycolysis and TCA cycle which hindered the efficient production of succinic acid. PDH bypass was interfered to decrease the overflow of acetic acid and produce succinic acid under natural pH. Acetic acid was reduced to 4.6 g/L through heterologous expression of acetyl coenzyme A synthase from Salmonella enteric, which was 75.4% of the control strain. Deletion of CoA-transferase gene Ylach1 eliminated acetate formation and improved succinic acid production, and the resulting strain produced as high as 7.0 g/L succinic acid. Our study provides foundation for further construction of efficient cell factory of succinic acid production.

Enhanced production of erythritol and mannitol by Yarrowia lipolytica in media containing surfactants

Abstract Various chemical compounds, including surfactants, when introduced to culture media may increase the permeability of cellular membranes and thereby affect the quantity of metabolites excreted by cells. The aim of the present study was to evaluate the impact of detergents including Triton X-100, Span 20 and Tween 80 on erythritol production from glycerol by Yarrowia lipolytica Wratislavia K1 in a shake-flask experiment, batch and fed-batch cultures. When Span 20 was added to a fed-batch culture with glycerol as a carbon source (300 g L-1), erythritol production increased by 15% compared to the culture without the surfactant where it reached 142 g L-1 after 5 days, which corresponded to 0.47 g g-1 yield and productivity of 1.1 g L-1 h-1. Therefore, it was concluded that Span 20 considerably enhanced the production of this polyol from glycerol.

Electrostatic and Water Content Effects on Yarrowia lipolytica Lipase Immobilization by Adsorption.

Aims: In the present investigation, an attempt has been made to explain lipase immobilization by adsorption on three minerals matrixes, i.e. Celite 545, Silica gel (60G) and Avicel (PH 101). Study Design: immobilization by absorption on minerals matrixes, water content by volumetric karl Fischer titration and surface potentials using a particle charge detector Mutek PCD 03 were used. Place and Duration of Study: Walloon Centre of Industrial Biology (CWBI) Unit of Bio- Industries, University of Liege, Gembloux Agro-Bio Tech, Passage des Deportes 2, B- 5030 Gembloux, Belgium between Jun 2012 and jun 2013. Methodology: A methodical order was developed whereby the influences of water content, surface potentials and pH, on immobilization by adsorption were explored. Adsorbed YLL was used to understand an interesterification reaction between rapeseed oil and milk fat in comparison with a commercial silica-granulated Thermomyces lanuginosus lipase (Lipozyme TL IM). Results: Maximum immobilization yield was obtained with Celite (70%) and the lowest with silica gel (29%). Total water content of free and immobilized lipase was determined by volumetric Karl Fischer titration. The water content of Silica gel was higher than the one of other supports. Water content of silica gel could prevent the enzyme fixation. These results could be explained by the adsorption being governed mainly by electrostatic interactions between the enzyme and matrix. This hypothesis was further reinforced by measurements of electrical potential. They showed a lowest negative potential of Silica gel after enzyme adsorption in comparison to Celite. Conclusion: From these results celite was designated as an efficient matrix to immobilize Yarrowia lipolytica lipase (YLL) by adsorption. This performed system was used to realize an interesterification reaction between rapeseed oil and milk fat in comparison with a commercial silica-granulated Thermomyces lanuginosus lipase (Lipozyme TL IM).

Bioconversion of R-(+)-limonene to perillic acid by the yeast Yarrowia lipolytica

Perillyl derivatives are increasingly important due to their flavouring and antimicrobial properties as well as their potential as anticancer agents. These terpenoid species, which are present in limited amounts in plants, may be obtained via bioconversion of selected monoterpene hydrocarbons. In this study, seventeen yeast strains were screened for their ability to oxidize the exocyclic methyl group in the p-menthene moiety of limonene into perillic acid. Of the yeast tested, the highest efficiency was observed for Yarrowia lipolytica ATCC 18942. The conversion of R (+)-limonene by Y. lipolytica was evaluated by varying the pH (3 to 8) and the temperature (25 to 30 ºC) in a reaction medium containing 0.5% v/v limonene and 10 gµL of stationary phase cells (dry weight). The best results, corresponding to 564 mgµL of perillic acid, were obtained in buffered medium at pH 7.1 that was incubated at 25 ºC for 48 h. The stepwise addition of limonene increased the perillic acid concentration by over 50%, reaching 855 mgµL, whereas the addition of glucose or surfactant to the reaction medium did not improve the bioconversion process. The use of Y. lipolytica showed promise for ease of further downstream processing, as perillic acid was the sole oxidised product of the bioconversion reaction. Moreover, bioprocesses using safe and easy to cultivate yeast cells have been favoured in industry.

Use of Plackett-Burman design for rapid screening of nitrogen and carbon sources for the production of lipase in solid state fermentation by Yarrowia lipolytica from mustard oil cake (Brassica napus)

Mustard oil cake (Brassica napus), the residue obtained after extraction of mustard oil from mustard oil seeds, was investigated for the production of lipase under solid state fermentation (SSF) using the marine yeast Yarrowia lipolytica NCIM 3589. Process parameters such as incubation time, biomass concentration, initial moisture content, carbon source concentration and nitrogen source concentration of the medium were optimized. Screening of ten nitrogen and five carbon sources has been accomplished with the help of Plackett-Burman design. The highest lipase activity of 57.89 units per gram of dry fermented substrate (U/gds) was observed with the substrate of mustard oil cake in four days of fermentation.

Factors influencing crude oil biodegradation by Yarrowia lipolytica

Yarrowia lipolytica is unique strictly aerobic yeast with the ability to efficiently degrade hydrophobic substrates such as n-alkenes, fatty acids, glycerol and oils. In the present work, a 2(4) full factorial design was used to investigate the influence of the independent variables of temperature, agitation, initial cell concentration and initial petroleum concentration on crude oil biodegradation. The results showed that all variables studied had significant effects on the biodegradation process. Temperature, agitation speed and initial cell concentration had positive effects, and initial petroleum concentration had a negative effect. Among the crude oil removal conditions studied, the best temperature and agitation conditions were 28ºC and 250 rpm, respectively.

Evaluation of the composition of culture medium for yeast biomass production using raw glycerol from biodiesel synthesis

The work herewith investigated the production of yeast biomass as a source of protein, using Yarrowia lipolytica NRRL YB-423 and raw glycerol from biodiesel synthesis as the main carbon source. A significant influence of glycerol concentration, initial pH and yeast extract concentration on biomass and protein content was observed according to the 2v5-1 fractional design. These factors were further evaluated using a central composite design and response surface methodology, and an empirical model for protein content was established and validated. The biomass of Yarrowia lipolytica NRRL YB-423 reached 19.5 ± 1.0 g/L in shaken flasks cultivation, with a protein content of 20.1 ± 0.6% (w/w).

Isolation, Identification and Characterization of a Antidementia Acetylcholinesterase Inhibitor-Producing Yarrowia lipolytica S-3

This report describes the isolation and identification of a potent acetylcholinesterase (AChE) inhibitor-producing yeasts. Of 731 species of yeast strain, the S-3 strain was selected as a potent producer of AChE inhibitor. The selected S-3 strain was investigated for its microbiological characteristics. The S-3 strain was found to be short-oval yeast that did not form an ascospore. The strain formed a pseudomycelium and grew in yeast malt medium containing 50% glucose and 10% ethanol. Finally, the S-3 strain was identified by its physiological characteristics and 26S ribosomal DNA sequences as Yarrowia lipolytica S-3.

Optimization of biomass production of a mutant of Yarrowia lipolytica with an increased lipase activity using raw glycerol

The yeast Yarrowia lipolytica accumulates oils and is able to produce extracellular lipases when growing in different carbon sources including glycerol, the principal by-product of the biodiesel industry. In this study, biomass production of a novel mutant strain of Y. lipolytica was statistically optimized by Response Surface Methodology in media containing biodiesel-derived glycerol as main carbon source. This strain exhibited distinctive morphological and fatty acid profile characteristics, and showed an increased extracellular lipase activity. An organic source of nitrogen and the addition of 1.0 g/l olive oil were necessary for significant lipase production. Plackett-Burman and Central Composite Statistical Designs were employed for screening and optimization of fermentation in shaken flasks cultures, and the maximum values obtained were 16.1 g/l for biomass and 12.2 Units/ml for lipase, respectively. Optimized batch bioprocess was thereafter scaled in aerated bioreactors and the values reached for lipase specific activity after 95 % of the glycerol had been consumed, were three-fold higher than those obtained in shaken flasks cultures. A sustainable bioprocess to obtain biomass and extracellular lipase activity was attained by maximizing the use of the by-products of biodiesel industry.

Optimización de la producción de biomasa usando glicerol crudo, de una cepa mutante de Yarrowia lipolytica con actividad incrementada de lipasa. La levadura Yarrowia lipolytica acumula aceites y produce una lipasa extracelular al crecer en diferentes fuentes de carbono, entre ellas el glicerol, principal subproducto de la creciente industria del biodiésel. En el presente trabajo, se optimizó mediante la metodología de superficies de respuesta la producción de biomasa de una nueva cepa mutante de Y. lipolytica, empleando medios con glicerol derivado de la industria del biodiésel como principal fuente de carbono. Esta cepa presentó características morfológicas y perfil de ácidos grasos distintivos, y una mayor actividad de lipasa extracelular. Para obtener una producción significativa de lipasa extracelular, fue necesario el agregado de una fuente orgánica de nitrógeno y de 1 g/l de aceite de oliva. Se utilizaron los diseños estadísticos de Plackett-Burman y central compuesto para la selección y la optimización de las fermentaciones en frascos agitados; los máximos valores de biomasa y de lipasa obtenidos fueron de 16,1 g/l y 12,2 unidades/ml, respectivamente. Luego, el bioproceso en lote optimizado se escaló a biorreactores aireados, y los valores de actividad específica de lipasa alcanzados después de haberse consumido el 95 % del glicerol fueron tres veces más altos que los obtenidos en los cultivos en frascos agitados. En suma, se desarrolló un bioproceso sostenible para la obtención de biomasa y de una actividad de lipasa extracelular, que a la vez maximiza el uso de subproductos de la industria del biodiésel.

Valorization of raw glycerol for citric acid production by Yarrowia lipolytica yeast

In the present report, citric acid production from raw glycerol in two fed-batch systems by acetate negative-mutants of Yarrowia lipolytica: Wratislavia 1.31 and Wratislavia AWG7 was compared. In the system, in which the total glycerol concentration was 200 g∙L-1, the substrate was added by pulsed additions, and in the other, in which the total glycerol concentration was 300 g∙L-1, the substrate was added at a constant feeding rate of 1.4 g∙h-1. Despite high citric acid concentrations (155.2 and 157.5 g∙L-1 with Y. lipolytica Wratislavia 1.31 and Y. lipolytica Wratislavia AWG7, respectively) obtained from 300 g∙L-1 of glycerol, the yield of citric acid was similar, i.e. about 0.6 g∙g-1. The volumetric citric acid productivity was markedly higher (1.05 and 0.94 g∙L-1h-1 with Y. lipolytica Wratislavia 1.31 and Y. lipolytica Wratislavia AWG7 strains, respectively) in the cultures containing 200 g∙L-1 of carbon source.

Properties of alkaline protease genetically engineered on cell surface of the yeast Yarrowia lipolytica.

ALP2 gene encoding alkaline protease cloned from Aureobasidium pullulans HN2-3 was ligated into the surface display plasmid and expressed in the cells of the yeast Yarrowia lipolytica. The expressed alkaline protease was immobilized on the yeast cells. The activity of the immobilized enzyme with 6 His tag was found to be significantly higher than that of without 6 His tag. The immobilized enzyme showed lower optimal temperature and a lower affinity for azocasein than the free enzyme purified from A. pullulans HN2-3. The thermal stability of the immobilized enzyme enhanced and the pH stability decreased, compared to that of the free enzyme.

Production of the Acetylcholinesterase Inhibitor from Yarrowia lipolytica S-3

The acetylcholinesterase (AChE) inhibitor of Yarrowia lipolytica S-3 was maximally produced when it was incubated at 30degrees C for 36 h in an optimal medium containing 1% yeast extract, 2% peptone and 2% glucose, with an initial pH 6.0. The final AChE inhibitory activity under these conditions was an IC50 value of 64 mg/ml. After partial purification of the AChE inhibitor by means of systematic solvent extraction, the final IC50 value of the partially purified AChE inhibitor was 0.75 mg/ml. We prepared a test product by using the partially purified AChE inhibitor and then determined its stability for the development of a new antidementia commercial product. The test product was stable at room temperature for 15 weeks.

A case of Yarrowia lipolytica fungemia after raw beef ingestion / 대한내과학회지

Candida lipolytica is also called Yarrowia lipolytica, and this is now microbiologically classified as a member of the Yarrowia genus. Y. lipolytica is a rare opportunistic pathogen that was first isolated in 1976 from a patient with traumatic ocular candidiasis. Five cases of infant patients were reported in 2000 in Korea for the first time, and then in 2001 a case was reported from an AML patient. No more cases have been reported since then. The authors experienced the case of a patient with blood culture positivity for Y. lipolytica and this patient had a previous history of raw beef ingestion and long term antibiotics therapy. Thus, we report on this case along with a review of the literature.