Steroid biotransformations in biphasic systems with Yarrowia lipolytica expressing human liver cytochrome P450 genes (original) (raw)
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Engineering in Life Sciences, 2019
The non‐conventional oleaginous yeast Yarrowia lipolytica is able to utilize both hydrophilic and hydrophobic carbon sources as substrates and convert them into value‐added bioproducts such as organic acids, extracellular proteins, wax esters, long‐chain diacids, fatty acid ethyl esters, carotenoids and omega‐3 fatty acids. Metabolic pathway analysis and previous research results show that hydrophobic substrates are potentially more preferred by Y. lipolytica than hydrophilic substrates to make high‐value products at higher productivity, titer, rate, and yield. Hence, Y. lipolytica is becoming an efficient and promising biomanufacturing platform due to its capabilities in biosynthesis of extracellular lipases and directly converting the extracellular triacylglycerol oils and fats into high‐value products. It is believed that the cell size and morphology of the Y. lipolytica is related to the cell growth, nutrient uptake, and product formation. Dimorphic Y. lipolytica demonstrates th...
Hydrophobic substrate utilisation by the yeast , and its potential applications
FEMS Yeast Research, 2005
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 b-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.
Integrative transformation of the yeast Yarrowia lipolytica
Current Genetics, 1985
We have derived a DNA-mediated transformation system for the yeast Yarrowia lipolytica based on the lithium acetate method developed for Saccharomyces cerevisiae. The first plasmid used, pLD25, contains the Y. lipolytica LEU2 gene (coding for the enzyme beta-isopropylmalate dehydrogenase, EC 1.1.1.85) on a 6.6 kb piece of DNA inserted into pBR322. The recipient strain ATCC 20688 contains the rarely reverting mutation leu2-35. The Y. lipolytica LEU2 gene complements leuB mutants in Escherichia coli and leu2 mutants in S. cerevisiae and it also hybridizes weakly to the S. cerevisiae LEU2 gene. Y. lipolytica transformation frequencies of up to 104 Leu + cells per microgram of DNA, per 108 viable cells have been obtained from plasmids linearized with restriction enzymes. The more than 100-fold increase in transformation frequency obtained by using linearized DNA instead of intact plasmid resembles the situation seen in S. cerevisiae for sitedirected integrative transformation (Orr-Weaver et al. 1981). The transformants were stable when grown in non-selective medium. We found that pLD25 integrated at the leu2 region when either linear or intact plasmid was used to transform Y. lipolytica.
Journal of Industrial Microbiology, 1992
Biotransformation ofbenzaldehyde and pyruvate to (R)-phenylacetyl carbinol by Saccharomyces cerevisiae was investigated in two-phase aqueous-organic reaction media. With hexane as organic solvent, maximum biotransformation activity was observed with a moisture content of 10~o. Of the organic solvents tested, highest biotransformation activities were observed with hexane and hexadecane, and lowest activities occurred with chloroform and toluene. Biocatalyst samples from biphasic media containing hexane, decane and toluene manifested no apparent cell structural damage when examined using scanning electron microscopy. In contrast, cellular biocatalyst recovered from two-phase systems containing chloroform, butylacetate and ethylacetate exhibited damage in the form of cell puncturing after different incubation periods. Phospholipids were detected in reaction media from biocatalytic systems which exhibited cell damage in electron micrographs. Phospholipid release was much lower in the two-phase systems containing toluene or hexane or in 100% aqueous biocatalytic system.
Food Bioengineering
The oleaginous yeast, Yarrowia lipolytica, has garnered much attention and is gaining recognition as a potential industrial platform for nutraceutical production. The superior properties of Y. lipolytica include its transparent inherited background, broad substrate spectrum, and superior environmental tolerance. Specifically, advances in genetic tools and engineering strategies have further widened its applications in metabolic engineering and biomanufacturing. In this review, we summarized developments in synthetic biology and engineering strategies for constructing Y. lipolytica to produce nutraceuticals. We also proposed perspectives on the limitations and challenges in developing microbial Y. lipolytica factories.
A membrane bioreactor for biotransformations of hydrophobic molecules
Biotechnology and Bioengineering, 1998
This work reports the application of organic solvent nanofiltration (OSN) membranes to a membrane bioreactor for biotransformations (MBB). An organic solvent phase was employed, allowing high substrate loadings and efficient product removal. The aqueous and organic phases were separated by an OSN membrane. The biotransformation of geraniol to R-citronellol by baker's yeast was used as the model reaction, and n-hexadecane and toluene as the organic solvents. The performance of the MBB was compared to that of a direct contact biphasic (DCB) bioreactor. The MBB system resulted in lower productivities than the DCB system due to mass transfer limitations. For the n-hexadecane system, the membrane was the main mass transfer resistance, whereas for the toluene system the contribution of the aqueous liquid film mass transfer resistance became predominant. Further investigations are needed to improve the substrate transfer rates. Despite this, the MBB system prevented aqueous breakthrough, and thus the formation of two-phase emulsions. Toluene toxicity to the biocatalyst was also minimized, although it caused a reduction in the reaction enantiospecificity. This work showed that OSN-MBB systems avoid the formation of emulsions, thus reducing downstream separation and allowing increased substrate loadings.
Yarrowia lipolytica as a Cell Factory for Oleochemical Biotechnology
Handbook of Hydrocarbon and Lipid Microbiology, 2010
The yeast Y. lipolytica has developed a very efficient mechanism in the degradation and utilization of hydrophobic substrates. In addition, its capacity to accumulate great amounts of lipids places it among the oleaginous yeasts. The completion of the sequence of the Y. lipolytica genome and the existence of genetic tools for gene manipulation allow the use of its metabolic functions for biotechnological applications. Examples are presented demonstrating that wild type and genetically engineered strains of Y. lipolytica can be used for fatty acid bioconversion, substrate valorization, polyunsaturated fatty acids production, oil polluted water bioremediation and single cell oil production. This review also presents the potential uses of this yeast as an alternative production (cell) factory in the oleochemical field.
Applied Microbiology and Biotechnology, 2005
Carvone has previously been found to highly inhibit its own production at concentrations above 50 mM during conversion of a diastereomeric mixture of (−)-carveol by whole cells of Rhodococcus erythropolis. Adaptation of the cells to the presence of increasing concentrations of carveol and carvone in n-dodecane prior to biotransformation proved successful in overcoming carvone inhibition. By adapting R. erythropolis cells for 197 h, an 8.3-fold increase in carvone production rate compared to nonadapted cells was achieved in an air-driven column reactor. After an incubation period of 268 h, a final carvone concentration of 1.03 M could be attained, together with high productivity [0.19 mg carvone h −1 (ml organic phase) −1 ] and high yield (0.96 g carvone g carveol −1 ).