Production of fatty alcohols (Machine-translation by Google Translate, not legally binding)

专利摘要:
Production of fatty alcohols. The present invention relates to the production of fatty alcohols by the cultivation of a microorganism of the rhodosporidium toruloides species, by the insertion of a gene that encodes an enzyme with acyl-coa reductase activity that allows to produce fatty alcohols in the presence of different sources of carbon. (Machine-translation by Google Translate, not legally binding)
公开号:ES2579384A1
申请号:ES201530156
申请日:2015-02-10
公开日:2016-08-10
发明作者:Sandy Christiane FILLET；Mª Del Carmen RONCHEL BARRENO；Beatriz Suárez González；Armando Lara Cambil；José Luis Adrio Fondevila；Javier Velasco Álvarez
申请人:Neol Biosolutions SA；
IPC主号:

专利说明:

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i) culturing the microorganism of the invention in a culture medium comprising at least one carbon source and at least one nitrogen source under conditions suitable for the growth of said microorganism;
5 ii) separate microbial biomass from the culture broth; and iii) extract the fatty alcohols from the microbial biomass and / or from the
culture obtained in step (ii) In another aspect, the invention relates to a process for obtaining biosurfactants comprising:
I) cultivating a microorganism of the invention in a culture medium comprising at least one carbon source and at least one nitrogen source under conditions suitable for the growth of said microorganism;
ii) separate the microbial biomass from the culture broth; Iii) extract the fatty alcohols from the microbial biomass and / or from the culture broth obtained in step (ii); and iv) converting the mixture of fatty alcohols obtained in step (iii) into biosurfactants. In another aspect, the present invention relates to the use of the microorganism according to the invention to obtain a microbial biomass enriched in fatty alcohols according to the first process of the invention.
In another aspect, the present invention relates to the use of the microorganism according to the invention to obtain a preparation rich in fatty alcohols according to the second process of the invention.
In another aspect, the present invention relates to the use of the microorganism according to the invention to obtain biosurfactants according to the third method of the invention.
30 BRIEF DESCRIPTION OF THE FIGURES
Figure 1: pNEOL102 integrative plasmid Figure 2: Production of fatty alcohols in different culture media.
35 DETAILED DESCRIPTION OF THE INVENTION
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Microorganism of the invention
In a first aspect, the present invention relates to a microorganism, hereinafter "microorganism of the invention", wherein the microorganism belongs to the genetically modified Rhodosporidium toruloides species with a gene encoding an enzyme with acyl-CoA reductase activity, in where said enzyme has the capacity to produce fatty alcohols.
The term "microorganism" or "microbe", as used herein, refers to a microscopic organism capable of producing fatty alcohols, which can be unicellular or multicellular. In particular, the microorganism of the invention is a yeast of the Rhodosporidium toruloides species.
The term "gene", as used herein, refers to a deoxyribonuclotide chain that encodes a protein. In the specific case of the invention, said term refers to a deoxyribonucleotide chain that encodes an acyl-CoA reductase enzyme capable of producing fatty alcohols.
The term "enzyme", in the context of the present invention, refers to a protein that functions as a highly selective catalyst, accelerating both the speed and the specificity of the metabolic reaction for which it is specific.
In particular, the microorganism of the invention is a genetically modified microorganism. The term "genetically modified microorganism" as used herein, refers to a microorganism whose genetic material has been altered using genetic engineering techniques. Accordingly, said genetically modified microorganism expresses an enzyme that has an acyl-CoA reductase activity, compared to a corresponding unmodified control microorganism of the same strain. In the context of the present invention, the enzyme having acyl-CoA reductase activity may be encoded by a gene encoding said enzyme in another organism. Generally, the gene encoding an enzyme that has acyl-CoA reductase activity can be introduced into the microorganism in any suitable form, for example, comprised in a vector, a plasmid or as a naked nucleic acid. After being introduced into the microorganism, the gene can be expressed exogenously if it is expressed in a vector / plasmid in the microorganism [ie, outside of the microbial chromosome (s)], or it can be incorporate into the microbial chromosome (s) by random (ectopic) recombination or
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homologous or any other suitable method known in the state of the art. Appropriate techniques that allow genetic transformation in yeasts include but are not limited to:
-Transformation of spheroplasts that involves eliminating the cell wall of yeast 5 and putting the spheroplasts in contact with the plasmid in the presence of PEG.
-Transformation with Li +, which involves the treatment of yeast cells with monovalent alkali cations (Na +, K +, Rb +, Cs + and Li +) in combination with PEG to stimulate the uptake of DNA by intact cells.
-Genic bombing that involves bombing the cells with microprojectiles 10 coated with the exogenous DNA.
-Electroporation, which involves administering electrical pulses to the yeasts that produces the opening of pores in the spheroplast membrane and intact yeast cells.
-Transformation mediated by Agrobacterium tumefaciens (ATMT) is based on the use of the gene transfer capacity that A. tumefaciens has in a way
natural. Transformants are grown in a suitable nutrient medium and under selection conditions to ensure retention of endogenous DNA. The insertion of the gene encoding an acyl-CoA reductase into said transformants can be determined by any
20 appropriate molecular biology technique for this, for example, by Southern blot or PCR. Conventional methods of detecting and quantifying the expression of a gene can be found, for example, in Sambrook et al., 2001. "Molecular cloning: a Laboratory Manual", 3rd ed., Cold Spring Harbor Laboratory Press, NY, Vol. 1 -3.
The term "acyl-CoA reductase", or "fatty acyl-CoA reductase" as used in the
This document refers to polypeptides that have the ability to reduce fatty acyl-CoA molecules to the corresponding alcohols (E.C. 1.2.1.50). The acyl-CoA that reduce the fatty acyl-CoA molecules to the corresponding fatty alcohols catalyze the reaction by reducing four electrons of the active forms of the fatty acids to give rise to fatty alcohols.
The term "fatty alcohol", as used herein, refers to chain alcohols of between 4 and 26 carbon atoms, derivatives of natural oils and fats. Illustrative, non-limiting examples of fatty alcohols according to the present invention include, 1-hexanol (or hexyl alcohol), hepathanol (or heptyl alcohol). 1-octanol (or alcohol
Caprylic), 2-ethylhexanol, 1-nonanol (or pelargonic alcohol), 1-decanol (or capric alcohol or
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decyl alcohol), dodecanol (lauric alcohol), 1-tridecanol (tridecyl alcohol), 1tetratdecanol (myristyl alcohol), 1-pentadecanol (pentadecyl alcohol), 1-hexadecanol (cetyl alcohol or palmitic alcohol), cis-9-hexadecen-1 -ol (or palmitoleic alcohol, 1heptadecanol (or pentadecyl alcohol), 1-octadecanol (or stearyl alcohol or octadecyl alcohol), cis-9-octadecen-1ol (or oleic alcohol), 1 nonadecanol (or nonadecyl alcohol), 1-eicosanol (or arachidyl alcohol), docosanol (or behenic alcohol), 1-tetracosanol (lignoceric alcohol) or 1 hecacosanol (or serum alcohol).
In a particular and preferred embodiment of the invention, the gene encoding the enzyme having acyl-CoA reductase activity has optimized codons, for expression in the recombinant microorganism, that is in R. toruloides. Codons are known in the art that can be used for gene expression in R. toruloides. Illustrative non-limiting examples of optimized codons that can be employed for gene expression in include: UUU, UUC, UUA, UUG, CUU, CUC, CUA, AUU, AUC, AUG, GUU, GUC, GUA or GUG (Codon usage database , data source: NCBI-GenBank). In this context of the invention, the genetically modified microorganism is grown under the appropriate conditions that allow the expression of the gene having acyl-CoA reductase activity and thus produce fatty alcohols. Suitable culture media for the appropriate growth of different microorganisms are well known in the art. Normally said culture media comprise carbon sources, such as glucose, xylose, sucrose, glycerin, and appropriate nitrogen sources such as, for example, yeast extract, peptone, ammonium salts, macerated corn liquid, urea or sodium glutamate
Preferably, said gene is introduced into a replicative DNA expression or construct vector that allows expression of the gene encoding an enzyme with acyl-CoA reductase activity according to the present invention and that includes a transcriptional unit comprising the assembly of (1) genetic element (s) that plays a regulatory role in gene expression, for example, promoters, operators or enhancers, operably linked to (2) the sequence of the gene encoding an enzyme with acyl-CoA reductase activity according to the invention and which is transcribed into messenger RNA and translated into protein and (3) sequences suitable for initiating and terminating transcription and translation. Vectors that can be used in the context of the present invention typically include a genetic marker, an origin of replication in bacteria or yeasts, multiple cloning sites and a genetic marker. The genetic marker is usually a gene that confers resistance to an antibiotic, for example ampicillin or geneticin, or
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US5063154, the TET promoter, whose expression is regulated in the presence of tetracyclines, the GAL1-10, GALL, GALS promoters that are activated in the presence of galactose, the estrogen-inducible VP16-ER promoter, and the phosphatase promoter (PH05) whose expression is activated in the presence of phosphate and the protein promoter
5 HSP150 thermal shock, whose expression is activated at high temperature.
-Repressible promoters such as, for example, the promoter of the enolase gene (ENO-1) of S. cerevisiae, whose expression can be repressed when the microorganism is grown in a non-fermentable carbon source, as well as promoters whose expression is subjected to glucose repression so that the
The expression will be repressed when part of the lactose has been hydrolyzed and the concentration of glucose in the medium begins to increase, the glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP) promoter from R.toruloides, and the galactokinase promoter ( GAL1).
Preferably, in those cases where heterologous protein is suspected of being toxic to the host cell, the promoter used to regulate its expression is preferably an inducible promoter so that the expression of the protein of interest can be delayed until it is delayed. have reached sufficient levels of biomass. In a preferred embodiment, the gene encoding an acyl-CoA reductase enzyme capable of
Producing fatty alcohols according to the present invention, is expressed under the control of a constitutive promoter. When constructing suitable expression plasmids, the termination sequences associated with these genes are also ligated into the 3 ’expression vector of the desired sequence to be expressed to provide mRNA polyadenylation and termination. Other promoters, which have the additional advantage of controlled transcription
25 due to growth conditions are the promoter region for alcohol dehydrogenase-2, isocytochrome C, acid phosphatase, degrading enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for the use of maltose and galactose Any plasmid vector containing a promoter, origin of replication and termination compatible with yeast, is
30 suitable.
Yeast vectors suitable for the present invention can be based on the following types of plasmids:
- Multicopy autonomous plasmids: these plasmids contain sequences that allow multiple copies of these vectors to be generated. These sequences can be called 2µ, such as the one that appears in episomal plasmids (YEp or
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one or more amino acids as long as the function of said enzyme is substantially maintained. Specifically, the functionally equivalent variants will maintain the ability to produce fatty alcohols from the corresponding fatty acyl-CoA. Methods for determining the production of fatty alcohols from fatty acyl-CoA are known in the state of the art. By way of illustration, the determination of fatty acid production can be carried out by any method that allows the detection of organic components in a sample, such as chromatographic methods including gas chromatography and mass chromatography. Example 5 of the present application details the determination of fatty alcohols in a sample by gas-mass chromatography. If necessary, prior extraction of fatty alcohols from the cell interior can be carried out by any method appropriate for this. Techniques that allow the extraction of fatty alcohols from the cell interior are known in the state of the art and include mechanical extraction methods such as filtration or centrifugation and solid-liquid extraction methods.
Functionally equivalent variants of said acyl-CoA reductase include those that show at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least one 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity with respect to the acyl-CoA reductase sequence indicated above. The degree of identity between two amino acid sequences can be determined by conventional methods mentioned in the context of the first method of the invention such as, for example, BLAST (Altschul SF et al. Basic Local Alignment Search Tool. J Mol Biol. 1990 Oct 5; 215 (3): 403-10). The person skilled in the art will understand that the amino acid sequences referred to in this description can be chemically modified, for example, by chemical modifications that are physiologically relevant, such as phosphorylations, acetylations, etc.
In a preferred embodiment of the invention, said gene encoding an enzyme with acyl-CoA reductase activity consists of the sequence shown in SEQ ID NO: 1
In a particular embodiment, the microorganism of the invention refers to a mutant of the Rhodosporidium toruloides strain CECT 13085. The term "strain", as used herein, refers to a genetic variant or subtype of an organism. determined.
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DAG1 gene in R. toruloides, preferably in R. toruloides CECT 13085, can be carried out by any method known in the state of the art that allows to detect the enzymatic activity of DAG1.
The term "mutation" refers to substitutions, insertions or deletions that occur at the level of the nucleotide sequence. By "insertion", the gain of one or more nucleotides is understood. Within the insertion there are duplications that consist of the repetition of a segment of DNA inside a sequence, which can occur three (triplication) or more times. By "deletion" as used herein, the loss of one or more nucleotides is understood. Deletions can be total or partial. By "total deletion" of the sequence of a gene, as used in the present invention, it refers to the loss of 100% of the nucleotides that constitute the nucleotide sequence of said gene. By "partial deletion" of the sequence of a gene, as used in the present invention it refers to the loss of at least 0.5%, 1%, 5%, 10%, 20%, 40%, 50%, 60%, 70%, 80%, 90% or 99% of the nucleotides that make up the nucleotide sequence of said gene. As one skilled in the art knows, there are appropriate methods in the art for performing mutations in the sequence of a gene such as site-directed mutagenesis by PCR or site-directed mutagenesis by PCR on a plasmid.
In another particular embodiment, the LRO1P gene of the microorganism of the invention is not functional. The term "LRO1P", as used herein refers to the phospholipid acylglycerol acyltransferase. The reaction catalyzed by said enzyme comprises transferring an acyl-CoA residue from a phospholipid to a diacylglycerol molecule resulting in a triacylglycerol molecule and a lysophospholipid. The sequence of the LRO1P protein from R. toruloides is deposited in the Uniprot database under accession number RHTO_01945 (May 29, 2013 version).
In a preferred embodiment, the expression "the LROP1 gene is not functional" as used herein, refers to said gene encoding an LROP1 protein whose ability to perform triglyceride synthesis from a phospholipid and acyl -CoA fatty, is diminished with respect to the ability to carry out said synthesis by a protein encoded by said functional LROP1 gene. According to the present invention, the microorganism of the invention has a non-functional LROP1 gene if the ability to synthesize triglycerides by the LROP1 protein encoded by said non-functional LROP1 gene is reduced by at least 50%, at least 60%, by at least 70%, at least 80% at
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at least 90%, at least 95% or more with respect to said synthesis performed by a functional LROP1 gene. The LROP1 gene of R. toruloides, preferably of R. toruloides CECT 13085 may be non-functional due to a mutation in the sequence of said gene.
In another preferred embodiment, the expression "LROP1 gene is not functional" also refers to said gene being absent in the genome of the microorganism of the invention, a consequence of a total deletion of the sequence of said gene. In a preferred embodiment of the invention, the microorganism of the invention has the entire deleted LROP1 gene. The determination of the functionality of the LROP1 gene in R. toruloides, preferably in R. toruloides CECT 13085, can be carried out by any method known in the state of the art that allows to detect the enzymatic activity of LROP1.
In another particular embodiment, the DAG3 gene of the microorganism of the invention is not functional. The term "DAG3", "DGAT3", or "delta-1-pyrroline carboxylate dehydrogenase" as used herein, refers to the soluble isoform of the enzyme diacylglycerol acyl transferase that catalyzes the final stage of the synthesis of triglycerides from fatty diacylglycerol and acyl-CoA and whose location is cytosolic. The sequence of the DAG3 protein of R. toruloides is deposited in the Uniprot database under the accession number RTHO_07378 (May 29, 2013 version).
In a preferred embodiment the expression "the DAG3 gene is not functional" as used herein, means that said gene encodes a DAG3 protein whose ability to perform triglyceride synthesis from a phospholipid and acyl- Fatty CoA is diminished with respect to the ability to carry out said synthesis by a protein encoded by said functional DAG3 gene. According to the present invention, the microorganism of the invention has a non-functional DAG3 gene if the ability to synthesize triglycerides by the DAG3 protein encoded by said non-functional DAG3 gene is reduced by at least 50%, at least 60%, by at least 70%, at least 80% at least 90%, at least 95% or more with respect to said synthesis performed by a functional DAG3 gene. The DAG3 gene of R. toruloides, preferably of R. toruloides CECT 13085 may be non-functional as a result of a mutation in the sequence of said gene.
In another preferred embodiment, the expression "DAG3 gene is not functional" also refers to said gene being absent in the genome of the microorganism of the invention, a consequence of a total deletion of the sequence of said gene. In one embodiment
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权利要求:
Claims (1)
[1]
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ES201530156A|ES2579384B1|2015-02-10|2015-02-10|Production of fatty alcohols|ES201530156A| ES2579384B1|2015-02-10|2015-02-10|Production of fatty alcohols|

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PCT/ES2016/070075| WO2016128602A1|2015-02-10|2016-02-09|Production of fatty alcohols|