116 results about "Yarrowia sp." patented technology

Engineered strains of the oleaginous yeast Yarrowia lipolytica capable of producing greater than 10% arachidonic acid (ARA, an ω-6 polyunsaturated fatty acid) in the total oil fraction are described. These strains comprise various chimeric genes expressing heterologous desaturases, elongases and acyltransferases, and optionally comprise various native desaturase and acyltransferase knockouts to enable synthesis and high accumulation of ARA. Production host cells are claimed, as are methods for producing ARA within said host cells.

Cytochrome b5 (Cb5) is a haem-binding protein located in the endoplasmic reticulum (ER) and the outer mitochondrial membranes of higher eukaryotes. In higher plants, animals, and fungi, the ER resident Cb5 has been shown to play a role in desaturation of acyl CoA fatty acids. Higher plants Cb5 isoforms from plants such as soybean or Arabidopsis are capable of modulating omega-3 desaturation. Co-expression of certain Cb5 isoforms with FAD3 in a host plant results in increased production of seed oil content as well as altered ratio between different fatty acids. It is also disclosed here that overexpression of Yarrowia ACL enzymes in the plastids of a host plant helps boost the synthesis of acetyl CoA, which in turn, may lead to increased synthesis of fatty acids and enhanced oil accumulation in the seeds.

The regulatory sequences associated with the Yarrowia lipolytica glyceraldehyde-3-phosphate dehydrogenase (gpd) and phosphoglycerate mutase (gpm) genes have been found to be particularly effective for the expression of heterologous genes in oleaginous yeast. The promoter regions of the invention, intron and enhancer have been shown to drive high-level expression of genes involved in the production of ω-3 and ω-6 fatty acids.

Two acyltransferases are provided, suitable for use in the manufacture of microbial oils enriched in omega fatty acids in oleaginous yeast (e.g., Yarrowia lipolytica). Specifically, the genes encoding phophatidylcholine-diacylglycerol acyltransferase (PDAT) and diacylglycerol acyltransferase (DGAT2) have been isolated from Y. lipolytica. These genes encode enzymes that participate in the terminal step in oil biosynthesis in yeast. Each is expected to play a key role in altering the quantity of polyunsaturated fatty acids produced in oils of oleaginous yeasts.

The present invention relates to methods for the production of γ-linolenic acid (GLA) in oleaginous yeast. Thus, Δ12 and Δ6 desaturases able to catalyze the conversion of oleic acid to GLA have been introduced into the genome of Yarrowia, using zeta-directed integration. Transformed strains produced over 25% GLA in the total lipids, as opposed to wildtype Yarrowia that is unable to synthesize any GLA. Metabolic engineering and fermentation methods are provided to further enhance GLA productivity in oleaginous yeast.

This invention provides microbial organisms, particularly yeasts such as Yarrowia lipolytica, that have one or more disrupted genes. The gene disruption(s) may yield improved production of fatty acyl-CoA derivatives.

The invention discloses a mutant of glutamine transaminase expressed by an active form, and belongs to the field of gene engineering and fermentation engineering. A genetically engineered bacterium po1h / hpro-mTG of high-output glutamine transaminase is structured by using yarrowia lipolytica as a host. The bacterial strain is high in enzyme production level; the fermenting enzyme activity of a shake flask is up to 11.7 U / mL, and improved by 106 times in comparison to that before transformation; the fermenting enzyme activity of a fermenting tank is up to 43.7 U / mL. Through co-expressing proteases TAMEP and hpro-mTG, the activity expression of glutamine transaminase is realized; the fermenting enzyme activity of the shake flask can reach 6.7U / mL, and the fermenting enzyme activity of the fermenting tank can reach 21.4U / mL. The fermenting enzyme production level of the recombinant bacteria is high, the production cost of the glutamine transaminase is reduced; the mutant is good for the industrial production of the glutamine transaminase.

Methods of increasing the total lipid content in a eukaryotic cell, the total content of polyunsaturated fatty acids [“PUFAs”], and / or the ratio of desaturated fatty acids to saturated fatty acids by reducing the activity of the heterotrimeric SNF1 protein kinase are disclosed. Preferably, the chromosomal genes encoding the Snf1 α-subunit, Gal83 β-subunit or Snf4 γ-subunit of the SNF1 protein kinase, the upstream regulatory genes encoding Sak1, Hxk2, Glk1 or Reg1, or the downstream genes encoding Rme1, Cbr1 or Snf3 are manipulated in a PUFA-producing strain of the oleaginous yeast Yarrowia lipolytica, resulting in increased total lipid content, as compared to the parent strain comprising the heterotrimeric SNF1 protein kinase not having reduced activity.

The invention provides a yarrowia lipolytica genetic engineering bacterium for producing beta-carotene. The yarrowia lipolytica genetic engineering bacterium for producing beta-carotene is prepared bythe following steps: knocking a carRP gene and a carB gene into chromosomes of uracil and leucine auxotrophic yarrowia lipolytica, then knocking a GGGS1 gene, a HMG gene, two copied carRP gene, one copied of carB gene, and an ERG13 gene into the chromosomes, and finally performing backfilling by two auxotrophic screening markers of uracil and leucine. Continuous feeding fermentation is performed,and the yield of beta-carotene can reach 4.5g / L. The yarrowia lipolytica genetic engineering bacteria constructed according to the invention has the advantages of simple and convenient operation andstable and reliable performance, and can be applied to large-scale commercial production, and the obtained beta-carotene can be safely used, so that a good prospect is achieved.

The invention discloses a method for constructing recombinant yarrowia lipolytica for synthesizing erythritol and a strain of the yarrowia lipolytica. The yarrowia lipolytica is adopted as a syntheticchassis, the yeast is subjected to gene editing through a metabolic engineering improvement means, the capability of erythritol synthesizing is enhanced, and the route of synthesizing by-products isblocked. The yeast strain is yarrowia lipolytica ery949-4 delta CGMCC No. 19351. Glucose with the initial concentration of 50-350 g / L is adopted as a carbon source, and a nitrogen source with the initial concentration of 5-30g / L and inorganic salts are taken as raw materials, high-temperature sterilization and cooling are carried out to be incubated with the yarrowia lipolytica yeast strain, and continuous fermentation or batch supplemented fermentation is carried out under the aerobic conditions; and after fermentation is finished, erythritol is purified from the fermentation broth. The strain can efficiently synthesize erythritol by taking glucose and other carbon source as the raw materials, does not synthesize by-products (such as mannitol and the like), and does not consume erythritol.

The invention discloses a construction method of recombinant Yarrowia lipolytica for synthesis of xylitol and a strain thereof. Yarrowia lipolytica is used as a synthetic chassis. Through the improvement means of metabolic engineering, the yeast undergoes gene splicing, genes for synthesizing xylitol from glucose, fructose, glycerol and starch as carbon sources are introduced, and metabolic pathways of synthesis by-products are blocked, so that recombinant Yarrowia lipolytica can synthesize xylitol from glucose, fructose, glycerol and starch as carbon sources by fermentation, and an engineering strain for synthesizing xylitol from carbon sources such as glucose and the like is obtained. After fermentation, xylitol crystals are obtained by separation of a bacterial liquid, ion exchange of aclarified fermentation liquid, decolorization, concentration, crystallization and other processes. The construction method of the engineering yeast for synthesizing xylitol from carbon sources such as glucose and the Yarrowia lipolytica strain obtained by the method and for synthesizing xylitol from carbon sources such as glucose can simplify existing methods for chemically synthesizing xylitol and have better application value.

The invention discloses a construction method of a recombinant Yarrowia lipolytica for producing Valencene and (+)-Nootkatone. The method is characterized in that a Valencene synthase coding gene CVSexpression cassette, a (+)-Nootkatone synthase coding gene CYP706M1 expression cassette and a cytochrome P450 reductase coding gene AtCPR expression cassette are introduced into the rDNA site of Yarrowia lipolytica through a homologous recombination technology to obtain the recombinant Yarrowia lipolytica 1 for producing Valencene and (+)-Nootkatone. Experiments prove that the recombinant Yarrowialipolytica for producing Valencene and (+)-Nootkatone, obtained by the homologous recombination technology, can increase the yield of the Valencene and the (+)-Nootkatone, and allows the yield of theValencene to be 6.5 to 18 mg / L and the yield of the (+)-Nootkatone to be 30 [mu]g / L to 0.5 mg / L. The method provides a basis for the artificial synthesis of the Valencene and (+)-Nootkatone.

The invention discloses recombinant Yarrowia lipolytica for heterogeneous synthesis of beta-amyrin and oleanolic acid and a construction method. The construction method comprises the step of introducing an optimized beta-amyrin synthase coding gene, an optimized oleanolic acid synthase coding gene and an optimized cytochrome-NADPH-reductase 1 coding gene into Yarrowia lipolytica, thereby obtaininga recombinant strain 2. Proven by experiments, the recombinant Yarrowia lipolytica capable of yielding the beta-amyrin and the oleanolic acid is obtained through a homologous recombination method, and thus, the yield of the beta-amyrin and the oleanolic acid is increased; and the method disclosed by the invention provides a foundation for artificial synthesis of the beta-amyrin and the oleanolicacid.

The current methods and compositions provide for nucleotide sequences of promoters from Yarrowia lipolytica which may be used to drive gene expression in a cell. In some aspects, the promoters are useful for modulating lipid production in oleaginous organisms such as yeast.

Disclosed herein are transformed Yarrowia lipolytica comprising an exogenous polynucleotide encoding a polypeptide having sucrose invertase activity. Also disclosed are methods of using the transformed Y. lipolytica.

Genetically engineered Saccharomyces cerevisiae and Yarrowia lipolytica for the production of fatty alcohols, including hexadecanol and octadecanol, are provided. The S. cerevisiae and Y. lipolytica can be genetically engineered to express or over-express a plurality of enzymes involved in fatty alcohol synthesis, such as acyl-CoA thioesterase, fatty acyl reductase, fatty acyl activating enzyme and fatty acyl synthetase, and can be further genetically engineered with a deficiency in one or more enzymes involved in fatty acid oxidation, such as fatty acyl-CoA oxidase. Methods of producing fatty alcohols, such as hexadecanol and octadecanol, by fermenting the genetically engineered S. cerevisiae and Y. lipolytica are also described.

The invention discloses recombinant yarrowia lipolytica for heterogeneous synthesis of alpha-amyrin and ursolic acid and a construction method. The construction method comprises steps as follows: an optimized mixed amyrin synthase encoded gene, an optimized ursolic acid synthase encoded gene and an optimized cytochrome-NADPH-reductase encoded gene are imported into yarrowia lipolytica, and recombinant bacteria 2 are obtained; and experiments prove that the recombinant yarrowia lipolytica for producing alpha-amyrin and ursolic acid is obtained with a homologous recombination method, so that theyield of alpha-amyrin and ursolic acid is improved. the method provides a basis for artificial synthesis of alpha-amyrin and ursolic acid.

This invention provides microbial organisms, particularly yeasts such as Yarrowia lipolytica, that have one or more disrupted genes. The gene disruption(s) may yield improved production of fatty acyl-CoA derivatives.

The invention discloses genetically engineered bacteria of yarrowia lipolytica capable of producing bisabolene and use of the genetically engineered bacteria, and belongs to the technical field of molecular biology. The genetically engineered bacteria are obtained by introducing an alpha-bisabolene synthase gene, a beta-bisabolene synthase gene or a gamma-bisabolene synthase gene into a host of the yarrowia lipolytica and by overexpression of a 3-hydroxy-3-methyl glutaryl coenzyme A reductase gene (HMGR gene). After the genetically engineered bacterium undergo shake-flask fermentation in respective yeast extract peptone dextrose (YPD) mediums, the yield of the bisabolene produced by genetically engineered bacteria Po1g Delta ku70 alpha-HR is 100.22 mg / L, the yield of the bisabolene produced by genetically engineered bacteria Po1g Delta ku70 beta-HR is 5.66 mg / L, and the yield of the bisabolene produced by genetically engineered bacteria Po1g Delta ku70 gamma-HR is 3.55 mg / L. After themediums are optimized, the yield of the bisabolene produced by the genetically engineered bacteria Po1g Delta ku70 alpha-HR is 162.24 mg / L, the yield of the bisabolene produced by genetically engineered bacteria Po1g Delta ku70 beta-HR is 20.811 mg / L, and the yield of the bisabolene produced by genetically engineered bacteria Po1g Delta ku70 gamma-HR is 6.25 mg / L.

The invention discloses a Yarrowia lipolytica genetically engineered bacterium for producing linalool and application of the Yarrowia lipolytica genetically engineered bacterium. The genetically engineered bacterium is formed by the following steps: optimizing a linalool synthetase gene LIS from Actinidia arguta and transforming the optimized linalool synthetase gene LIS into Yarrowia lipolytica Polf; and on the basis, over expressing one or more of endogenous genes HMG1, IDI1 or ERG synchronously. Additionally, a carbon source such as glucose, glycerin, fructose, citric acid and pyruvic acidadded when in fermentation of the genetically engineered bacterium and the concentration of the carbon source are selected, so that the yield of linalool is improved further. The highest yield of linalool can be 6.96mg / L, the content of linalool in a unit cell is 939.04 [mu]g / g DCW (dry cell weight), and the yield is extremely high. The genetically engineered bacterium can be used for large-scalecommercialized production and is good in prospect.

The invention discloses a yarrowia lipolytica genetic engineering strain, as well as a preparation method and an application thereof. The preparation method comprises the following steps of: selecting a POX3 gene in a chromosome of a wild strain as a target gene, knockouting the POX3 gene, simultaneously inserting POX2 and CRF1 genes into the chromosome to enable the chromosome to completely lose short-chain specific acyl-CoA oxidation ability (POX3 gene coding), and simultaneously performing multiple coping of the POX2 genes of coded long-chain specific acyl-CoA oxidase so as to greatly enhance the capability of producing gamma-decalactone of the obtained engineering strain. The labeled gene CRF1 is also derived from the initial strain, so that the biological safety is ensured. A converter Tp-12 is stable in heredity; and furthermore, as the degradation efficiency of the yield of the gamma-decalactone is not obvious during the fermentation process, the converter Tp-12 can be used for industrial continuous fermentation culture of the gamma-decalactone by utilizing castor oil.

A method for producing Yarrowia lipolytica acid-resistant recombinant lipase utilizes a culture medium without any products of animal origin or non-characterized mixtures such as tryptone, peptone or lactoserum. A recombinant strain of Yarrowia lipolytica capable of producing an excessive amount of the lipase Lip2 is referred to as YL-LIP2-6C and filed with the Collection Nationale de Cultures de Microorganismes (C. N. C. M.) under a number of I-3542 on December 15 2005. The invention further relates to uses of the recombinant strain.

Two acyltransferases are provided, suitable for use in the manufacture of microbial oils enriched in omega fatty acids in oleaginous yeast (e.g., Yarrowia lipolytica). Specifically, the genes encoding phophatidylcholine-diacylglycerol acyltransferase (PDAT) and diacylglycerol acyltransferase (DGAT2) have been isolated from Y. lipolytica. These genes encode enzymes that participate in the terminal step in oil biosynthesis in yeast. Each is expected to play a key role in altering the quantity of polyunsaturated fatty acids produced in oils of oleaginous yeasts.

The invention discloses a yarrowia lipolytica genetically engineered bacterium for producing limonene. A method comprises the following steps: introducing an optimized xylose reductase gene XR, an optimized xylitol dehydrogenase gene XDH and an optimized xylose assimilation enzyme gene XK into chromosomes of uracil and leucine auxotrophic yarrowia lipolytica to construct a yarrowia lipolytica genetically engineered bacterium YBX06; transforming a recombinant vector containing two copy-optimized limonene synthase genes LS and one copy-optimized neroli diphosphate synthase 1 gene NDPS1 into theyarrowia lipolytica genetically engineered bacterium YBX06, and performing construction to obtain the yarrowia lipolytica genetically engineered bacterium YBX07. The yarrowia lipolytica genetically engineered bacterium constructed by the invention can utilize lignocellulose hydrolysate as a raw material and synchronously utilize xylose and glucose as a mixed carbon source to produce the limonene through biological fermentation, so that the yarrowia lipolytica genetically engineered bacterium has a good application prospect.

The invention particularly relates to yarrowia lipolytica with high yield of [beta]-carotene and application thereof. The strain can utilize common carbon sources and nitrogen sources, after high-density culture, the yield of [beta]-carotene reaches 7.8 g / L, the content reaches 161 mg / g DCW, and the strain is an excellent strain for high yield of [beta]-carotene. According to the engineering strain, a low-yield [beta]-carotene yarrowia lipolytica strain T1 which integrates CarRP and CarB genes to a zeta site at a fixed point is taken as a starting strain, endogenous key enzymes HMGs, HMGCR, ERG20, GGS1, ERG8, ERG12, ERG19 and IDI are subjected to multiple rounds of random integration, and exogenous GGPP synthetase GPS (which can directly catalyze IPP and DMAPP into GGPP) and hemoglobin VHb are introduced; and finally, the key gene AAL8 for reducing lipidosome degradation is knocked out to obtain the high-yield strain. As a [beta]-carotene production engineering bacterium, the strain has a good application prospect in the fields of food, feed, medicine and cosmetics.

The invention discloses a method for integrating a yarrowia lipolytica genome based on a non-homologous end-linking mechanism. The method comprises the following steps of: integrating yarrowia lipolytica endogenous genes DL4 and XRCC4 and an exogenous wisdom gene PAXX into a genome rDNA site of a yarrowia lipolytica ATCC201249 strain in an rDNA site integration manner, so as to obtain a recombinant strain YNH01; linking a to-be-transferred gene expression cassette with a URA3 nutrition tag U11 truncated to 11bp by a promoter, and transforming an obtained linking fragment into the strain YNH01,so as to obtain a high-pressure screening tag strain; transforming the linking fragment into the high-pressure screening tag strain, so as to obtain a recombinant strain subjected to two-round transformation; and transforming the linking fragment into the recombinant strain subjected to two-round transformation again, so as to obtain a transferred gene high-expression strain. According to the method, a large number of diversified strains are obtained within a short time, and the efficient application of an NHEJ integration technology in microbial synthesis of natural products is realized.

The invention discloses a recombinant glucose-oxidase-expressing Yarrowia lipolytica and application thereof, belonging to the technical field of biology. By constructing a recombinant plasmid pINA1297-GOD, a lithium acetate conversion method is utilized to convert Yarrowia lipolytica po1h, and screening is performed to obtain the recombinant strain with favorable enzyme producing capacity. After the strain is fermented for 168 hours by using sucrose as the unique carbon source, the enzyme activity of the glucose oxidase reaches 13.9 U / ml, and is enhanced by two times as compared with the original Aspergillus niger.

The invention provides recombinant Yarrowia lipolytica to produce (-)-alpha-bisabolol and a construction method and application thereof and relates to the field of biological engineering. The recombinant Yarrowia lipolytica to produce (-)-alpha-bisabolol is constructed by inserting an (-)-alpha-bisabolol synthase, farnesyl pyrophosphate synthase and 3-hydroxy-3-methylglutaryl CoA synthase expression kit into the genome of Yarrowia lipolytica. The recombinant Yarrowia lipolytica is suitable for efficiently synthesizing (-)-alpha-bisabolol; the construction method is efficient and is simple to perform.