Patent Grant
10640796
The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: PRVI_018_02US_SeqList.txt, date recorded: Aug. 13, 2018, file size 75 kilobytes).
The invention relates to various yeast strains of the type Yarrowia lipolytica as well as relevant methods for the biocatalytic preparation of ω-hydroxy fatty acids or dicarboxylic acids with the aid of these strains.
Dicarboxylic acids comprise carboxylic acids having two carboxy groups (general structure: HOOC—(CH2)n—COOH), which have diverse applications in the chemical industry, e.g. for the preparation of fragrances, adhesives, Nylon and other polyamides, resins, corrosion inhibitors or lubricants. In particular, the biotechnological production of long-chain dicarboxylic acids is of particular interest since a number of undesired side products are formed during the chemical synthesis of the same.
ω-hydroxy fatty acids contain at the Cω atom (C atom having the furthest possible distance from the carboxyl group) a hydroxyl group (general structure: HOOC—(CH2)n—CH2OH). ω-hydroxy fatty acids are also of great importance in the chemical industry since they are used, inter alia, in lubricants, adhesives, cosmetics and cancer therapeutic agents. Furthermore, ω-hydroxy fatty acids could acquire great importance in future since they can be used as monomers for the synthesis of bioplastics.
In order to obtain ω-hydroxy fatty acids and dicarboxylic acids from n-alkanes and fatty acids, the non-conventional yeast Yarrowia (Y.) lipolytica is to be used within the framework of the invention. The yeast Y. lipolytica was selected as host organism for the bioconversion since it is able to use a plurality of substrates as carbon source. In addition to glucose, glycerol, proteins, alcohols and acetate, this also includes a plurality of hydrophobic substrates such as vegetable oils, fats, fatty acids and n-alkanes (Barth G & Gaillardin C (1997) FEMS Microbiol Rev 19: 219-237). The hydrophobic substrates are emulsified by the yeasts and assimilated into the cell interior with the aid of specialized membrane transporters (Fickers P, Benetti P H, Waché Y, Marty A, Mauersberger S, Smit M S & Nicaud J M (2005) FEMS Yeast Res 5: 527-543). n-alkanes assimilated into the cell are converted stepwise to fatty acid having the same chain length (
Various yeasts such as, for example, Candida (C.) tropicalis and Y. lipolytica are capable of converting long-chain alkanes or fatty acids to α,ω-dicarboxylic acids. In order that the hydrophobic substrates can be converted at all to the respective dicarboxylic acid, their metabolization in the course of the β-oxidation must be prevented. This can be accomplished, for example, by deletion of the POX gene coding for the acyl-CoA-oxidase. If the β-oxidation is eliminated, this leads to an increased ω-oxidation of the fatty acids (Smit et al. (2005) Biotechnol. Lett. 27: 859-864).
The microbial production of long-chain dicarboxylic acids has already been carried out with the aid of the yeast C. tropicalis:
In this case, the production of the dicarboxylic acids was primarily achieved by deletion of the genes POX4 and POX5. There are a number of patents which protect corresponding C. tropicalis production strains and relevant production methods (e.g. U.S. Pat. No. 4,339,536A, EP296506A2 and WO200017380A1).
In a 2013 patent specification it is described how yeasts in general (in a manner known per se) can be induced to produce increased quantities of dicarboxylic acids. In the exemplary embodiments however, only C. tropicalis is specifically discussed here (WO201306730A2, WO201306733A2).
The microbial production of long-chain ω-hydroxy fatty acids has also already been carried out with the aid of the yeast C. tropicalis:
For this purpose, in addition to the genes POX4 and POX5, six cytochrome P450, four alcohol oxidase and six alcohol dehydrogenase genes were deleted. These strains and production methods are already protected under patent law (WO2011008232A2).
Furthermore, it has already been described how Y. lipolytica can be induced to form dicarboxylic acids:
In this case, all the presently known acyl-CoA-oxidase genes (POX1, POX2, POX3, POX4, POX5 and POX6) as well as the acyl-CoA-diacylglycerol-acyltransferase gene (DGA1) and the lecithin-cholesterol-acyltransferase gene (LRO_1) were deleted. Furthermore, the NADPH-cytochrome P450-reductase gene (CPR1) was overexpressed (WO200664131A8).
In addition to the biotechnological production of ω-hydroxy fatty acids and dicarboxylic acids with the aid of genetically modified yeasts using the intracellular ω-oxidation of fatty acids, there are also other biotechnological methods based on other enzymatic conversions. An example of this is the use of Baeyer-Villiger monooxygenase, which catalyzes the conversion of a ketone to the ester (WO2013151393A1).
It is the object of the present invention to provide new types of yeast strains of the type Y. lipolytica which are suitable for the microbial production of ω-hydroxy fatty acids or dicarboxylic acids in larger quantities than previously known as well as a method for cultivating precisely these strains.
The object is solved by a yeast strain according to claim 1 and by methods according to claims 8 and 11. Advantageous embodiments are given in the dependent claims.
According to a first aspect of the invention, for the microbial production of ω-hydroxy fatty acids from hydrophobic substrates, a yeast strain of the type Y. lipolytica is provided, which exhibits a reduction in the activities of acyl-CoA oxidases (namely POX1: YALI0E32835g, POX2: YALI0F10857g, POX3: YALI0D24750g, POX4: YALI0E27654g, POX5: YALI0C23859g, POX6: YALI0E06567g), wherein in this yeast strain the activities of relevant (fatty) alcohol dehydrogenases (=(fatty acid) alcohol dehydrogenases, namely selected from FADH: YALI0F09603g, ADH1: YALI0D25630g, ADH2: YALI0E17787g, ADH3: YALI0A16379g, ADH4: YALI0E15818g, ADH5: YALI0D02167g, ADH6: YALI0A15147g and/or ADH7: YALI0E07766g) are reduced and the activities of relevant (fatty) alcohol oxidases (=(fatty acid) alcohol oxidases, namely FAO1: YALI0B14014g) are reduced, which leads to an increased formation of ω-hydroxy fatty acids, preferably linear ω-hydroxy fatty acids according to the general structure: HOOC—(CH2)n—CH2OH, HOOC—(CnH2n-2)—CH2OH, COOC—(CnH2n-4)—CH2OH or HOOC—(CnH2n-6)—CH2OH, wherein n is in the range from 6 to 18, preferably in the range from 8 to 16, from a hydrophobic substrate, in particular a corresponding n-alkane or a fatty acid (and other hydrophobic substrates).
Particularly preferably the ω-hydroxy fatty acid is a linear ω-hydroxy fatty acid according to the general structure: HOOC—(CH2)n—CH2OH where n is in the range from 8 to 16.
The formation of ω-hydroxy fatty acids or dicarboxylic acids is increased in the sense of the present invention if this is increased compared with yeast cells without the corresponding genetic modification by at least 10 mol. %, particularly preferably at least 20 mol. %.
The invention is based on the recently obtained finding that the gene which codes for the fatty alcohol oxidase FAO1: YALI0B14014g in the yeast strain of the type Y. lipolytica, could be identified.
The inventors have additionally now surprisingly found that the deletion of the gene which codes for the enzyme Faolp reduces the further ω-oxidation of the ω-hydroxy fatty acids to the fatty acid aldehyde or to the dicarboxylic acid in the course of the ω-oxidation of fatty acids.
The reduction of the activity of the products of FADH, ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7 and/or FAO1 particularly advantageously prevents or reduces the further oxidation of ω-hydroxy fatty acids to the fatty acid aldehyde or to the dicarboxylic acid in the course of the ω-oxidation of fatty acids.
The fatty alcohol oxidase gene has not yet been described so far in Y. lipolytica. Its use within the framework of biotechnological fields of application is also part of this invention.
A reduction in the activity of a protein is preferably to be understood that the activity of the protein per cell is reduced. Particularly preferably this is to be understood as the lack of activity of the protein in the yeast cell.
The reduction in the activity of proteins can be achieved by various methods known to the person skilled in the art such as, for example:
The inhibition or reduction of the expression of a gene coding for a protein can, for example, be accomplished by inhibition or reduction of the transcription of the coding gene or the translation of the mRNA formed. The deletion of the coding genes can be performed, for example, by a removal of the genes by means of deletion cassettes. The expression of a dysfunctional or activity-reduced gene product can be accomplished, for example, by insertion, substitution or point mutation in the gene coding for the protein.
The deletion of a coding gene is preferred for the reduction in activity.
The yeast strain according to the invention for the production of ω-hydroxy fatty acids thus exhibits a deletion of the genes for the acyl-CoA oxidases (POX1, POX2, POX3, POX4, POX5 and POX6) as well as for the (fatty) alcohol dehydrogenases (FADH, ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7) and/or for the (fatty) alcohol oxidases (FAO1). Furthermore, the formation of lipid bodies is reduced, preferably due to the reduction in the activity of the gene products of PAH1 and/or SCT1.
The deletion of the genes POX1, POX2, POX3, POX4, POX5 and POX6 prevents the β-oxidation and thus inhibits the breakdown of fatty acids (to acetyl-CoA or propionyl-CoA).
Preferably in the yeast strain according to the invention for the microbial production of ω-hydroxy fatty acids, the activities of individual or all cytochrome P450-gene products, in particular of ALK1: YALI0E25982g, ALK2: YALI0F01320g, ALK3: YALI0A20130g, ALK4: YALI0B13816g, ALK5: YALI0B13838g, ALK6: YALI0B01848g, ALK7: YALI0A15488g, ALK8: YALI0C12122g, ALK9: YALI0B06248g, ALK10: YALI0B20702g, ALK11: YALI0C10054g and/or ALK12: YALI0A20130g are additionally increased. Advantageously the increase in individual or all cytochrome P450-gene products results in an increased formation of ω-hydroxy fatty acids from the corresponding hydrophobic salt, in particular n-alkanes and fatty acids.
It can be advantageous that in the yeast strain according to the invention for the microbial production of ω-hydroxy fatty acids, the activity of the NADPH-cytochrome P450-reductase (CPRI: YALI0D04422g) is additionally increased, which leads to an increased formation of ω-hydroxy fatty acids from the corresponding hydrophobic substrate, in particular n-alkanes and fatty acids.
Preferably in the yeast strain according to the invention for the microbial production of ω-hydroxy fatty acids, the activity of the phosphatidic acid-dephosphohydrolase (PAH1: YALI0D27016p) is additionally reduced. Advantageously this leads to a reduction in the size of the lipid bodies and therefore to an increased formation of ω-hydroxy fatty acids or dicarboxylic acids from the corresponding hydrophobic substrate, in particular n-alkanes and fatty acids.
According to a preferred embodiment of the present invention, in the yeast strain according to the invention for the microbial production of ω-hydroxy fatty acids, the activity of the glycerol-3-phosphate-acyltransferase (SCT1: YALI0C00209g) is additionally reduced, which advantageously leads to a reduction in the size of the lipid bodies and therefore to an increased formation of ω-hydroxy fatty acids from the corresponding hydrophobic substrate, in particular n-alkanes and fatty acids (and other hydrophobic substrates).
It can also be further provided that in the yeast strain according to the invention for the microbial production of ω-hydroxy fatty acids, the activity of the phospholipid-diacylglycerol-acyltransferase (LRO1: YALI0E16797g) is additionally reduced, which leads to a reduction in the size of the lipid bodies and therefore to an increased formation of ω-hydroxy fatty acids from the corresponding hydrophobic substrate, in particular n-alkanes and fatty acids.
According to a preferred embodiment of the present invention, in the yeast strain according to the invention for the microbial production of ω-hydroxy fatty acids, the activity of the diacylglycerol-acyltransferase (DGAJ: YALI0E32769g) is additionally reduced, which leads to a reduction in the size of the lipid bodies and therefore to an increased formation of ω-hydroxy fatty acids from the corresponding hydrophobic substrate, in particular n-alkanes and fatty acids.
According to a preferred embodiment of the present invention, in the yeast strain according to the invention for the microbial production of ω-hydroxy fatty acids, the activity of the glycerol-3-phosphate-dehydrogenase (GUT2: YALI0B13970g) is additionally increased, which leads to a reduction in the size of the lipid bodies and therefore to an increased formation of ω-hydroxy fatty acids from the corresponding hydrophobic substrate, in particular n-alkanes and fatty acids.
It was particularly advantageously now found that the additional increase in the activities of relevant triacylglycerol lipases (TGL3/TGL4: YALI0F10010g, YALI0D16379g, YALI0D17534g) in the yeast strain according to the invention for the microbial production of ω-hydroxy fatty acids, leads to a reduction in the size of the lipid bodies and therefore to an increased formation of ω-hydroxy fatty acids from the corresponding hydrophobic substrate, in particular n-alkanes and fatty acids.
The reduction in the activity of the citrate and oxalacetate transporters (YHM2: YALI0B10736g) and/or the reduction in the activity of the citrate transport protein (CTP1: YALI0F26323g) in the yeast strain according to the invention for the microbial production of ω-hydroxy fatty acids has proved to be particularly advantageous since as a result, a smaller quantity of organic acids is produced and/or secreted into the culture medium.
It can optionally be provided that in the yeast strain according to the invention for the microbial production of ω-hydroxy fatty acids, the transport of ω-hydroxy fatty acids from the yeast cell or from the endoplasmic reticulum into the cytoplasm is additionally increased, e.g. due to the increased activity of specific membrane transporters, which leads to an increased formation of ω-hydroxy fatty acids from the corresponding hydrophobic substrate, in particular n-alkanes and fatty acids.
Preferably in the yeast strain according to the invention for the microbial production of ω-hydroxy fatty acids, the transport of the hydrophobic substrate into the yeast cell or the respective organelles (peroxisomes, endoplasmic reticulum), is additionally increased, e.g. due to the increased activity of specific membrane transporters, which leads to an increased formation of ω-hydroxy fatty acids from the corresponding hydrophobic substrate, in particular n-alkanes and fatty acids.
The invention also relates to the use of the yeast strain according to the invention for the microbial production of ω-hydroxy fatty acids having the general structure: HOOC—(CH2)n—OH, HOOC—(CnH2n-2)—CH2OH, COOC—(CnH2n-4)—CH2OH or HOOC—(CnH2n-6)—CH2OH, where n is in the range from 6 to 18, from a hydrophobic substrate.
The invention also comprises a yeast strain of the type Y. lipolytica for the microbial production of dicarboxylic acids from n-alkanes and fatty acids (and other hydrophobic substrates) which exhibit a reduction in the activities of acyl-CoA oxidases (namely POX1: YALI0E32835g, POX2: YALI0F10857g, POX3: YALI0D24750g, POX4: YALI0E27654g, POX5: YALI0C23859g, POX6: YALI0E06567g) and is characterized in that in this yeast strain the activities of relevant (fatty) alcohol dehydrogenases (namely selected from FADH: YALI0F09603g, ADH1: YALI0D25630g, ADH2: YALI0E17787g, ADH3: YALI0A16379g, ADH4: YALI0E15818g, ADH5: YALI0D02167g, ADH6: YALI0A15147g and/or ADH7: YALI0E07766g) and the activities of relevant (fatty) alcohol oxidases (namely FAO1: YALI0B14014g) are additionally increased, which leads to an increased formation of dicarboxylic acids, preferably according to the general structure: HOOC—(CH2)n—COOH, HOOC—(CnH2n-2)—COOH, COOC—(CnH2n-4)—COOH or HOOC—(CnH2n-6)—COOH, where n is in the range from 6 to 18, preferably in the range from 8 to 16, from n-alkanes and fatty acids (and other hydrophobic substrates).
The yeast strain according to the invention for the production of dicarboxylic acids preferably exhibits a deletion of the genes POX1, POX2, POX3, POX4, POX5 and POX6. In addition, it exhibits an increased activity of the products of FADH, ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7 and/or FAO1. Furthermore, the formation of lipid bodies is reduced, preferably due to the reduction in the activity of gene products of PAH1 and/or SCT1.
Particularly advantageously, the increase in activity of the gene products of FADH, ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7 and/or FAO1 intensifies the further oxidation of ω-hydroxy fatty acids to fatty acid aldehyde or to dicarboxylic acid in the course of the ω-oxidation of fatty acids, so that the desired dicarboxylic acids are increasingly formed.
An increased activity of a protein is preferably to be understood that the activity per cell is increased which can be achieved by the increase in the quantity of protein or by the increase in the protein's own activity. The increase in the quantity of protein is characterized in that more protein is formed than for an unmodified strain under the same conditions. The increase in the protein's own activity is characterized in that the same quantity of protein has a higher activity in a modified strain than in an unmodified strain.
An increased activity of the proteins can be produced in various ways:
For the increase in activity, the additional introduction of the relevant gene under control of a strong promoter is particularly preferred, preferably the TEF1 promoter from Yarrowia lipolytica.
According to the invention, the yeast strains of the type Y. lipolytica for the production of ω-hydroxy fatty acids or dicarboxylic acids are not capable of breaking down fatty acids in the course of the β-oxidation. This can be ensured, for example, by the following measures:
For the production of ω-hydroxy fatty acids, in addition to the prevention of β-oxidation, the oxidation of ω-hydroxy fatty acids to fatty acid aldehyde or to dicarboxylic acid in the course of ω-oxidation must be prevented or reduced. This can be achieved as follows:
The product yield is preferably further increased by the direct influencing of relevant metabolic pathways, in particular these are:
Only for the production of dicarboxylic acids:
In the course of our investigations, it was established that the cells form very large lipid bodies in the course of cultivation. It can be assumed that the fatty acids (or intracellular derivatives thereof) are esterified with glycerol and stored intracellularly in the lipid bodies. This reduces the product yield ultimately achieved:
Genetic modifications preferably result in the formation of smaller lipid bodies and therefore in an increased product yield, in particular these are:
In particular, the reduction in the activity of phosphatidic acid dephosphohydrolase, diacyl-glycerol acyltransferase and/or glycerol-3-phosphate acyltransferase result in an increased product yield, i.e. production of ω-hydroxy fatty acids or dicarboxylic acids.
Furthermore, transport of the substrates into the cell or the respective organelles (peroxisomes, endoplasmic reticulum) as well as transport of the products (ω-hydroxy fatty acids and dicarboxylic acids) from the cell or from the respective organelles is increased. This can be ensured, for example, by the increase in the activity of specific transporters.
According to a particularly preferred embodiment of the present invention, the combination of several of the aforesaid genetic modifications results in an increased product yield, an increased output and/or an increased space-time yield.
A wild type isolate of the yeast Y. lipolytica, preferably of the strain H222 can be used as the starting strain for the construction of a strain according to the invention. The strain H222 was deposited on 29 Apr. 2013 at the DSMZ (Deutsche Sammlung für Mikroorganismen and Zellkulturen GmbH, D-38142 Braunschweig) under the number DSM 27185 according to the Budapest Treaty on the international Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
A selection marker is required for the use of a strain for further genetic processing. This selection marker can be introduced into the strain in a manner known per se, e.g. in the form of the uracil auxotroph. Alternatively, already known uracil auxotrophic strains can be used, preferably the strain H222-S4 (Mauersberger S, Wang H J, Gaillardin C, Barth G & Nicaud J M (2001) J Bacteriol 183: 5102-5109).
Additional genes or constructs are introduced, which replace the existing genes, preferably by means of usual methods known in the prior art. In order to ensure an efficient transcription of the introduced genes, it is preferable to link promoters functioning for the organism to the open reading frame of the genes. In this case, both constitutive promoters and also promoters dependent on the cultivation conditions can be used.
Preferably the introduced genes which code for the said enzymes are placed functionally under the control of a constitutive promoter, where the promoters can be identical or different from one another.
Preferably constitutive promoters are used. Particularly preferably the promoter of the translation elongation factor 1 alpha (pTEF1) is used.
Alternatively preferably the introduced genes are placed under the control of a promoter which is dependent on the cultivation conditions. Preferably activators or inductors activate the promoter which is dependent on the cultivation conditions (in the sense of the application also called operator) by interacting directly with a controllable promoter or by binding to a repressor protein which then detaches from the promoter. As a result of the contact of the yeast strain according to the invention with the activator and/or inductor, the aforesaid enzymes are synthesized in the yeast strain according to the invention and are thus available for the biocatalytic conversion of a hydrophobic substrate, in particular of a corresponding n-alkane or a fatty acid.
In one embodiment of the invention, the promoters dependent on the cultivation conditions differ from one another so that the promoters are activatable in a primary-signal specific manner. The presence of different activators and/or inductors in the yeast strain can thus advantageously lead to the expression of selected genes, whereby the stress for a recombinant yeast cell is reduced.
The corresponding nucleic acid sequences for promoters (for example, pTEF1, pPOX2, pPOT1, pXPR1, pICL1), which can be used for a method according to the invention are extremely well known to the person skilled in the art or can be deduced from known databases. Advantageously for a method according to the invention, natural promoters can also be used in addition to promoters introduced artificially into the organisms.
Likewise it is preferable to provide the open reading frame of the genes with a terminator. Preferably the terminator of the gene of the isocitrate lyase (ICL1t) is used.
The subject matter of the invention is also a method for the biocatalytic production of ω-hydroxy fatty acids, preferably according to the general structure: HOOC—(CH2)n—CH2OH, HOOC—(CnH2n-2)—CH2OH, COOC—(CnH2n-4)—CH2OH or HOOC—(CnH2n-6)—CH2OH, where n is in the range from 6 to 18, preferably in the range from 8 to 16, from a hydrophobic substrate, which comprises the following steps:
Particularly preferably the ω-hydroxy fatty acid is a linear ω-hydroxy fatty acid having the general structure HOOC—(CH2)n—CH2OH where n is in the range from 8 to 16.
According to a preferred embodiment of the method according to the invention for the biocatalytic production of ω-hydroxy fatty acids, it can be provided that the reaction to give the reaction product ω-hydroxy fatty acid additionally takes place with (fatty) alcohol oxidases, (fatty) alcohol dehydrogenases remaining in the yeast cells used.
Preferably the yeast cells of at least one yeast strain in the method according to the invention for the biocatalytic production of ω-hydroxy fatty acids of the type Y. lipolytica are selected from the yeast strain according to the invention for the microbial production of ω-hydroxy fatty acids.
It has surprisingly been shown that after the biocatalytic conversion of the hydrophobic substrate to the desired reaction product ω-hydroxy fatty acids, these are not further metabolized in the yeast cells according to the invention of a yeast strain of the type Y. lipolytica (i.e. broken down in the metabolism of the whole cell catalyst) and accumulate in the aqueous component. Advantageously the ω-hydroxy fatty acid accumulates as a consequence of the discharge from the yeast cells in the cultivation medium.
Preferably the contacting of the yeast strain with a hydrophobic substrate takes place by direct addition to the liquid component in the form of liquid and/or solid. During direct addition to the yeast strain, an organic phase can additionally be used as substrate reservoir, whereby the process run time can be optimized.
In the sense of the present invention, a hydrophobic substrate is to be understood as a per se water-insoluble substance having an optionally branched and/or unsaturated hydrocarbon scaffold with 8 to 20 carbon atoms. Preferably the hydrophobic substrate is selected from the group of n-alkanes having the general structure: H3C—(CH2)n—CH3 (for example, dodecane, pentadecane, hexadecane) or alkenes having the general structure H3C—(CH2)n—CH3, H3C—(CnH2n-4)—CH3 or H3C—(CnH2n-6)—CH3, where n is an integer in the range from 6 to 18, the optionally branched saturated fatty acids having the general structure: HOOC—(CH2)n—CH3, where n is an integer in the range from 6 to 18 (for example, dodecanoic acid, hexadecanoic acid, pentadecanoic acid, phytanic acid), the unsaturated fatty acids having the general structure: HOOC—(CnH2n-2)—CH3, COOC—(CnH2n-4)—CH3 or HOOC—(CnH2n-6)—CH3, where n is an integer in the range from 6 to 18 (for example, oleic acid, linoleic acid, arachidonic acid), and fatty acid alkyl esters having the general structure: ROOC—(CH2)n—CH3, where n is in the range from 6 to 18 and where R is an optionally branched C1-4-alkyl group.
Particularly preferably but not exclusively aforesaid n-alkanes, alkenes or saturated or unsaturated fatty acids having a chain length (number of carbon atoms of the longest chain) from eight to eighteen carbon atoms, i.e., n in the range from 6 to 16 serve as hydrophobic substrate for the bioconversion.
According to an alternatively preferred embodiment of the present invention, preferably but not exclusively n-alkanes and fatty acids having a chain length of eight to eighteen carbon atoms, i.e. n in the range from 6 to 16, serve as hydrophobic substrate for the bioconversion.
According to a preferred embodiment of the method according to the invention, the concentration of the hydrophobic substrate at the beginning of production is 1 to 50 grams per litre of culture medium (g 1−1), particularly preferred is 5 to 40 g 1−1.
The invention further relates to a method for the production of dicarboxylic acids, preferably according to the general structure: HOOC—(CH2)n—COOH, HOOC—(CnH2n-2)—COOH, COOC—(CnH2n-4)—COOH or HOOC—(CnH2n-6)—COOH, where n is in the range from 6 to 18, preferably in the range from 8 to 16, from a hydrophobic substrate, in particular selected from n-alkanes and fatty acids, comprising the following steps:
Particularly preferably the dicarboxylic acid is a linear dicarboxylic acid according to the general structure: HOOC—(CH2)n—CH2OH where n is in the range from 8 to 16.
Preferably in the yeast cells for the method according to the invention for the production of ω-hydroxy fatty acids or dicarboxylic acids, the activities of individual or all cytochrome P450-gene products, in particular of ALK1: YALI0E25982g, ALK2: YALI0F01320g, ALK3: YALI0A20130g, ALK4: YALI0B13816g, ALK5: YALI0B13838g, ALK6: YALI0B01848g, ALK7: YALI0A15488g, ALK8: YALI0C12122g, ALK9: YALI0B06248g, ALK10: YALI0B20702g, ALK11: YALI0C10054g and/or ALK12: YALI0A20130g are additionally increased. Advantageously the increase of individual or all cytochrome P450-gene products leads to an increased formation of ω-hydroxy fatty acids or dicarboxylic acids from the corresponding hydrophobic substrate, in particular n-alkanes and fatty acids.
It can advantageously be provided that in the yeast cells for the method according to the invention for the production of ω-hydroxy fatty acids or dicarboxylic acids, the activity of the NADPH-cytochrome P450-reductase (CPR1: YALI0D04422g) is additionally increased, which leads to an increased formation of ω-hydroxy fatty acids or dicarboxylic acids from the corresponding hydrophobic substrate, in particular n-alkanes and fatty acids.
According to a preferred embodiment of the method according to the invention for the production of dicarboxylic acids, in the yeast cells the activities of relevant (fatty) alcohol dehydrogenases (namely FADH: YALI0F09603g, ADH1: YALI0D25630g, ADH2: YALI0E17787g, ADH3: YALI0A16379g, ADH4: YALI0E15818g, ADH5: YALI0D02167g, ADH6: YALI0A15147g, ADH7: YALI0E07766g) can be additionally increased, which advantageously leads to an increased formation of dicarboxylic acids from the corresponding hydrophobic substrate, in particular n-alkanes and fatty acids.
Preferably in the yeast cells of the method according to the invention for the production of dicarboxylic acids, the activities of relevant (fatty) alcohol oxidases (FAO1: YALI0B14014g) are additionally increased, which leads to an increased formation of dicarboxylic acids from the corresponding hydrophobic substrate, in particular n-alkanes and fatty acids.
According to a preferred embodiment of the method according to the invention for the production of dicarboxylic acids, in the yeast cells the activities of relevant (fatty) alcohol dehydrogenases (FALDH1: YALI0A17875g, FALDH2: YALI0E15400g, FALDH3: YALI0B01298g, FALDH4: YALI0F23793g) are additionally increased, which leads to an increased formation of dicarboxylic acids from the corresponding hydrophobic substrate, in particular n-alkanes and fatty acids.
Preferably the yields of ω-hydroxy fatty acids or dicarboxylic acids in the method according to the invention over a period of one day (i.e. 24 h) lie in the range from 1 to 20 g 1−1, particularly preferably in the range from 4 to 15 g 1−1.
According to a preferred embodiment of the invention, the production of ω-hydroxy fatty acids and dicarboxylic acids takes place in a bioreactor (fermenter) in which a yeast strain according to the invention is fermented under suitable growth conditions in a suitable cultivation medium.
The invention further relates to method for producing ω-hydroxy fatty acids and dicarboxylic acids which are characterized in that a micro-organism according to the invention is cultivated in a manner known per se in a bioreactor (fermenter).
Preferably the reaction product ω-hydroxy fatty acids and dicarboxylic acids, are isolated by extraction of the aqueous component with an organic solvent selected from the group of phthalic acid esters, particularly preferably ethylacetate, 1,2-cyclo-hexane dicarboxylic acid diisononylester and Mesamoll®, and/or the aliphatic branched and/or linear hydrocarbons, preferably with 5 to 16 carbon atoms such as, for example, n-pentane, cyclopentane, n-hexane, cyclohexane, n-heptane, n-octane, cyclooctane, n-decane, n-dodecane or n-hexadecane. Preferably the said organic solvents are used in a single-phase aqueous system for extraction after conversion of the hydrophobic substrate. Alternatively the said organic solvents are used in a two-phase system in the form of a second phase in addition to the cultivation medium as a reservoir for the hydrophobic substrate and/or for separation of the reaction product.
The isolation of the dicarboxylic acids and/or ω-hydroxy fatty acids by extraction of the aqueous component with an organic solvent preferably takes place after separation of the yeast cells in the form of biomass from the cultivation medium, where the separation of the yeast cells in the form of biomass from the cultivation medium is preferably accomplished by centrifugation or filtration.
Preferably the extraction of the reaction products ω-hydroxy fatty acids and dicarboxylic acids is accomplished with an organic solvent with a pH between 0 and 8, particularly preferably between 1 and 7, quite particularly preferably between 1 and 6.
Optionally a distillation is accomplished following the extraction for purification of the dicarboxylic acids and/or ω-hydroxy fatty acid, where preferably the organic solvent is separated. Preferably the organic solvent is separated by evaporation at a pressure between 0.1 and 1000 mbar, particularly preferably between 0.1 and 750 mbar, quite particularly preferably between 1 and 400 mbar.
Preferably in a yeast strain for the method according to the invention for the production of ω-hydroxy fatty acids or dicarboxylic acids, the activity of the phosphatidic acid dephosphohydrolase (PAHL YALI0D27016p) is additionally reduced. Advantageously this leads to a reduction in the size of the lipid bodies and therefore to an increased formation of ω-hydroxy fatty acids or dicarboxylic acids from the corresponding hydrophobic substrate, in particular n-alkanes and fatty acids.
According to a preferred embodiment of the method according to the invention for the production of ω-hydroxy fatty acids or dicarboxylic acids, in the yeast strain used the activity of the glycerol-3-phosphate acyltransferase (SCT1: YALI0C00209g) is additionally reduced, which advantageously leads to a reduction in the size of the lipid bodies and therefore to an increased formation of ω-hydroxy fatty acids or dicarboxylic acids from the corresponding hydrophobic substrate, in particular n-alkanes and fatty acids (and other hydrophobic substrates).
It can further be provided that in the method according to the invention for the microbial production of ω-hydroxy fatty acids or dicarboxylic acids, in the yeast cells of the yeast strain used, the activity of the phospholipid diacylglycerol acyltransferase (LRO1: YALI0E16797g) is additionally reduced, which leads to a reduction in the size of the lipid bodies and therefore to an increased formation of ω-hydroxy fatty acids or dicarboxylic acids from the corresponding hydrophobic substrate, in particular n-alkanes and fatty acids.
According to a preferred embodiment of the present invention, in the yeast strain according to the invention for the microbial production of ω-hydroxy fatty acids or dicarboxylic acids, the activity of the diacylglycerol acyltransferase (DGA1: YALI0E32769g) is additionally reduced, which leads to a reduction in the size of the lipid bodies and therefore to an increased formation of ω-hydroxy fatty acids or dicarboxylic acids from the corresponding hydrophobic substrate, in particular n-alkanes and fatty acids.
According to a preferred embodiment of the method according to the invention for the production of ω-hydroxy fatty acids or dicarboxylic acids, in the yeast cells of the appropriately used yeast strain, the activity of the glycerol-3-phosphate dehydrogenase (GUT2: YALI0B13970g) is additionally increased, which leads to a reduction in the size of the lipid bodies and therefore to an increased formation of ω-hydroxy fatty acids or dicarboxylic acids from the corresponding hydrophobic substrate, in particular n-alkanes and fatty acids.
Particularly advantageously, it was now found that the additional increase in the activities of relevant triacylglycerol lipases (TGL3/TGL4: YALI0F10010g, YALI0D16379g, YALI0D17534g) in the yeast cells used for the microbial production of ω-hydroxy fatty acids or dicarboxylic acids, leads to a reduction in the size of the lipid bodies and therefore to an increased formation of ω-hydroxy fatty acids or dicarboxylic acids from the corresponding hydrophobic substrate, in particular n-alkanes and fatty acids.
Preferably in the yeast cells of the yeast strain used for the method according to the invention for the production of ω-hydroxy fatty acids or dicarboxylic acids, the transport of the hydrophobic substrates into the yeast cell or the respective organelles (peroxisomes, endoplasmic reticulum), is additionally increased, e.g. due to the increased activity of specific membrane transporters, which leads to an increased formation of ω-hydroxy fatty acids or dicarboxylic acids from the corresponding hydrophobic substrate, in particular n-alkanes and fatty acids.
Optionally it can be provided that in the yeast cells for the microbial production of ω-hydroxy fatty acids or dicarboxylic acids, the transport of ω-hydroxy fatty acids and dicarboxylic acids from the yeast cell or from the endoplasmic reticulum into the cytoplasm is additionally increased, e.g. due to the increased activity of specific membrane transporters, which leads to an increased formation of ω-hydroxy fatty acids or dicarboxylic acids from the corresponding hydrophobic substrate, in particular n-alkanes and fatty acids and enrichment in the culture medium.
The reduction in the activity of the citrate and oxalacetate transporter (YHM2: YALI0B10736g) and/oor the reduction in the activity of the citrate transport protein (CTP1: YALI0F26323g) in the yeast cells has proved to be particularly advantageous for the method according to the invention for the microbial production of ω-hydroxy fatty acids or dicarboxylic acids since as a result, a smaller quantity of organic acids is produced and/or is secreted in the culture medium.
Preferably the cultivation medium is a minimal medium. A minimal medium is understood in the sense of the present invention as an aqueous culture medium which contains all the substrates required for the growth of yeast cells (for example, C source, mineral salts, trace elements, vitamins) in dissolved form but which at the same time has no complex ingredients or complex biomolecules (e.g. yeast extract, peptone, tryptone etc.). The person skilled in the art is familiar with a plurality of minimal media for the cultivation of yeast cells from the prior art.
In addition to the hydrophobic substrate supplied for the bioconversion, another carbon source (C source; substrate for energy recovery) on the basis of which the cell can acquire energy, needs to be provided for the cultivation.
Preferably therefore mono- or disaccharides, for example, glucose, D-fructose, xylose, maltose, saccharose and/or polyvalent alcohols, such as for example, glycerol, raw glycerol and mannitol are added to the cultivation medium as C source, glucose being particularly preferred as the added carbon source.
In particular, ammonium/ammonia or amino acids such as glutamine/glutamate serve as nitrogen source (N source) for the cultivation of yeast strains in the method according to the invention.
For the cultivation of a strain according to the invention, cultivation conditions under which the lipid bodies are comparatively weakly defined and the conversion rate of the supplied substrates are comparatively strongly defined are to be preferred.
A limitation of the cell growth prevents too large quantities of the substrate used being stored in the cells, which reduces the product yield, and prevents the occurrence of oxygen limitation which lowers the productivity.
A possible form of limitation is nitrogen limitation. This limitation in the yeast Y. lipolytica results in a stoppage of growth after consumption of the nitrogen source and in the formation of citric acid and iso-citric acid. Preferably the nitrogen content in the medium during the production phase, i.e. after contact of the yeast cells with the hydrophobic substrate, should be less than 2 g 1−1, preferably less than 1 g 1−1, highly preferably less than 0.5 g 1−1, most preferably 0 g 1−1. The production phase is characterized in that it follows a phase of strong growth (growth phase) and only exhibits (compared to the growth rate) low growth but increased product formation.
Preferably cultivation takes place in the fermenter under controlled process parameters, where particularly preferably the pH is kept constant in the cultivation medium (during a specific growth phase). In particular, the fermentation takes place under limitation conditions.
The pH in the cultivation medium after contact, i.e. during the growth phase, should correspond to the physiologically preferred value of 5.5 and during the production phase, i.e. after contact of the yeast cells with the hydrophobic substrate, should preferably be 5.5-9.0, particularly preferably 7.0-9.0 and especially preferably 8.0.
Preferably the yeast cells are cultivated under physiological conditions at a temperature between 0 and 60° C., preferably between 10 and 50° C., particularly preferably between 20 and 40° C., where the pH of the cultivation medium lies between 2.0 and 9.0, preferably between 5.5 and 9.0, particularly preferably between 7.0 and 9.0, especially preferably around 8.0.
Preferably in the method according to the invention, the said limitation conditions are combined with one another.
The subject matter of the invention is also a kit for the biocatalytic production of dicarboxylic acids or ω-hydroxy fatty acids containing:
The biomass in the form of yeast cells for the method according to the invention for the biocatalytic production of dicarboxylic acids or ω-hydroxy fatty acids can be obtained in a manner known to the person skilled in the art involving pre-cultivation of the corresponding yeast cells to be used (propagation on full medium and/or minimal medium, for example, by cultivation in full medium such as YPD-Medium (DSM-Medium No. 393), but preferably by cultivation in a medium which allows the production of high cell densities, for example, by cultivation in minimal medium (for example, 5% (w/v) glucose, 20 g CaCO3, 1 g 1−1 KH2PO4, 0.16 g 1−1 K2HPO4×3 H2O, 3 g 1−1 (NH4)2SO4, 0.7 g 1−1 MgSO4×7 H2O, 0.5 g 1−1 NaCl, 0.4 g 1−1 Ca(NO3)2×4 H2O, 0.5 mg 1−1 H3BO3, 0.0 mg 1−1 CuSO4×5 H2O, 0.1 mg 1−1 KI, 0.4 mg 1−1 MnSO4×4 H2O, 0.2 mg 1−1 Na2MoO4×2 H2O, 0.4 mg 1−1 ZnSO4×7 H2O, 6 mg 1−1 FeCl3×6 H2O, 0.3 mg 1−1 thiamine hydrochloride) in particular, greater than 1×109 cells per ml. The propagation of this pre-culture of the yeast cells of the kit according to the invention is preferably accomplished in conventional laboratory shaking flasks, but propagation under controlled conditions in the fermenter is also possible to produce larger quantities of biomass.
A further aspect of the invention also relates to the use of yeast cells modified according to the invention of the yeast strain of the type Y. lipolytica, a method according to the invention or a kit according to the invention.
Preferred further developments of the invention are obtained from the combinations or the claims or individual features thereof.
The invention will be explained in detail hereinafter with reference to some exemplary embodiments and relevant figures. The following examples are used to further explain the invention without restricting the invention to these.
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For construction of the deletion cassettes for the POX genes, the respective promoter and terminator region was amplified by PCR, where the primers pXXX_fw/pXXX_rv and XXXt_fw/XXXt_rv were used (XXX stands for the gene to be deleted). Genomic DNA of Y. lipolytica H222 was used as template here.
By using overhang primers, an I-SceI-restriction interface was inserted at the end of the promoter and at the beginning of the terminator region. Promoter and terminator fragment were fused together by overlap-PCR with the primers pXXX_fw and XXXt_rv. The overlap fragment was then ligated into the vector pJET1.2/blunt (Fermentas; SEQ ID NO 1). The resulting plasmid was cut with I-SceI and the loxP-URA3-loxP cassette, which was obtained from the pJMP113 plasmid (Fickers P, Le Dall M T, Gaillardin C, Thonart P & Nicaud J M (2003) New disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia lipolytica. J Microbiol Methods 55: 727-737) using the restriction enzyme I-SceI was inserted.
The respective deletion cassette was obtained by PCR or restriction and transformed in Y. lipolytica H222-S4, which can be produced from Y. lipolytica H222 (Mauersberger, S., H. J. Wang, et al. (2001), J Bacteriol 183(17): 5102-5109), according to Barth and Gaillardin (Barth G & Gaillardin C (1996) Yarrowia lipolytica. Springer-Verlag, Berlin, Heidelberg, New York).
Before a renewed transformation, the marker was recovered with the aid of the Cre-lox recombination systems (Fickers P, Le Dall M T, Gaillardin C, Thonart P & Nicaud J M (2003) New disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia lipolytica. J Microbiol Methods 55: 727-737).
The successful deletion of a gene was confirmed by PCR where the primers pXXX_fw and XXXt_rv as well as pXXX_fw and XXXt_rv2. XXXt_rv2 binds in the region of the gene to be deleted but outside the deletion cassette.
The strain which carried the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 was called H222ΔP and forms the basis for the construction of strains which can be used for the biotechnological production of ω-hydroxy fatty acids and dicarboxylic acids.
attaccctattatccctaTGAAGGTTGCAGTCGTAGTC
tagggataacagggtaatTGCGATCTCGATGAGTGATG
attaccctgttatccctaCGTCGAGGAAGTAGGICATC
tagggataacagggtaatGCGAGCTTGATGAGGAATAG
tagggataacagggtaatAGGACGCACAACGCCATCAC
attaccctgttatccctaCGCTCCCATTGGAAACTA
tagggataacagggtaatATGTCTCTAGGGTCGAAGTC
attaccctattatccctaTGGCAAGCCTCACTACTACG
tagggataacagggtaatACGTCTCGGACCTTGAATTG
attaccctgttatccctaCCTTCAACCTGTCCGACTTC
tagggataacagggtaatGGTCCATAAGCAGAGTGTTC
attaccctgttatccctaACCCTCGACCTCCTTATTAC
For construction of the deletion cassettes for the (fatty) alcohol dehydrogenase and oxidase genes, the respective promoter and terminator region was amplified by PCR, where the primers pXXX_fw/pXXX_rv and XXXt_fw/XXXt_rv were used (XXX stands for the gene to be deleted). Genomic DNA of Y. lipolytica H222 was used as template here.
For the construction of the deletion cassettes for the genes ADH1-ADH6 and FAO1 a BamHI restriction interface was inserted by using overhang primers at the end of the promoter and at the beginning of the terminator region. In addition, an HindIII-restriction interface was attached at the beginning of the promoter region and an NdeI-restriction interface was attached at the end of the terminator region (ADH1: NotI, FAO1: EcoRI). The fragments were ligated into the vector pJET1.2/blunt (Fermentas; SEQ ID NO 1) or pUCBM21 (Boehringer Ingelheim; SEQ ID NO 2). The plasmids thus obtained were linearized with BamHI and the URA blaster (TcR-URA3-TcR-Kassette), which was obtained from the plasmid pUC-Lys2-DK2 (SEQ ID NO 3) by restriction with BamHI and BglII, was inserted.
For the construction of the ADH7 deletion vector, the complete gene (including promoter and terminator region) was amplified by means of PCR, where the primers pADH7_fw and ADH7t rv were used. The fragment obtained was ligated into the vector pJET1.2/blunt (Fermentas; SEQ ID NO 1), the open reading frame of the ADH7 gene was removed by restriction with SanDI and NsiI and the URA blaster, which was obtained from the plasmid pUC-Lys2-DK2 by restriction with SanDI and NsiI, was inserted.
The respective deletion cassette was obtained by PCR or restriction and transformed in Y. lipolytica H222-S4, which can be produced from Y. lipolytica H222 (Mauersberger, S., H. J. Wang, et al. (2001), J Bacteriol 183(17): 5102-5109), according to Barth and Gaillardin (Barth G & Gaillardin C (1996) Yarrowia lipolytica. Springer-Verlag, Berlin, Heidelberg, New York).
Before a renewed transformation, the marker was recovered by FOA selection (Boeke J D, La-Croute F & Fink G R (1984) Mol Gen Genet 197: 345-346).
The successful deletion of a gene was confirmed by PCR, where the primers pXXX_fw and XXXt_rv as well as pXXX_fw and XXXt_rv2 were used. XXXt_rv2 binds in the region of the gene to be deleted but outside the deletion cassette.
The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also FADH, AHD1, AHD2, AHD3, AHD4, AHD5, AHD6 and AHD7 was called H222ΔPΔA.
The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also FAO1 was called H222ΔPΔF.
In order to check whether the initial strains produced Yarrowia lipolytica H222ΔP, H222ΔPΔA and H222ΔPΔF are suitable for the production of larger quantities of ω-hydroxy fatty acids and dicarboxylic acids, these were cultivated in minimal medium with glycerol and various n-alkanes (dodecane, pentadecane, hexadecane) and fatty acids (dodecanoic acid, pentadecanoic acid, hexadecanoic acid) (3% (v/v) glycerol+0.5% (v/v) glycerol after 48 h, 1% (v/v) n-alkane or 1% (v/v) fatty acid, 20 g 1−1 CaCO3, 17.3 g 1−1 KH2PO4, 1.35 g 1−1 K2HPO4×3 H2O, 3 g 1−1 (NH4)2SO4, 0.7 g 1−1 MgSO4×7 H2O, 0.5 g 1−1 NaCl, 0.4 g 1−1 Ca(NO3)2×4 H2O, 0.5 mg 1−1 H3BO3, 0.04 mg 1−1 CuSO4×5 H2O, 0.1 mg 1−1 KI, 0.4 mg 1−1 MnSO4×4 H2O, 0.2 mg 1−1 Na2MoO4×2 H2O, 0.4 mg 1−1 ZnSO4×7 H2O, 6 mg 1−1 FeCl3×6 H2O, 0.3 mg 1−1 thiamine hydrochloride). The quantities of ω-hydroxy fatty acids (ω-hydroxydodecanoic acid, ω-hydroxypentadecanoic acid, ω-hydroxyhexadecanoic acid) and dicarboxylic acids formed (dodecanoic diacid, pentadecanoic diacid, hexadecanoic diacid) were determined by means of gas chromatography 96 h after cultivation (
Y. lipolytica H222ΔP here formed relatively large quantities of dicarboxylic acids and can thus be used as initial strain for the construction of a corresponding production strain for dicarboxylic acids.
Both Y. lipolytica H222ΔPΔA and also H222ΔPΔF formed increased quantities of ω-hydroxy fatty acids and can thus be used as initial strains for the construction of corresponding production strains for ω-hydroxy fatty acids. Y. lipolytica H222ΔPΔF is to be preferred to H222ΔPΔA here.
Both n-alkanes and also fatty acids can be converted from all strains to ω-hydroxy fatty acid or dicarboxylic acid having the same chain length.
Different strains (e.g. Yarrowia lipolytica H222ΔP and H222ΔPΔF) were cultivated in different media under various conditions, where it was found that the media and conditions used are variously well suited.
The yeasts were cultivated comparatively in minimal medium with glucose (3% (w/v) glucose, 1% (w/v) pentadecanoic acid, 17.3 g 1−1 KH2PO4, 1.35 g 1−1 K2HPO4×3 H2O, 3 g 1−1 (NH4)2SO4, 0.7 g 1−1 MgSO4×7 H2O, 0.5 g 1−1 NaCl, 0.4 g 1−1 Ca(NO3)2×4 H2O, 0.5 mg 1−1 H3BO3, 0.04 mg 1−1 CuSO4×5 H2O, 0.1 mg 1−1 KI, 0.4 mg 1−1 MnSO4×4 H2O, 0.2 mg 1−1 Na2MoO4×2 H2O, 0.4 mg 1−1 ZnSO4×7 H2O, 6 mg 1−1 FeCl3×6 H2O, 0.3 mg 1−1 thiamine hydrochloride) and glycerol (3% (v/v) glycerol, 1% (w/v) pentadecanoic acid, 17.3 g 1−1 KH2PO4, 1.35 g 1−1 K2HPO4×3 H2O, 3 g 1−1 (NH4)2SO4, 0.7 g 1−1 MgSO4×7 H2O, 0.5 g 1−1 NaCl, 0.4 g 1−1 Ca(NO3)2×4 H2O, 0.5 mg 1−1 H3BO3, 0.04 mg 1−1 CuSO4×5 H2O, 0.1 mg 1−1 KI, 0.4 mg 1−1 MnSO4×4 H2O, 0.2 mg 1−1 Na2MoO4×2 H2O, 0.4 mg 1−1 ZnSO4×7 H2O, 6 mg 1−1 FeCl3×6 H2O, 0.3 mg 1−1 thiamine hydrochloride) as carbon source, where after 48 h 1% (w/v) glucose, or 1% (v/v) glycerol were after-fed. After 96 h, all the strains studied (e.g. Y. lipolytica H222ΔP, H222ΔPΔA and H222ΔPΔF) formed larger quantities of ω-hydroxy fatty acid or pentadecanoic diacid for growth in minimal medium with glucose than for growth in minimal medium with glycerol.
The yeasts were then cultivated in full medium with glucose and pentadecanoic acid (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose and 1% (w/v) pentadecanoic acid in 1% Tween 80), where 1% (w/v) glucose was after-fed when this was used up. After 4 days, 1% (w/v) of pentadecanoic acid in 1% Tween 80 was after-fed. Under these conditions, almost no ω-hydroxy pentadecanoic acid or pentadecanoic diacid was formed (each<0.1 g 1−1).
The yeasts were cultivated in minimal medium with glucose and pentadecanoic acid (3% (v/v) glucose, 1% (w/v) pentadecanoic acid in 1 Tween 80, 17.3 g 1−1 KH2PO4, 1.35 g 1−1K2HPO4×3 H2O, 3 g 1−1 (NH4)2SO4, 0.7 g 1−1 MgSO4×7 H2O, 0.5 g 1−1 NaCl, 0.4 g 1−1 Ca(NO3)2×4 H2O, 0.5 mg 1−1 H3BO3, 0.04 mg 1−1 CuSO4×5 H2O, 0.1 mg 1−1 KI, 0.4 mg 1−1 MnSO4×4 H2O, 0.2 mg 1−1 Na2MoO4×2 H2O, 0.4 mg 1−1 ZnSO4×7 H2O, 6 mg 1−1 FeCl3×6 H2O, 0.3 mg 1−1 thiamine hydrochloride), where 1% (w/v) glucose was after-fed when this was used up. After 4 days, 1% (w/v) pentadecanoic acid in 1% Tween 80 was after-fed. Under these cultivation conditions, the strain Y. lipolytica H222ΔPΔF after 10 days formed around 0.7 g 1−1ω-hydroxy pentadecanoic acid and 2.4 g 1−1 pentadecanoic diacid.
The yeasts were cultivated in minimal medium with glucose and pentadecanoic acid (3% (v/v) glucose, 1% (w/v) pentadecanoic acid in 1 Tween 80, 17.3 g 1−1 KH2PO4, 1.35 g 1−1K2HPO4×3 H2O, 3 g 1−1 (NH4)2SO4, 0.7 g 1−1 MgSO4×7 H2O, 0.5 g 1−1 NaCl, 0.4 g 1−1 Ca(NO3)2×4 H2O, 0.5 mg 1−1 H3BO3, 0.04 mg 1−1 CuSO4×5 H2O, 0.1 mg 1−1 KI, 0.4 mg 1−1 MnSO4×4 H2O, 0.2 mg 1−1 Na2MoO4×2 H2O, 0.4 mg 1−1 ZnSO4×7 H2O, 6 mg 1−1 FeCl3×6 H2O, 0.3 mg 1−1 thiamine hydrochloride), where the glucose was after-fed so that its concentration in the medium was between 1 and 3% (w/v). After 5 days 1% (w/v) pentadecanoic acid in 1% Tween 80 was after-fed. Under these cultivation conditions the strain Y. lipolytica H222ΔPΔF after 7 days formed around 3.9 g 1−1 ω-hydroxy pentadecanoic acid and 1.3 g 1−1 pentadecanoic diacid.
To sum up, it should be noted that minimal medium is better suited for the cultivation than full medium. Furthermore, glucose is more suitable than glycerol as carbon source for the energy preparation. The glucose should be after-fed in the course of the cultivation so that the glucose is not used up (i.e. its concentration does not drop to 0 g 1−1).
For construction of the deletion cassettes for the genes DGA1, PAH1 and SCT1 the respective promoter and terminator region was amplified by PCR where the primers pXXX_fw/pXXX_rv and XXXt_fw/XXXt_rv were used (XXX stands for the gene to be deleted). Genomic DNA of Y. lipolytica H222 was used as template here.
For the construction of deletion cassettes for the genes DGA1, PAH1 and SCT1 a BamHI-restriction interface was inserted by using overhang primers at the end of the promoter region and at the beginning of the terminator region. In addition a HindIII-restriction interface was attached at the beginning of the promoter region and an EcoRI-restriction interface was attached at the end of the terminator region. The fragments were ligated into the vector pUCBM21 (Boehringer Ingelheim; SEQ ID NO 2). The plasmids thus obtained were linearized with BamHI and the URA blaster (TcR-URA3-TcR-cassette), which was obtained from the plasmid pUC-Lys2-DK2 (SEQ ID NO 3) by restriction with BamHI and BglII was inserted. The respective deletion cassette was obtained by PCR or restriction and transformed in Y. lipolytica H222-S4, which can be produced from Y. lipolytica H222 (Mauersberger, S., H. J. Wang, et al. (2001), J Bacteriol 183(17): 5102-5109), according to Barth and Gaillardin (Barth G & Gaillardin C (1996) Yarrowia lipolytica. Springer-Verlag, Berlin, Heidelberg, New York).
Before a renewed transformation, the marker was recovered by FOA selection (Boeke J D, La-Croute F & Fink G R (1984) Mol Gen Genet 197: 345-346).
The successful deletion of a gene was confirmed by PCR, where the primers pXXX_fw and XXXt_rv as well as pXXX_fw and XXXt_rv2 were used. XXXt_rv2 binds in the region of the gene to be deleted but outside the deletion cassette.
The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also DGA1 was called H222ΔPΔD.
The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also PAH1 was called H222ΔPΔH.
The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also SCT1 was called H222ΔPΔS.
The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also FAO1 and DGA1 was called H222ΔPΔFΔD.
The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also FAO1 and PAH1 was called H222ΔPΔFΔH.
The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also FAO1 and SCT1 was called H222ΔPΔFΔS.
The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also FADH, ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, FAO1 and DGA1 was called H222ΔPΔAΔFΔD.
The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also FADH, ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, FAO1 and PAH1 was called H222ΔPΔAΔFΔH.
The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also FADH, ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, FAO1 and SCT1 was called H222ΔPΔAΔFΔS.
The strains Y. lipolytica H222ΔPΔFΔD, H222ΔPΔFΔH and H222ΔPΔFΔS as well as the initial strain Y. lipolytica H222ΔPΔF were cultivated in minimal medium with glucose and pentadecanoic acid (3% (v/v) glucose, 1% (w/v) pentadecanoic acid, 17.3 g 1−1 KH2PO4, 1.35 g 1−1 K2HPO4×3 H2O, 3 g 1−1 (NH4)2SO4, 0.7 g 1−1 MgSO4×7 H2O, 0.5 g 1−1 NaCl, 0.4 g 1−1 Ca(NO3)2×4 H2O, 0.5 mg 1−1 H3BO3, 0.04 mg 1−1 CuSO4×5 H2O, 0.1 mg 1−1 KI, 0.4 mg 1−1 MnSO4×4 H2O, 0.2 mg 1−1 Na2MoO4×2 H2O, 0.4 mg 1−1 ZnSO4×7 H2O, 6 mg 1−1 FeCl3×6 H2O, 0.3 mg 1−1 thiamine hydrochloride). Every 24 h the glucose concentration was adjusted to 3% (w/v) and after 3 days 1% (w/v) of pentadecanoic acid was after-fed. The quantities of ω-hydroxy fatty acid and pentadecanoic acid were determined after cultivation for 8 days by means of gas chromatography. The cells were examined under the microscope, where the lipid bodies were stained with Nile red and could thus be detected by fluorescence microscopy (
Here it can be identified that the size of the lipid bodies was significantly reduced due to deletions of PAH1 and SCT1 under the said cultivation conditions. As already shown, this could not be determined for the already described deletion of DGA1 (Thevenieau F (2006) Institut National Agronomique Paris-Grignon, PhD thesis; Athenstaedt K (2011) Biochim Biophys Acta 1811: 587-596).
The availability of strains with reduced lipid bodies forms the basis for the development of strains for the efficient production of ω-hydroxy fatty acids and dicarboxylic acids since the esterification of the supplied substrates with glycerol and its incorporation into the lipid bodies reduces the conversion rate.
For construction of a vector for overexpression of FAO1 a part of the constitutive promoter of the translation elongation factor 1 alpha-gene (TEF1: YALI0C09141g) was obtained by means of PCR, where the primers pTef_SpeI_fw3 and pTef_FAOo_ol_rv were used. The plasmid pINTB_HMG1 (SEQ ID NO 5) was used as template here.
Furthermore, the FAO1-gene (YALI0B14014g) was amplified by means of PCR using the primers pTef_FAOo_ol_fw and FAO1o_SphI_rv (template: genomic DNA of Y. lipolytica H222).
By means of the primers pTef_FAOo_ol_rv and pTef_FAOo_ol_fw an overhang region was attached, with the aid of which both PCR fragments were linked by means of overlap PCR using the primers pTef_SpeI_fw3 and FAO1o_SphI_rv. The overlap fragment was then ligated into the vector pJET1.2/blunt (Fermentas; SEQ ID NO 1), cut out from this using the restriction enzymes SpeI and SphI and ultimately ligated into the backbone of the plasmid pINTB_HMG1 (SEQ ID NO 5) cut with SpeI and SphI, where the plasmid pINTB-FAO1 was formed.
Furthermore, an integration platform was obtained by means of PCT using the primers INT_AscI_fw KpnI and INT_AscI_rv_KpnI, cut with KpnI and ligated into the backbone of the vector pINTB-FAO1 obtained by restriction digestion with Kpnl, where the plasmid pINTC-FAO1 was formed.
The plasmid pINTC-FAO1 was ultimately linearized with AscI and transformed into the desired Y. lipolytica recipient strain.
Successful integration of the FAO1 gene was confirmed by PCR, where the primers INT_AscI_fw_out and INT_AscI_rv_out were used which bind in the genomic DNA outside the integrated construct.
The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also the additional copy of FAO1 under control of the TEF1-promoter was called H222ΔPoF and was used as initial strain for construction of further strains for the production of dicarboxylic acids.
gcatgcTTAGATTCGAGGTCGGAGAT
ggtacCACGCACGGATAGTTTATCCA
ggtacCCAAAGTCAACTAATGTCAAG
For construction of a vector for overexpression of FAO1 a part of the constitutive promoter of the translation elongation factor 1 alpha-gene (TEF1: YALI0C09141g) was obtained by means of PCR, where the primers pTef_SpeI_fw3 and pTEF_CPR1_ol_fw were used. The plasmid pINTB_HMG1 (SEQ ID NO 5) was used as template here.
Furthermore, the CPR1-gene (YALI0D04422g) was amplified by means of PCR using the primers pTEF_CPR1_ol_fw and CPR1_SphI_rv (template: genomic DNA of Y. lipolytica H222).
By means of the primers pTEF_CPR1_ol_rv and pTEF_CPR1_ol_fw an overhang region was attached, with the aid of which both PCR fragments were linked by means of overlap PCR using the primers pTef_SpeI_fw3 and CPR1_SphI_rv. The overlap fragment was then ligated into the vector pJET1.2/blunt (Fermentas; SEQ ID NO 1), cut out from this using the restriction enzymes SpeI and SphI and ultimately ligated into the backbone of the plasmid pINTB_HMG1 (SEQ ID NO 5) cut with SpeI and SphI, where the plasmid pINTB-CPR1 was formed.
The resulting plasmid was ultimately linearized with NotI and transformed into the desired Y. lipolytica recipient strain.
The successful integration of the CPR1-gene was confirmed by PCR, where the primers INTB_out_fw and INTB_out_rv were used, which bind in the genomic DNA outside the integrated construct.
The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also the additional copy of CPR1 under control of the TEF1-promoter was called H222ΔPoC and was used as initial strain for construction of further strains for the production of dicarboxylic acids.
The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5, PPX6, FADH, ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, FAO1 and also the additional copy of CPR1 under control of the TEF1-promoter was called H222ΔPΔAΔFoC.
In order to check how far different genetic modifications affect the production of ω-hydroxy fatty acids and/or dicarboxylic acids, the strains Yarrowia lipolytica H222ΔP, H222ΔPΔA, H222ΔPΔF, H222ΔPΔAΔF, H222ΔPoF, H222ΔPoC, H222ΔPΔD, H222ΔPΔH, H222ΔPΔS, H222ΔPΔAΔFoC, H222ΔPΔAΔFΔD, H222ΔPΔAΔFΔH and H222ΔPΔAΔFΔS, whose construction was described in Examples 1, 2, 5, 6 and 7 were cultivated in the fermenter. The cultivation medium in this case was minimal medium with glucose (5% (w/v) glucose, 1 g 1−1 KH2PO4, 0.16 g 1−1 K2HPO4×3 H2O, 3 g 1−1 (NH4)2SO4, 0.7 g 1−1 MgSO4×7 H2O, 0.5 g 1−1 NaCl, 0.4 g 1−1 Ca(NO3)2×4 H2O, 0.5 mg 1−1 H3BO3, 0.04 mg 1−1 CuSO4×5 H2O, 0.1 mg 1−1 KI, 0.4 mg 1−1 MnSO4×4 H2O, 0.2 mg 1−1 Na2MoO4×2 H2O, 0.4 mg 1−1 ZnSO4×7 H2O, 6 mg 1−1 FeCl3×6 H2O, 0.3 mg 1−1 thiamine hydrochloride). Cultivation was carried out in the fermenter at 28° C. The oxygen saturation was set at 55%. The cells were inoculated with an OD600 of 1 and incubated for 24 h at pH 5.5 (automated titration of HCl and NaOH). Then 15 g 1−1 DD was added and the pH was set to 8.0. Glucose was added every 24 h to adjust a final concentration of 5% (w/v).
Y. lipolytica H222ΔP here formed relatively large quantities of dicarboxylic acids and can thus be used as initial strain for the construction of a corresponding production strain for dicarboxylic acids (
Y. lipolytica H222ΔPΔA and also H222ΔPΔF formed increased quantities of ω-hydroxy fatty acids and can thus be used as initial strains for the construction of corresponding production strains for ω-hydroxy fatty acids. Here Y. lipolytica H222ΔPΔF is to be preferred compared with H222ΔPΔA (
Y. lipolytica H222ΔPΔAΔF formed only small quantities of dicarboxylic acids and is thus the preferred initial strain for the construction of production strains for ω-hydroxy fatty acids (
The overexpressions of FAO1 and CPR1 in the strain Y. lipolytica H222ΔP (resulting strains: Y. lipolytica H222ΔPoF and H222ΔPoC) in each case resulted in an increased production of dicarboxylic acids (
The deletion of DGA1 in the strain Y. lipolytica H222ΔP (resulting strain: Y. lipolytica H222ΔPΔD) resulted in an increased production of dicarboxylic acids whereas the deletions of PAH1 and SCT1 (resulting strains: Y. lipolytica H222ΔPΔH and H222ΔPΔS) did not significantly increase the production of dicarboxylic acids (
The overexpression of CPR1 in the strain Y. lipolytica H222ΔPΔAΔF (resulting strain: Y. lipolytica H222ΔPΔAΔFoC) resulted in an increased production of dicarboxylic acids but not of ω-hydroxy fatty acids (
The deletions of DGA1, PAH1 and SCT1 in the strain Y. lipolytica H222ΔPΔAΔF (resulting strains: Y. lipolytica H222ΔPΔAΔFΔD, H222ΔPΔAΔFΔH and H222ΔPΔAΔFΔS) did not result in an increased production of ω-hydroxy fatty acids (
The enzyme activities of the (fatty) alcohol dehydrogenases and oxidase were determined in cell lysates of the strains Yarrowia lipolytica H222ΔP, H222ΔPΔA, H222ΔPΔF, H222ΔPΔAΔF and H222ΔPoF. The cells were cultivated as described in Example 8 and harvested and macerated after three days.
The (fatty) alcohol dehydrogenase and oxidase activities were performed as described in Matatiele (2005) (Matatiele P R (2005) PhD thesis, University of the Free State, Republic of South Africa).
The (fatty) alcohol dehydrogenase assay (50 mM Tris-HCl pH 8.5, 1.3 mM dodecan-1-ol in DMSO, 2 mM NAD+, 2 mM NAP+, 1.5% (v/v) cell extract) was measured using a recording spectrophotometer at 30° C. and λ=340 nm and the enzyme activity was calculated with the aid of the determined increase in extinction (εNAD(P)H=6.3 mM−1 cm−1).
The (fatty) alcohol oxidase assay (50 mM glycine NaOH pH 9.0, 0.35 mM dodecan-1-ol in DMSO, 0.013% (w/v) peroxidase (150 U/mg), 0.044% (w/v) ABTS, 6 mM sodium azide, 1-5% (v/v) cell extract) was measured using a recording spectrophotometer at λ=405 nm and the enzyme activity was calculated with the aid of the determined increase in extinction (εABTSox=18.4 mM−1 cm−1).
Surprisingly no clear difference of the (fatty) alcohol dehydrogenase was determined between the strains (
As was expected, the (fatty) alcohol oxidase activity could only be detected in the strains Y. lipolytica H222ΔP and H222ΔPΔA and not in the FAO1-deletion strains Y. lipolytica H222ΔPΔF and H222ΔPΔAΔF (
In order to check whether the production strains are capable of producing larger quantities of dicarboxylic acids and/or ω-hydroxy fatty acids and accumulating in the culture medium, the strain Y. lipolytica H222ΔPΔF was cultivated for 7 d in the fermenter. The cultivation medium here was minimal medium with glucose and an increased quantity of mineral salts, trace elements and vitamins (5% (w/v) glucose, 2 g 1−1 KH2PO4, 0.32 g 1−1 K2HPO4×3 H2O, 6 g 1−1 (NH4)2SO4, 1.4 g 1−1 MgSO4×7 H2O, 1 g 1−1 NaCl, 0.8 g 1−1 Ca(NO3)2×4 H2O, 1 mg 1−1 H3BO3, 0.08 mg 1−1 CuSO4×5 H2O, 0.2 mg 1−1 KI, 0.8 mg 1−1 MnSO4×4 H2O, 0.4 mg 1−1 Na2MoO4×2 H2O, 0.8 mg 1−1 ZnSO4×7 H2O, 12 mg 1−1 FeCl3×6 H2O, 0.6 mg 1−1 thiamine hydrochloride). Cultivation was carried out in a fermenter at 28° C. The oxygen saturation was adjusted to 55%. The cells were inoculated with an OD600 of 1 and incubated for 2 d at pH 5.5 (automated titration of HCl and NaOH). After 2 d the pH was adjusted to 8.0 and 15 g 1−1 of dodecane was added after 2 d and 3 d. Glucose was added every 24 h in order to adjust a final concentration of 5-10% (w/v).
After cultivation for 6 d (i.e. after 4 d in the production phase), Y. lipolytica H222ΔPΔF formed 29.5 g 1−1 of ω-hydroxydodecanoic acid and 3.5 g 1−1 of dodecanoic acid (
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2013 022 175 | Dec 2013 | DE | national |
This application is a continuation of U.S. patent application Ser. No. 15/103,191, filed on Jun. 9, 2016, which is itself a U.S. national stage of International Application No. PCT/EP2014/077237 filed on Dec. 10, 2014, which itself claims the benefit of priority under 35 U.S.C. § 119 of German Application No. DE 102013022175.7, filed on Dec. 12, 2013; all applications are incorporated by reference herein in their entirety.
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| Number | Date | Country | |
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| 20180371510 A1 | Dec 2018 | US |
| Number | Date | Country | |
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| Parent | 15103191 | US | |
| Child | 16102213 | US |
