OF FATTY ACIDS

TECHNICAL FIELD

The application provides genetically engineered microorganisms for use in the production of fatty acids, alcohols and esters and their applications.

BACKGROUND

At present, renewable fatty acids are obtained solely from plant oils. Medium chain fatty acids (C8-C14) are typically sourced from coconut and palm oil, whereas longer chain saturated and unsaturated fatty acids are typically sourced from tallow, soy, corn or sunflower oil. Fatty acids are widely used for food, personal care products, industrial applications (e.g., lubricants, adhesives, detergents and plastics), as well as increasingly as biofuels. The demand for renewable fatty acids is rising and expanding.

With the current understanding of biological pathways it becomes possible to utilize other organisms, especially microorganisms, for the production of renewable chemicals such as fatty acids.

The natural process of microbial fatty acid synthesis proceeds via a stepwise addition of two carbon units onto a growing acyl chain bound to acyl carrier protein (ACP). The process begins as a condensation of acetyl-ACP and malonyl-ACP into acetoacetyl-ACP liberating C0 ₂ which drives the reaction forward. The second step involves reduction of acetoacetyl-ACP to D-3-hydroxybutyryl-ACP using NADPH. Following a dehydration to crotonyl-ACP and another reduction using NADPH, butyryl-ACP is formed. The chain elongation typically continues with further addition of malonyl-ACP until a C16 acyl chain is formed, which is then hydrolyzed by a thioesterase into a free C16 fatty acid.

Dellomonaco et al. (201 1 "Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals", Nature, 476: 355) suggest the engineering of a fatty acid biosynthesis pathway in Escherichia coli, called the "reversed beta oxidation pathway", that is believed to be more efficient than the native pathway in producing fatty acids and alcohols of various lengths and functionalities, which can in their turn be catalytically converted to chemicals and fuels. Dellomonaco et al., however, do not provide a system to implement fatty acid production based on this pathway in a micro-organism.

SUMMARY OF THE INVENTION

Disclosed herein are genetically altered micro-organisms that can be used for the production of fatty acids, alcohols and esters and products derived therefrom, such as chemicals, fuels, lipids and/or oils. Also disclosed herein are novel and improved methods for the production of fatty acids and products derived therefrom, such as chemicals, fuels, lipids and/or oils which make use of these organisms.

The application provides micro-organisms that have been genetically modified for increased production of fatty acids by transformation with different genes involved in fatty acid metabolism of cellular organisms.

More particularly, the application provides an alternative pathway to ensure fatty acid production in micro-organisms using the reversed beta-oxidation pathway. It has been determined that the gene ter from E. gracilis, encoding an enoyl-CoA reductase, in combination with the overexpression of recombinant genes fadB, fadA, and a thioesterase gene (such as for instance but not limited to fadM, tesA, tesB or yciA) allows to efficiently produce fatty acids by means of the reversed beta-oxidation cycle, without requiring the artificial production of stoechiometric amounts of reduced ferredoxin. Instead, this enzyme uses NADH as a cofactor, which is naturally synthesized in sufficient amounts in the microorganisms by means of the glycolysis pathway. Indeed, while it was suggested in the prior art that beta oxidation of fatty acids in E. coli can be reversed by overexpression of the recombinant genes fadB, fadA, a thioesterase gene and ydiO (the latter being a ferredoxin-dependent trans enoyl CoA reductase), this upstream fatty acid synthesis route only (efficiently) works when sufficient amounts of cofactor, i.e. reduced ferredoxin, are produced to catalyze the action of trans enoyl CoA reductase (encoded by ydiO). However, in most micro-organisms (i.e. yeasts, E. coli), such amounts of ferredoxin are not naturally produced in stoechiometric amounts via the upstream pathway.

The application also provides alternative embodiments, wherein overexpression of the pyruvate:ferredoxin oxidoreductase gene or PFOR gene encoding a pyruvate ferredoxin oxidoreductase, in combination with the overexpression of recombinant genes fadB, fadA, a thioesterase gene (such as for instance but not limited to fadM, tesA, tesB or yciA) and ydiO in a micro-organism results in the production of sufficient amounts of reduced ferredoxin to catalyze trans enoyl CoA reductase and thereby complete the reversed beta- oxidation pathway. However, the use of the ter gene as described above was found to be significantly more effective.

The application further provides strategies for optimizing the efficiency of fatty acid production in a micro-organism. Indeed, it has been found that introducing enzymes involved in the reversed beta oxidation pathway in oleaginous micro-organisms, more particularly oleaginous yeast, more particularly in yeast strains in which the endogenous fatty acid production and/or acetyl-CoA consumption pathways are blocked, ensures high yield production of fatty acids. Moreover, further reduction of fatty acid oxidation in said organism, stimulates the reverse beta oxidation pathway.

Thus, in particular embodiments, envisaged micro-organisms are yeast or fungi, whereby the yeast or fungi comprise at least one engineered gene deletion and/or inactivation in endogenous fatty acid accumulation, production, or consumption. In particular embodiments, the micro-organisms contain an engineered gene deletion and/or inactivation in a gene encoding an enzyme such as but not limited to those selected from the group consisting of diacylglyceroltransferase (DGAT), phospholipid diacylglycerol transferase (PDAT), acyl-CoA dependent triacylglycerol synthase (DGA), acyl-CoA independent acyltransferase (LRO), acyl-CoA sterol acyltransferase (ARE) phospholipid- diacylglycerol acyltransferase (PLH), Diglyceride acyltransferase (or O-acyltransferase) (DGAT), diacylglycerol acyltransferase (DAGAT), fatty acid synthase (FAS), and any combination thereof. In particular embodiments, the micro-organisms contain an engineered gene deletion and/or inactivation in one or more genes encoding enzymes involved in the triacylglycerol biosynthesis, such as phospholipid diacylglycerol transferase (PDAT), diacylglyceroltransferase (DGAT), or fatty acid synthase (FAS). In particular embodiments, the micro-organisms contain an engineered gene deletion and/or inactivation in one or more genes encoding enzymes involved in the degradation of fatty acids, such as acyl-CoA oxidase (AOX) or acyl-CoA dehydrogenase, to stimulate the reverse beta-oxidation pathway.

In particular embodiments, the micro-organisms envisaged herein are genetically modified yeasts or fungi, such as but not limited to S. cerevisiae and Schizosaccharomyces pombe, and more particularly oleaginous yeasts or fungi, such as Mortierella ramanniana, Rhodutorula glutinis, Yarrowia lipolytica and Cryptococcus curvatus.

Thus, the application provides different ways in which micro-organisms can be engineered to ensure efficient production of fatty acids, involving the modification of expression of one or more genes involved in fatty acid production pathway. In particular embodiments this involves the introduction of one or more recombinant genes encoding enzymes typically involved in fatty acid production pathway, such as fadB, fadA and a thioesterase gene, more particularly the introduction of a recombinant fadB , fadA and a thioesterase gene (such as for instance but not limited to fadM, tesA, tesB or yciA).

Additionally the micro-organisms may be modified to eliminate repression of endogenous enzymes involved in beta oxidation by the product of the fadR gene. This is achieved by inactivation of the fadR gene. Isolated micro-organisms are thus provided, which have been genetically modified for increased production of fatty acids, by introduction of and/or modification of expression of one or more genes of the beta oxidation pathway for the formation of fatty acids selected from a recombinant fadB gene, a recombinant fadA gene, a recombinant thioesterase gene, and whereby, in addition, production of the substrate for said thioesterase is ensured by the introduction of a recombinant gene encoding an NADH dependent trans- enoyl-CoA reductase. This pathway ensures a functional reversal of the beta-oxidation cycle of fatty acids and can be performed anaerobically which significantly increases the yield of fatty acid production.

It has been found that the ter gene of Euglena gracilis is particularly suitable in the context of the effects envisaged herein. Accordingly, in particular embodiments, the genetically modified micro-organisms envisaged herein comprise, in addition to one or more genes encoding enzymes from the beta oxidation pathway selected from a recombinant fadB gene, a recombinant fadA gene, a recombinant gene encoding a thioesterase, and a recombinant ter gene from Euglena gracilis.

In particular embodiments, the micro-organism is further modified to eliminate repression of the enzymes involved in beta oxidation by the product of the fadR gene. Thus in particular embodiments, the microorganisms are FadR negative strains.

Alternatively, in certain specific embodiments, the genetically engineered micro-organisms of the present invention comprise in addition to one or more genes selected from a recombinant fadB gene, a recombinant fadA gene and a recombinant thioesterase gene, a recombinant gene encoding an enoyl-CoA reductase, such as the ydiO gene and a recombinant pyruvate:ferredoxin oxidoreductase gene. It is demonstrated herein that the yield of fatty acids can be further enhanced in the organisms described above comprising genes encoding enzymes involved in the reversed beta oxidation pathway, by inactivating endogenous genes of said organisms encoding proteins involved in an endogenous metabolic pathway which produces a metabolite other than the fatty acid of interest and/or wherein the endogenous metabolic pathway consumes the fatty acid. In particular embodiments, the production of the metabolite other than the fatty acid of interest is reduced. According to particular embodiments, the microorganisms envisaged herein comprise, in addition to enzymes involved in the reversed beta oxidation pathway, at least one engineered gene deletion and/or inactivation of an endogenous pathway in which the fatty acid is consumed or produced or a gene encoding a product involved in an endogenous pathway which produces a metabolite other than the fatty acid of interest. Elimination of expression of such a gene increases fatty acid synthesis yield.

In more particular embodiments, the micro-organisms envisaged herein are recombinant micro-organisms which comprise the above-mentioned combinations of recombinant foreign genes involved in fatty acid production and/or at least one engineered gene deletion and/or inactivation of an endogenous pathway in which the fatty acid is consumed or produced and/or a gene encoding an enzyme involved in an endogenous pathway which produces a metabolite other than the fatty acid of interest.

According to particular embodiments, the micro-organisms envisaged herein further comprise at least one engineered gene deletion and/or inactivation of an endogenous lactic acid production pathway and/or an endogenous acetate production pathway, which for example may be an engineered gene deletion and/or inactivation in a gene encoding an enzyme such as those selected from the group consisting of D-lactate dehydrogenase (d-ldh), L-lactate dehydrogenase (l-ldh), phosphotransacetylase (pta), pyruvate decarboxylase (pdc) and pyruvate dehydrogenase (pdh) and any combination thereof.

Thus, in particular embodiments, the microorganisms further comprise at least one engineered gene deletion and/or inactivation of an endogenous lactic acid production pathway and/or an endogenous acetate production pathway, such as but not limited to at least one engineered gene deletion and/or inactivation of one or more lactate dehydrogenase (Idh) genes, phosphotransacetylase (pta), pyruvate decarboxylase (pdc) and/or pyruvate dehydrogenase (pdh) genes.

Accordingly, in particular embodiments, the micro-organisms envisaged herein are genetically modified bacteria. The micro-organisms may be applied to produce fatty acids on a large scale and with high yields. More particularly, the micro-organisms may be capable of producing fatty acids at a yield of at least 0.5 g/L from sugars, more particularly at least 2 g/L, or at least 5g/L from sugars under anaerobic conditions. In particular embodiments, the micro-organisms envisaged herein are capable of converting monosaccharides, such as but not limited to glucose, xylose, arabinose, galactose, disaccharides, such as but not limited to maltose, sucrose, lactose, cellobiose, polyalcohols, such as but not limited to mannitol, sorbitol, glycerol, and mixtures thereof at a concentration of at least 10 g/L into fatty acids. In a further aspect, the application provides for the use of the genetically engineered micro-organisms described herein in the industrial production of one or more fatty acids, alcohols and/or esters or products derived therefrom. In certain embodiments, the fatty acid production by the micro-organisms of the present invention is performed under anaerobic conditions.

In a further aspect, the application provides methods for production of a composition comprising one or more fatty acids and/or one or more products derived therefrom involving the microorganisms disclosed herein. In particular embodiments, such methods may comprise the steps of:

(i) providing a genetically modified micro-organism according to an embodiment as described herein; and

(ii) culturing the micro-organism in the presence of pentose or hexose sugars under conditions suitable for the expression of said recombinant genes. In specific embodiments, the culturing step (ii) of the methods for production described herein may be performed either completely or partially under anaerobic conditions.

In a further aspect, the compositions obtainable by methods described herein for increased production of a composition comprising one or more fatty acids and/or one or more products derived therefrom are provided.

In a further aspect, the methods for producing one or more purified fatty acids, alcohols or esters are provided, which methods may comprise the steps of:

(i) providing one or more genetically modified micro-organism comprising one or more recombinant genes as described herein; and (ii) culturing the micro-organism in the presence of pentose or hexose sugars under conditions suitable for the expression of said recombinant genes, and

(iii) recovering the one or more fatty acids from the cultivation medium. BRIEF DESCRIPTION OF THE FIGURES

The teaching of the application is illustrated by the following Figures which are to be considered as illustrative only and do not in any way limit the scope of the claims.

Figure 1. Illustration of the use of the reversed beta oxidation pathway for fatty acid production according to a particular embodiment of the invention whereby genes fadA, fadB, fadM and yidO are overexpressed. This approach requires the production of reduced ferredoxin stream upwards in order to catalyze the action of the ferredoxin-dependent trans enoyl CoA reductase. In particular embodiments of the present invention, this is ensured by introduction of a gene encoding a pyruvate ferredoxin oxidoreductase, such as the ydbK gene from E. coli; catalyzing a reaction with a sufficient rise in redox potential in order to ensure the production of reduced ferredoxin streamupwards in the reversed beta oxidation cycle.

Figure 2. Illustration of the use of the reversed beta oxidation pathway for fatty acid production according to a particular embodiment of the invention with indicated genes fadA, fadB, fadM and ter to be overexpressed and candidate genes for inactivation or gene deletion marked by red crosses.

Figure 3. Illustration of the use of the reversed beta oxidation pathway in yeast according to a particular embodiment of the invention, with indicated genes fadA, fadB, fadM and ter to be overexpressed and candidate genes for inactivation or gene deletion marked by red crosses.

Figure 4. Illustration of an exemplary method of a step-wise construction of a transformation vector pASK-IBA3C which can be used for the transformation of E. coli strains according to particular embodiments of the invention. The four genes fadA, fadB, fadM and ter are cloned step by step into the expression vector. The fragment fadBA cloned with into the MCS2 of the vector (Figure 4a), the ter gene cloned into the MCS1 of the plasmid (Figure 4b), the combination of the cloning of fadAB in MCS2 and ter in MCS1 (Figure 4c) and the full construct comprising fadAB in MCS2 and ter+fadM in MCS1 (Figure 4d).

Figure 5. Illustration of exemplary thioesterase activity of extracts of micro-organisms transformed with codon optimized fadM (codFADM) according to particular embodiments of the invention, confirming the efficient transformation with a thioesterase gene. The incubation started at t=0 seconds, the measurement started at t=60 seconds. Circles: not IPTG induced; squares: induced with 1 mM IPTG, open circles and open squares: 1 μΙ cell free extract; filled circles and filled squares: 100 μΙ cell free extract. Grey triangles: control. Figure 6. Illustration of the use of the reversed beta oxidation pathway in yeast according to a particular embodiment of the invention, further illustrating the envisaged modifications in metabolic pathways.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.

As used herein, the singular forms "a", "an", and "the" include both singular and plural referents unless the context clearly dictates otherwise.

The terms "comprising", "comprises" and "comprised of as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. Where reference is made to embodiments as comprising certain elements or steps, this implies that embodiments are also envisaged which consist essentially of the recited elements or steps.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

The term "about" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/-10% or less, preferably +1-5% or less, more preferably +/-1 % or less, and still more preferably +/-0.1 % or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" refers is itself also specifically, and preferably, disclosed.

As used herein, the term "homology" denotes structural similarity between two macromolecules, particularly between two polypeptides or polynucleotides, from same or different taxons, wherein said similarity is due to shared ancestry. Hence, the term "homologues" denotes so-related macromolecules having said structural similarity.

All documents cited in the present specification are hereby incorporated by reference in their entirety.

As used herein, sequence identity between two polypeptides can be determined by optimally aligning (optimal alignment of two protein sequences is the alignment that maximises the sum of pair-scores less any penalty for introduced gaps; and may be preferably conducted by computerised implementations of algorithms, such as "Clustal" W using the alignment method of Wilbur and Lipman, 1983 (Proc. Natl. Acad. Sci. USA, 80: 726-730). Alternative methods include "Gap" using the algorithm of Needleman and Wunsch 1970 (J Mol Biol 48: 443-453), or "Bestfit", using the algorithm of Smith and Waterman 1981 (J Mol Biol 147: 195—197), as available in, e.g. , the GCG™ v. 1 1.1 .2 package from Accelrys) the amino acid sequences of the polypeptides and scoring, on one hand, the number of positions in the alignment at which the polypeptides contain the same amino acid residue and, on the other hand, the number of positions in the alignment at which the two polypeptides differ in their sequence. The two polypeptides differ in their sequence at a given position in the alignment when the polypeptides contain different amino acid residues at that position (amino acid substitution), or when one of the polypeptides contains an amino acid residue at that position while the other one does not or vice versa (amino acid insertion or deletion). Sequence identity is calculated as the proportion (percentage) of positions in the alignment at which the polypeptides contain the same amino acid residue versus the total number of positions in the alignment. In particular embodiment the algorithm for performing sequence alignments and determination of sequence identity is one based on the Basic Local Alignment Search Tool (BLAST) originally described by Altschul et al. 1990 (J Mol Biol 215: 403-10), more particularly the "Blast 2 sequences" algorithm described by Tatusova and Madden 1999 (FEMS Microbiol Lett 174: 247-250), such as using defaults settings thereof.

As used herein, "sequence similarity" between two polypeptides can be determined by optimally aligning (see above) the amino acid sequences of the polypeptides and scoring, on one hand, the number of positions in the alignment at which the polypeptides contain the same or similar (i.e., conservatively substituted) amino acid residue and, on the other hand, the number of positions in the alignment at which the two polypeptides otherwise differ in their sequence. The two polypeptides otherwise differ in their sequence at a given position in the alignment when the polypeptides contain non-conservative amino acid residues at that position, or when one of the polypeptides contains an amino acid residue at that position while the other one does not or vice versa (amino acid insertion or deletion). Sequence similarity is calculated as the proportion (percentage) of positions in the alignment at which the polypeptides contain the same or similar amino acid residue versus the total number of positions in the alignment.

The term "fatty acid" as used herein refers to any of a large group of organic acids, especially those found in animal and vegetable fats and oils. Characteristically made up of saturated or unsaturated aliphatic compounds with an even number of carbon atoms, this group of acids includes palmitic, stearic, and oleic acids.

The term "saturated fatty acid" as used herein refers to any fatty acid in which all bonds between carbon atoms are all single bonds.

The term "unsaturated fatty acid" as used herein refers to any fatty acid in which at least one of the bonds between carbon atoms is a double bond.

The term "organic acid" as used herein refers to an organic compound with acidic properties. More particularly in the context of the present invention organic acid compounds are selected from the group consisting of acetic acid, lactic acid (2- hydroxypropionic acid), succinic acid, furandicarboxylic acid, fumaric acid, maleic acid, citric acid, glutamic acid, aspartic acid, acrylic acid, oxalic acid, and glucanic acid.

The term "carboxylic acid" refers to an organic acid characterized by the presence of at least one carboxyl group. Acids with two or more carboxyl groups are also referred to as dicarboxylic, tricarboxylic, etc. Examples of carboxylic acids include but are not limited to acetic acid, oxalic acid and mal(e)ic acid, and succinic acid. The term "lactic acid" in this application refers to 2-hydroxypropionic acid in either the free acid or salt form. The salt form of lactic acid is referred to as "lactate" regardless of the neutralizing agent, i.e. calcium carbonate or ammonium hydroxide. As referred to herein, lactic acid can refer to either stereoisomeric form of lactic acid (L-lactic acid or D-lactic acid). The term lactate can refer to either stereoisomeric form of lactate (L-lactate or D- lactate). When referring to lactic acid production this includes the production of either a single stereoisomer of lactic acid or lactate or a mixture of both stereoisomers of lactic acid or lactate.

The term "acetic acid" in this application refers to the carboxylic acid with the chemical formula CH ₃ C0 ₂ H (also written as CH ₃ COOH) in either the free acid or salt form. The salt form of acetic acid is referred to as "acetate".

"Lactate dehydrogenase activity" as used herein refers to the ability of the enzyme lactate dehydrogenase to catalyze the reaction of pyruvate to lactate. It will be clear to the skilled person that this enzyme is also able to catalyze this reaction in the opposite direction under suitable conditions. Lactate dehydrogenase enzymes include (but are not limited to) the enzymes categorized by the Enzyme Commission numbers EC1.1.1.27 and EC1.1.1.28.

"Phosphotransacetylase activity" used herein refers to the ability of the enzyme phosphotransacetylase (also named phosphate acetyltransferase, acetyl-CoA: phosphate acetyltransferase, phosphoacylase, and PTA) to catalyze the reversible transfer of an acetyl group from acetyl coenzyme A to a phosphate, with formation of acetyl phosphate. It will be clear to the skilled person that this enzyme is also able to catalyze this reaction in the opposite direction under suitable conditions. Phosphotransacetylase enzymes include (but are not limited to) the enzymes categorized by the Enzyme Commission numbers EC2.3.1.8.

"Pyruvate decarboxylase activity" used herein refers to the ability of the enzyme pyruvate decarboxylase (also named 2-oxo-acid carboxylase, alpha-ketoacid carboxylase, pyruvic decarboxylase, and PDC) to catalyze the conversion of pyruvate into acetaldehyde and carbon dioxide. Pyruvate decarboxylase depends on cofactors thiamine pyrophosphate (TPP) and magnesium. It will be clear to the skilled person that this enzyme is also able to catalyze this reaction in the opposite direction under suitable conditions. Pyruvate decarboxylase enzymes include (but are not limited to) the enzymes categorized by the Enzyme Commission numbers EC4.1.1.1.

"Pyruvate dehydrogenase activity" used herein refers to the ability of the enzyme pyruvate dehydrogenase (PDH), an oxidoreductase (EC 1.2.4.1 ), that catalyzes the oxidative decarboxylation of pyruvate into acetyl-CoA. It will be clear to the skilled person that this enzyme is also able to catalyze this reaction in the opposite direction under suitable conditions.

The term "fadA (gene)" as used herein refers to a gene encoding the protein FadA, which is a thiolase (3-ketoacyl-CoA thiolase (thiolase I) EC 2.3.1.16) involved in the beta oxidation of fatty acids. FadA cleaves 3-ketoacyl-CoA, resulting in shortening of the input acyl-CoA by two carbon atoms to give acetyl-CoA, in the beta oxidation of fatty acids. The term "fadB (gene)" as used herein refers to a gene encoding the protein FadB having L-3-hydroxyacyl-CoA dehydrogenase (EC1.1.1.35) and small-chain-length enoyl-CoA hydratase (cronotase) (EC4.2.1.17) activity. FadB converts enoyl-CoA to 3-ketoacyl-CoA via 3-hydroxylacyl-CoA through hydration and oxidation in the beta oxidation of fatty acids. The term "fadM (gene)" as used herein refers to the gene encoding the protein FadM, which is thioesterase III (long-chain acyl-CoA thioesterase) (EC3.1 .2.-) FadM is involved in the beta oxidation of fatty acids.

The term "Escherichia coli fatty acid oxidation complex" refers to the tetrameric multienzyme complex FadA ₂ FadB ₂ formed by the proteins FadA and FadB in E.coli.

The term "fadAB operon" as used herein refers to a nucleotide sequence contained in the

DNA insert of plasmid pK52 coding for the Escherichia coli fatty acid oxidation complex.

The term "ferredoxin-dependent acyl-CoA dehydrogenase gene" as used herein refers to the gene encoding the protein YdiO, a putative ferredoxin-dependent acyl-CoA dehydrogenase (EC1.3.1.8), an enzyme that catalyzes the conversion of enoyl-CoA to acylCoA. The term "ydiO (gene)" refers to a ferredoxin-dependent acyl-CoA dehydrogenase gene from E. coli, which is a possible AraC-like regulator of the ydiQRSTD operon in E. coli. Other names include trans enoyl CoA reductase.

The term "pyruvate:ferredoxin oxidoreductase gene" or "PFOR gene" as used herein refers to the gene encoding a pyruvate ferredoxin oxidoreductase (EC1.2.7.1 ), an enzyme that catalyzes the conversion of pyruvate into acetyl-CoA. The three substrates of this enzyme are pyruvate, CoA, and oxidized ferredoxin, whereas its four products are acetyl-CoA, C0 ₂ , reduced ferredoxin, and H ⁺ . This enzyme belongs to the family of oxidoreductases, specifically those acting on the aldehyde or oxo group of donor with an iron-sulfur protein as acceptor. The systematic name of this enzyme class is pyruvate:ferredoxin 2- oxidoreductase (CoA-acetylating). Other names in common use include pyruvate oxidoreductase, pyruvate synthetase, pyruvate:ferredoxin oxidoreductase, and pyruvic- ferredoxin oxidoreductase. The "ydbK (gene) " is a gene encoding a pyruvate :ferredoxin oxidoreductase in E. coli (Akthar et al. (2009) Metabolic Engineering 1 1 (3): 139-147). "ter (gene)" as used herein refers to the gene encoding the protein TER, which is a trans- 2-enoyl-CoA reductase (EC1.3.1.44), an enzyme that catalyzes the conversion of enoyl- CoA to acyl-CoA under oxidation of NAD(P)H to NAD(P). The terms "recombinant" or "genetically modified" as used herein with reference to host organisms, microorganisms or cells, encompass such host organisms, microorganisms or cells into which a recombinant nucleic acid molecule has been introduced or which has been in another way genetically modified, as well as the recombinant progeny of such host organisms, micro-organism or cells. This includes both organisms in which endogenous gene sequences are introduced at a position other than their natural position in the genome and organisms in which endogenous gene sequences have been modified or deleted.

The term "transformation" encompasses the introduction or transfer of a foreign nucleic acid such as a recombinant nucleic acid into a host organism, microorganism or cell. The so-introduced nucleic acid or the resulting deletion of endogenous nucleic acid is preferably maintained throughout the further growth and cell division of said host organism, microorganism or cell. Any conventional gene transfer or genetic modification methods may be used to achieve transformation, such as without limitation electroporation, electro-permeation, chemical transformation, lipofection, virus- or bacteriophage-mediated transfection, etc.

The term "gene" as generally used herein refers to a nucleic acid sequence which contains a coding sequence, a promoter and any other regulatory regions required for expression in a host cell. The term "recombinant gene" as generally used herein in the context of a micro-organism refers to a sequence which contains a coding sequence a promoter and any other regulatory regions required for expression in said host cell which is not present as such in said host cells. This includes endogenous gene sequences which are introduced at a position other than their natural position in the genome and/or which have been modified in the coding and/or regulatory sequence.

As used herein, the term "promoter" refers to an untranslated sequence located within 50 bp upstream the transcription start site and which controls the start of transcription of the structural gene. Generally it is located within about 1 to 1000 bp, preferably 1-500 bp, especially 1-100 bp upstream (i.e., 5') to the translation start codon of a structural gene. Similarly, the term "terminator" refers to an untranslated sequence located downstream (i.e., 3') to the translation stop codon of a structural gene (generally within about 1 to 1000 bp, more typically 1 -500 base pairs and especially 1-100 base pairs) and which controls the end of transcription of the structural gene. A promoter or terminator is "operatively linked" to a structural gene if its position in the genome relative to that of the structural gene is such that the promoter or terminator, as the case may be, performs its transcriptional control function.

As used herein, the term "exogenous" refers to the fact that the gene or coding sequence under consideration originates from outside of the host organism of concern or study. As used herein, the term "heterologous" refers to the fact that the gene or coding sequence under consideration is not native or endogenous to the host but rather originates or has been cloned from a different cell type or from an organism of a different species than the recipient host.

The term "native" or "endogenous" is used herein with respect to genetic materials (e.g., a gene, promoter or terminator) that are found (apart from individual-to-individual mutations which do not affect function) within the genome of wild-type cells of the host strain.

The term "overexpression" as used herein when referring to expression of a gene in a host cell, refers to the fact that it is expressed at a higher level than naturally in said host cell. This may imply that it is a foreign gene, not naturally expressed in the host cell or that the endogenous gene has been modified so as to increase expression in said host cell.

The term "inactivation" as used herein when referring to a gene present in a host cell, refers to the fact that the gene product is either not expressed or not active in said host cell. This typically implies that the endogenous gene has been modified so as to no longer allow expression in said host cell but may also imply that the gene has been modified to ensure that the gene product is no longer active.

By "encoding" is meant that a nucleic acid sequence or part(s) thereof corresponds, by virtue of the genetic code of an organism in question, to a particular amino acid sequence, e.g., the amino acid sequence of a desired polypeptide or protein. By means of example, nucleic acids "encoding" a particular polypeptide or protein may encompass genomic, hnRNA, pre-mRNA, mRNA, cDNA, recombinant or synthetic nucleic acids.

Preferably, a nucleic acid encoding a particular polypeptide or protein may comprise an open reading frame (ORF) encoding said polypeptide or protein. An "open reading frame" or "ORF" refers to a succession of coding nucleotide triplets (codons) starting with a translation initiation codon and closing with a translation termination codon known per se, and not containing any internal in-frame translation termination codon, and potentially capable of encoding a polypeptide. Hence, the term may be synonymous with "coding sequence" as used in the art.

When referring to the term "anaerobic conditions" in the context of the present application in fact refer to conditions in which no air or free oxygen is present or provided.

When referring to an "anaerobic" micro-organism or reaction this typically implies that it does not require the presence of molecular oxygen serving as the terminal electron acceptor in respiration or in metabolic oxygenation.

On the other hand, when referring to the term "aerobic conditions" in the context of the present application, this refers to conditions whereby free oxygen or air is present or introduced. When referring to a chemical reaction or micro-organism the term "aerobic" typically implies that it requires the presence of molecular oxygen serving as the terminal electron acceptor in respiration or in metabolic oxygenation.

In a first aspect, genetically engineered micro-organisms are provided that are capable of increased production of fatty acids, alcohols, esters or other products derived there from. In particular embodiments, the microorganism ensure a rate of fatty acid production which is sufficiently high to be industrially valuable.

The envisaged micro-organisms have been genetically modified to affect expression of one or more genes involved in fatty acid synthesis. More particularly they have been engineered to modify the expression of one or more genes encoding enzymes which ensure the reversed beta oxidation pathway, which operates with coenzyme A (CoA) thioester intermediates and directly uses acetyl-CoA for acyl-chain elongation (rather than first requiring ATP-dependent activation to malonyl-CoA), which are characteristics that enable product synthesis at high carbon and energy efficiency .

In particular embodiments, the (increased) fatty acid synthesis is ensured in the microorganisms envisaged herein by overexpression of one or more genes selected from the fadB gene, the fadA gene, and a thioesterase gene. In particular embodiments, the microorganisms comprise a recombinant fadB gene, a recombinant fadA gene and/or a recombinant thioesterase gene. Suitable examples of fadA, fadB and thioesterase genes envisaged in the context of the present invention are the fadA, fadB, fadM, tesA, tesB or yciA genes from E. coli. In particular embodiments the thioesterase gene is selected from fadM, tesA, tesB and yciA. Methods for ensuring overexpression of endogenous genes and/or foreign genes are known to the skilled person and will be illustrated herein.

Additionally or alternatively, in particular embodiments of the micro-organisms envisaged in different aspect of the present invention, the modification of expression of these genes is ensured by eliminating the negative regulation of the beta oxidation enzymes by the FadR gene product. The FadR gene product represses the activity of the beta oxidation enzymes in the presence of e.g. sugars. Eliminating this negative regulation can be ensured by inactivating an endogenous fadR gene, such that the genes involved in beta oxidation are constitutively expressed. In particular embodiments, inactivation implies that all or part of the coding region of the gene is eliminated (deletion), or the gene or its promoter and/or terminator region is modified (such as by deletion, insertion, or mutation) so that the gene no longer produces an active enzyme, or produces an enzyme with severely reduced activity. Inactivation further includes silencing such as by antisense, triple helix, and ribozyme approaches, all known to the skilled person.

FadR negative strains of E. coli are easily obtainable (e.g. Keio collection). Moreover, methods for selectively knocking-out genes are well known in the art. Accordingly, in particular embodiments, the recombinant microorganisms are fadR mutants. Thus, in particular embodiments, the microorganisms are characterized in that they have an inactivated fadR gene. In addition, the microorganisms of the present invention in which one or more of the fadB gene, the fadA gene, and the thioesterase gene are overexpressed, further comprise one or more modifications which ensure the reduction of enoyl-CoA in order to generate acyl-CoA, which is a substrate for the thioesterase gene.

An alternative strategy is provided herein to genetically modify micro-organisms in order to allow the production of fatty acids using the reversed beta oxidation pathway. Indeed it is demonstrated that, a non ferredoxin-dependent enoyl-CoA reductase can be used, such as the NADH dependent trans-enoyl-CoA reductase enzymes identified in Euglena gracilis. Thus, it was found that fatty acid synthesis can also be achieved by microorganisms characterized by overexpression of one or more genes selected from the fadB gene, the fadA gene, and a gene encoding a thioesterase and further characterized by overexpression of an NADH dependent trans-enoyl-CoA reductase enzyme. Thus, in particular embodiments, the microorganisms may be characterized by overexpression of a ter gene encoding a trans-2-enoyl-CoA reductase (EC1.3.1.44), which catalyzes the conversion of enoyl-CoA to acetylCoA under oxidation of NAD(P)H to NAD(P).

Typically, overexpression is ensured by the presence of a recombinant ter gene. The recombinant ter gene may originate from any suitable source or organism. Non-limiting examples are genes encoding TER enzymes originating from the yeast Saccharomyces cerevisiae (Song et al. 2009, J Exp. Bot 2009. April 60(6): 1839-1848), the spirochete Trepanoma denticola (Sara Tucci, William Martin, FEBS letters 581 (2007) 1561 -1566), mammalian peroxisomes (Das et al. 2000 Das AK, Uhler MD, Hajra AK. 2000. J. Biol.Chem 275: 24333-24340), cotton (Song W-Q et al. 2009. J. Exp. Bot. 60(6): 1839- 1848) or rat liver microsomes (Prasad et al. 1985. Arch. Biochem Biophys 237(2):535- 544). However, in particular embodiments, the recombinant ter gene is a ter gene originating from Euglena gracilis (Hiroshi et al.1984 "Fatty acid synthesis in mitochondria of Euglena gracilis" Eur. J. Biochem. 142: 121-126), such as but not limited to one of the genes terl, having a high specificity for C4 compounds, ter2, having a high specificity for C6 and C8 compounds or ter3, having a high specificity for C6 compounds. A suitable ter gene may thus be chosen depending on the desired length of fatty acid that is to be produced. Indeed, by relying on the preferred specificity for each of the TER enzymes, the length of the fatty acid to be produced can be controlled by selecting a particular TER enzyme with a particular specificity.

In particular embodiments, the microorganisms may thus comprise one or more recombinant genes selected from a recombinant fadB gene, a recombinant fadA gene, and a recombinant gene encoding a thioesterase, such as but not limited to fadM, tesA, tesB or yciA and, in addition a recombinant ter gene. In further particular embodiments, the microorganisms may be characterized by a) a recombinant fadB gene, b) a recombinant fadA gene, c) a recombinant gene encoding a thioesterase, and d) a recombinant ter gene. Not only does this alternative approach require one less genetic modification because the ter gene product does not require the presence of reduced ferredoxin, but it also works highly efficient under anaerobic conditions, which is a major advantage when production on industrial scale is envisaged. The engineered micro-organisms envisaged herein can be of bacterial origin or of yeast or fungal origin. In principle, any species of micro-organism that can be suitably engineered with one or more of the above-disclosed combinations of recombinant genes, resulting in the (over)production of fatty acids, can be used in the context of the present invention. In particular embodiments, the micro-organisms of the present invention are genetically modified bacteria or genetically modified yeasts or fungi. More particularly, microorganisms which can be cultivated anaerobically are of interest.

In particular embodiments, the micro-organism is an enterobacterium, such as Escherichia coli.

As further detailed herein, it has further been determined that an efficient production of fatty acids can be obtained by using an oleaginous yeast, wherein the endogenous fatty acid production and/or consumption pathway has been modified and which is further modified by enzymes involved in the fatty acid production using the reversed beta oxidation pathway. Accordingly, in further particular embodiments, the host is selected from S. cerevisiae, Schizosaccharomyces pom be, Mortierella ramanniana, Rhodutorula glutinis, Yarrowia lipolytica and Cryptococcus curvatus. In particular alternative embodiments of the micro-organisms envisaged herein, the reversed beta-oxidation pathway is used by introduction a ferredoxin-dependent enoyl- CoA reductase. As this requires the presence of reduced ferredoxin, it is demonstrated herein that efficient production of fatty acids is further ensured by reduction of enoyl-CoA by overexpression of a ferredoxin-dependent trans enoyl CoA reductase. The nature of the ferredoxin-dependent trans enoyl CoA reductase is not critical to obtaining the envisaged effect. However, in particular embodiments, the gene encoding a ferredoxin-dependent enoyl-CoA reductase is the ydiO gene from E. coli (EcoGene Accession Number: EG13974;

K-12 Gene Accession Number: ECK1693; MG1655 Gene Identifier: b1695).

However, this approach requires the production of reduced ferredoxin stream upwards in order to catalyze the action of the ferredoxin-dependent trans enoyl CoA reductase. Indeed, in the absence of reduced ferredoxin, the ferredoxin-dependent trans-enoyl-CoA reductase will not function, preventing the synthesis of fatty acid by the recombinant microorganism, let alone fatty acid production on an industrial scale and in an economically efficient way.

It has however been found that overexpression of a specific combination of genes will allow to the microorganism to efficiently produce one or more fatty acids by means of the reversed beta-oxidation pathway. More particularly, the micro-organisms are engineered such that they are able to convert simple sugars to a fatty acid, and in particular embodiments to produce fatty acids at high yield. In particular embodiments these organisms are organisms which can be cultivated under anaerobic or quasi-anaerobic conditions, providing an additional advantage in the context of industrial production methods.

Thus, in certain embodiments, the microorganisms envisaged herein are characterized by overexpression of one or more genes selected from the fadB gene, the fadA gene, and a gene encoding a thioesterase and are further characterized by over-expression of a gene encoding an ferredoxin-dependent enoyl-CoA reductase and a pyruvate:ferredoxin oxidoreductase gene. Overexpression of these genes can be ensured by increasing expression of endogenous gene and/or expression of recombinant genes. Thus, in particular embodiments, the microorganisms may comprise one or more recombinant genes selected from a recombinant fadB gene, a recombinant fadA gene, and a recombinant gene encoding a thioesterase and, in addition a recombinant gene encoding an ferredoxin-dependent enoyl-CoA reductase and a recombinant pyruvate:ferredoxin oxidoreductase gene.

In particular embodiments, the recombinant pyruvate:ferredoxin oxidoreductase gene or PFOR gene is the ydbK gene of E. coli (Akthar et al. (2009) Metabolic Engineering 1 1 (3): 139-147)(EcoGene Accession Number: EG13183; K-12 Gene Accession Number: ECK1374; MG1655 Gene Identifier: b1378). Indeed, it was found that the recombinant gene ydbK from E. coli, encoding a pyruvate:ferredoxin oxidoreductase, catalyzes a reaction with a sufficient rise in redox potential in order to ensure the production of reduced ferredoxin stream upwards in the reversed beta oxidation cycle, namely the conversion of pyruvate into acetyl-CoA. The produced reduced ferredoxin is indispensable for the production of fatty acids by means of reversed beta-oxidation since it functions as a substrate for the conversion of enoyl-CoA to acetylCoA, which is in its turn needed as a direct substrate for fatty acid production by the fadM enzyme (see Figure 1 ).

In particular embodiments, the recombinant microorganism may comprise a) a recombinant fadB gene, b) a recombinant fadA gene, c) a recombinant gene encoding a thioesterase, d) a recombinant ydiO gene, and e) a recombinant ydbK gene.

The invention further envisages that the yield of fatty acids can be further enhanced by inactivating endogenous genes encoding proteins involved in an endogenous metabolic pathway which produces a metabolite other than the fatty acid of interest and/or wherein the endogenous metabolic pathway consumes or accumulates the fatty acid.

Thus, according to particular embodiments recombinant the micro-organisms engineered to produce fatty acids as described herein further comprise at least one engineered gene deletion and/or inactivation of an endogenous pathway in which the fatty acid is consumed and/or a gene encoding an enzyme involved in an endogenous pathway which produces a metabolite other than the fatty acid of interest. In more particular embodiments, the at least one engineered gene deletion or inactivation is in a gene encoding an enzyme selected from the group consisting of D-lactate dehydrogenase (d-ldh), L-lactate dehydrogenase (l-ldh), phosphotransacetylase (pta), pyruvate formate lyase (pfl), pyruvate decarboxylase (pdc), pyruvate dehydrogenase (pdh) and any combination thereof.

In more particular embodiments, the micro-organisms envisaged herein comprise at least one engineered gene deletion and/or inactivation in an endogenous gene encoding lactate dehydrogenase, thereby preventing the production of the metabolite lactate (or lactic acid). In further more particular embodiments, the micro-organisms envisaged herein comprise at least one engineered gene deletion or inactivation is in an endogenous gene encoding a phosphotransacetylase, thereby preventing the production of the metabolite acetate (or acetic acid).

In further more particular embodiments, the micro-organisms envisaged herein comprise at least one engineered gene deletion or inactivation is in an endogenous gene encoding a pyruvate formate lyase (pfl), thereby preventing the conversion of pyruvate into formate and acetyl CoA and channeling pyruvate to the beta-oxidation cycle.

In still particular embodiments of the micro-organisms as envisaged herein, the deletion or inactivation of the endogenous gene is ensured by introduction of a recombinant gene involved in the reversed beta oxidation pathway. For instance, in particular embodiments, the recombinant gene encoding a ferredoxin-dependent enoyl-CoA reductase is introduced into the endogenous gene encoding a pyruvate formate lyase (pfl) or a pyruvate dehydrogenase (pdh), thereby knocking out this gene.

In still further particular embodiments, the micro-organisms as envisaged herein comprise at least one engineered gene deletion and/or inactivation of an endogenous gene encoding a lactate dehydrogenase (Idh), a phosphotransacetylase (pta), or a pyruvate formate lyase (pfl) thereby preventing the production of the metabolites lactate (or lactic acid) and acetate (or acetic acid) and formate and channeling the substrate pyruvate to the beta-oxidation cycle.

In still further particular embodiments, the micro-organisms as envisaged herein are of bacterial origin and comprise at least one engineered gene deletion and/or inactivation in the endogenous genes encoding a pyruvate decarboxylase (pdc) and pyruvate dehydrogenase (pdh) thereby preventing the conversion of pyruvate into acetaldehyde and acetyl-CoA, respectively, and channeling the substrate pyruvate to the beta-oxidation cycle.

According to particular embodiments envisaged herein, strains are provided which are characterized by the feature that the lactic acid, acetic acid (acetate) and/or ethanol production by said strain is at least reduced, more particularly is significantly reduced, compared to the background production in the corresponding wild-type strain. Accordingly, in particular embodiments, the lactic acid, acetic acid or acetate production in the bacterial strains of the present invention is for instance at least 60% lower, preferably at least 80% lower and even more preferably at least 90% lower than in the corresponding wild-type strain.

In particular embodiments however, the product of interest is an alcohol or an ester and the micro-organisms of the present invention contain further modifications for converting the fatty acid to the alcohol or ester of interest.

According to particular embodiments, the micro-organisms envisaged herein are yeast or fungal micro-organisms, more particularly oleaginous yeast, which further comprise at least one engineered gene deletion and/or inactivation in an endogenous pathway, in which fatty acids are accumulated, consumed or which compete with the fatty acid synthesis pathway of interest.

Indeed, it has been determined that such organisms are particularly suited for efficient fatty acid production.

More particularly, the yeast micro-organism may be engineered to avoid the use of fatty acids in membrane production. In particular embodiments, the engineered gene deletion and/or inactivation is in a gene encoding an enzyme such as but not limited to those selected from the group consisting of diacylglyceroltransferase (DGAT), phospholipid diacylglycerol transferase (PDAT), acyl-CoA dependent triacylglycerol synthase (DGA), acyl-CoA independent acyltransferase (LRO), acyl-CoA sterol acyltransferase (ARE) phospholipid-diacylglycerol acyltransferase (PLH), Diglyceride acyltransferase (or O- acyltransferase) (DGAT), diacylglycerol acyltransferase (DAGAT), fatty acid synthase (FAS), and any combination thereof. In further particular embodiments, the yeast or fungal micro-organisms comprise at least one engineered gene deletion and/or inactivation in an endogenous pathway, in which fatty acids are degenerated, in order to stimulate the reversed beta oxidation pathway. In further particular embodiments, the engineered gene deletion and/or inactivation is in a gene encoding acyl-CoA oxidase (AOX) or a gene with similar function, e.g. acyl-CoA dehydrogenase (AcDH).

An illustration of the metabolic pathways which are envisaged for modification herein according to particular embodiments, is provided in Figure 6.

In particular embodiments, the micro-organisms envisaged herein comprise a gene activation and/or gene deletion of one or more genes selected from DGAT1, DGAT2, LR01, ARE1 and ARE2, plhl, FAS, AOX, AcDH.

In particular embodiments, the micro-organisms envisaged herein are capable of growing on sugars and convert these sugars into fatty acids. In more particular embodiments, the micro-organisms of the invention can use monosaccharides such as glucose, xylose, arabinose, galactose, disaccharides as maltose, sucrose, lactose, cellobiose, polyalcohols like mannitol, sorbitol, glycerol, and mixtures thereof at a concentration at least 10 g/L for conversion into fatty acids. In particular embodiments, the micro-organisms envisaged herein comprise additional genetic modifications which further the use of a particular substrate. More particularly, micro-organisms are envisaged which contain one or more genetic modifications furthering the consumption of arabinose.

In particular embodiments, the recombinant micro-organisms are capable of producing fatty acids when cultivated in a fermentable medium, even and especially under anaerobic or quasi-anaerobic conditions. In particular embodiments, the micro-organisms may be capable of producing the fatty acid at a theoretical maximal yield (TM) of at 0.66 C/C with a practical maximal yield (PM) of at least 0.55 C/C and a productivity of at least 20g/l/h, such as at least 25g/l/h, at least 30g/l/h, such as at least 35g/l/h or even 40g/l/h under anaerobic or quasi-anaerobic conditions from pentose or hexose sugars.

More particularly, the yield that can be obtained by the microorganisms is at least 0.5 g/L, most particularly at least 2 g/L. In particular embodiments, the conversion yield of consumed sugar to fatty acid is at least 50%. In particular embodiments genetically modified or recombinant strains are provided, which are capable of producing fatty acids from pentose or hexose sugars at a yield of at least 5 g/L.

Typically the fatty acids produced by the microorganisms envisaged herein are a mixture of different lengths. More particularly the fatty acids produced by the microorganisms may be any fatty acids ranging from C4 to C18, more particularly C4, C6, C8, C12, C14, C16, C18 fatty acid. As detailed above, when using recombinant ter genes, the length of the fatty acid produced by the micro-organism can be controlled by selection of the appropriate ter gene and/or thioesterase gene. In particular embodiments the fatty acids produced by the micro-organisms herein contain high amounts of C14 fatty acids.

In a further aspect, the application relates to the use of the micro-organisms provided herein in the industrial production of fatty acids and/or products derived therefrom, such as alcohols' or esters. In this context, genetically modified strains capable of producing fatty acids with increased yield or with a modified composition are of particular interest. Indeed, in particular embodiments the micro-organisms may be capable of ensuring a high yield at limited production costs. Accordingly, in particular embodiments high yield methods of producing a composition comprising a fatty acid are provided. In particular embodiments, methods are provided comprising the steps of (i) providing a genetically modified or recombinant micro-organism as described herein, and (ii) culturing said micro-organism in the presence of a feedstock under conditions suitable for the production of the fatty acid or the product derived therefrom.

In further particular embodiments methods of producing a composition comprising specified amounts of one or more particular fatty acids are envisaged, such as, but not limited to C12, C14, C16 and C18 fatty acids. In particular embodiments, methods are provided comprising the steps of (i) providing a genetically modified or recombinant microorganism as described herein, and (ii) culturing said micro-organism in the presence of a feedstock under conditions suitable for the production of the fatty acid or the product derived therefrom. Different types of feedstocks are envisaged in the context of the present invention including but not limited to purified sugars (e.g. hexose and/or pentose sugars), Cellulosic Sugars, Algae, Ag Residues & Waste, Agave, Camelina, Canola, C0 ₂ , Cyanobacteria, MSW, Micro-Crops, Jatropha, Wood and Forest Residues, Salicornia, Sorghum, Switchgrass, Sugarcane, Soy, and Industrial Waste Streams/

In particular embodiments, cultivation is envisaged on hexose and/or pentose sugars as the sole carbon source.

Compositions can be obtained by the methods envisaged herein which contain high levels of fatty acid and/or a specific composition of fatty acids. In particular embodiments, the microorganism is capable of secreting the fatty acid into the medium. In alternative embodiments, the fatty acids are accumulated by the recombinant microorganisms in lipid granules within the host, and obtaining the fatty acids from the microorganisms may be necessary.

In particular embodiments, the fatty acids are secreted into the medium such that a composition comprising a relatively high titer of fatty acids can be obtained directly, by separating the microorganisms from the medium. In further particular embodiments, the relative titer of proteins in the compositions is reduced.

In certain preferred embodiments of methods for the production of fatty acids as envisaged herein, the cultivation of the micro-organisms is performed at least partially under anaerobic conditions.

In particular embodiments, methods for producing one or more fatty acids or a product derived therefrom, more particularly at high yield, are provided which comprise the steps of (i) providing a genetically modified or recombinant micro-organism that has been modified to produce a fatty acid or a product derived therefrom at high yield from hexose or pentose sugars and (ii) culturing said micro-organism in the presence of hexose sugars. In particular embodiments, the methods according to the present invention further comprise the step of recovering the fatty acid or product derived therefrom.

Accordingly, in a further aspect, methods are provided for producing one or more purified fatty acids or one or more other products derived therefrom, which methods comprise the steps of:

(i) providing a genetically modified micro-organism as envisaged herein; and (ii) culturing the micro-organism in the presence of pentose or hexose sugars under conditions suitable for the expression of said recombinant genes, and

(iii) recovering the one or more fatty acids or products derived therefrom from the cultivation medium.

The methods for generating the recombinant microorganisms involve standard genetic modifications, for which well established methods are available to the skilled person. In particular embodiments, the coding sequence encoding the enzyme of interest is placed under the transcriptional control of one or more promoters and one or more terminators, both of which are functional in the modified host cell.

Promoters and terminator sequences may be native to the host strain or exogenous to the cell. Useful promoter and terminator sequences include those that are highly identical (i.e. have an identities score of 90% or more, especially 95% or more, most preferably 99% or more) in their functional portions compared to the functional portions of promoter and terminator sequences, respectively, that are native to the host strain, particularly when the insertion of the exogenous gene is targeted at a specific site in the strain's genome.

In particular embodiments the promoter has an identity score at least 90%, 95% or 99% relative to a promoter that is native to a fungal gene. More particularly the promoter has an identity score of at least 90%, 95% or 99% compared to a promoter for a gene that is native to the host strain. In particular embodiments, the terminator has an identity score of at least 90%, 95% or 99% compared to a terminator for a gene that is native to a fungus. The terminator may have an identity score of at least 90%, 95% or 99% with a terminator for a gene that is native to the host strain. The use of native (to the host strain) promoters and terminators, together with their respective upstream and downstream flanking regions, can permit the targeted integration of the recombinant gene into specific loci of the host strain's genome, and for simultaneous integration the recombinant gene and deletion of another native gene. It is possible for the different recombinant coding sequences to be placed under the control of different types of promoters and/or terminators.

The recombinant gene may be integrated randomly into the host strain's genome or inserted at one or more targeted locations. Examples of targeted locations include the loci of a gene that is desirably deleted or disrupted. Suitable promoter sequences in the context of the present invention are known in the art and include for example an inducible tet promoter (Skerra, 1994), an inducible IPTG promoter (Jacob and Monod, 1961 ; J Mol Biol. 3:318-356).

Genetic modification of the host strains is accomplished in one or more steps via the design and construction of appropriate vectors and transformation of the host strain with those vectors. Electroporation and/or chemical (such as calcium chloride- or lithium acetate-based) transformation methods or Agrobacterium tumefaciens-med\aled transformation methods as known in the art can be used. The vectors can either be cut with particular restriction enzymes or used as circular DNA. The vector used for genetic modification of the host strains may be any vector so long as it can integrate in the genome of the host strain. Vectors of the present invention can be operable as cloning vectors or expression vectors in the selected host strain. Numerous vectors are known to practitioners skilled in the art, and selection of an appropriate vector is a matter of choice. The vectors may, for example, be the pASK-IBA3C expression vector (IBA-life sciences), pUR5750 transformation vector (de Groot et al. 1998 Nature Biotechnology 16, 839 - 842), the pCGHT3 transformation vector (Chambers et al. 1988 Gene, Volume 68, Issue 1 : 15; Scholtmeyer etal. 2001 Appl. Environ. Microbiol. 67(1 ): 481 ).

In general, a vector is prepared that contains the (combination of) coding sequences of interest and associated promoter and terminator sequences. The vector may contain restriction sites of various types for linearization or fragmentation. Vectors may further contain a backbone portion (such as for propagation in E. coli) many of which are conveniently obtained from commercially available yeast or bacterial vectors. The vector preferably contains one or more selection marker gene cassettes. A "selection marker gene" is one that encodes a protein needed for the survival and/or growth of the transformed cell in a selective culture medium. Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins such as chloramphenicol, zeocin (sh ble gene from Streptoalloteichus hindustanus), genetecin, melibiase (MEL5), hygromycin (aminoglycoside antibiotic resistance gene from E. coli), ampicillin, tetracycline, or kanamycin (kanamycin resistance gene of Tn903), (b) complement auxotrophic deficiencies of the cell. Two prominent examples of auxotrophic deficiencies are the amino acid leucine deficiency (e.g. LEU2 gene) or uracil deficiency (e.g. URA3 gene). Cells that are orotidine-5'-phosphate decarboxylase negative (ura3-) cannot grow on media lacking uracil. Thus a functional URA3 gene can be used as a marker on a cell having a uracil deficiency, and successful transformants can be selected on a medium lacking uracil. Only cells transformed with the functional URA3 gene are able to synthesize uracil and grow on such medium. If the wild-type strain does not have a uracil deficiency (as is the case with /. orientalis, for example.), an auxotrophic mutant having the deficiency must be made in order to use URA3 as a selection marker for the strain. Methods for accomplishing this are well known in the art.

Preferred selection makers include the zeocin resistance gene, G418 resistance gene, hygromycin resistance gene. The selection marker cassette typically further includes a promoter and terminator sequence, operatively linked to the selection marker gene, and which are operable in the host strain.

Successful transformants can be selected for in known manner, by taking advantage of the attributes contributed by the marker gene, or by other characteristics (such as ability to produce fatty acids, inability to produce lactic acid or lactate, inability to produce acetic acid or acetate, or ability to grow on specific substrates) contributed by the inserted genes. Screening can be performed by PCR or Southern analysis to confirm that the desired insertions and deletions have taken place, to confirm copy number and to identify the point of integration of genes into the host strain's genome. Activity of the enzyme encoded by the inserted gene and/or lack of activity of enzyme encoded by the deleted gene can be confirmed using known assay methods.

The deletions or inactivations envisaged herein can be accomplished by genetic engineering methods, forced evolution or mutagenesis and/or selection or screening. Indeed, the present state of the art provides a wide variety of techniques that can be used for the inactivation, deletion or replacement of genes. Such molecular techniques include but are not limited to:

(i) gene inactivation techniques based on natural gene silencing methods including antisense RNA, ribozymes and triplex DNA formation,

(ii) techniques for single gene mutation such as gene inactivation by single crossing over with non-replicative plasmid and gene inactivation with a non-replicative plasmid or a linearized DNA fragment capable of double-crossover chromosomal integration (Finchham, 1989, Microbiological Reviews, 53: 148-170; Archer et a/., 2006, Basic Biotechnology: 95-126), and

(iii) techniques for multiple unmarked mutations in the same strain, such as but not limited to:

(a) deletion and replacement of the target gene by an antibiotic resistance gene by a double crossover integration through homologous recombination of an integrative plasmid, giving segregationally highly stable mutants;

(b) removing of the antibiotic resistance gene with the Flp recombinase system from Saccharomyces cerevisiae allowing the repeated use of the method for construction of multiple, unmarked mutations in the same strain, and

(c) generating a strain deleted for the upp gene, encoding uracil phosphoribosyl transferase, thus allowing the use of 5-fluorouracyl as a counter selectable marker and a positive selection of the double crossover integrants.

In particular embodiments the deletion or disruption of the endogenous gene is performed according to the method described by Oliveira et al (2008) (Appl Microbiol Biotechnol 80, 917-924)

In particular non-limiting embodiments envisaged herein, the deletion or disruption of the endogenous gene, may be performed by the simultaneous introduction of one or more functional structural genes, notably a gene encoding an enzyme involved in the production of the fatty acid of interest, such as, but not limited to one of the genes described above, inserted between the 5' and 3' flanking portions of one of the endogenous gene of the host strain. The functional gene preferably includes functional promoter and terminator sequences operatively linked to the structural gene. This approach allows for the simultaneous deletion of the endogenous gene and insertion of the functional exogenous or heterologous gene. The vector may include a selection marker gene instead of or in addition to the structural gene. Again, the selection marker gene is positioned on the vector between the 5' and 3' flanking portions of the endogenous gene(s) being targeted, and becomes inserted in the locus of the functional endogenous gene. The use of a selection marker gene has the advantage of introducing a means of selecting for successful transformants. However, it is also possible to select for successful transformants based on the resulting functional characteristics. For instance, depending on the genes deleted and introduced it may be possible to screen on reduced or eliminated ability to grow on specific building blocks, to produce the fatty acid of interest at high concentrations or on their reduced ability to produce specific metabolites.

Accordingly, a further aspect provides methods of obtaining micro-organisms with an increased and/or modified production of fatty acid, or a product derived therefrom, which methods comprise:

a) transforming the micro-organism with one or more recombinant nucleic acid sequences which ensure an increased and/or modified production of the fatty acid (and/or the product derived therefrom envisaged); and

b) selecting a micro-organism capable of high yield production of the fatty acid or product derived therefrom.

The step of transforming the micro-organism is described in detail hereinabove. As detailed above, different genetic modifications are envisaged which increase the yield of fatty acid production. In particular embodiments the step of transforming the microorganism with one or more recombinant nucleic acid sequences which ensure an increased production of the fatty acid (and/or the product derived therefrom includes the modification and/or inactivation of endogenous genes involved in the synthesis of other products the synthesis of which competes with fatty acid production in said organism, in order to increase the yield of fatty acid production.

The step of selecting a micro-organism capable of high yield fatty acid production (or high yield production of a product derived therefrom) is a selection step known to the skilled person and includes but is not limited to measuring activity of enzymes involved in the production of the fatty acid of interest. Additionally or alternatively in the methods according to the invention, the selection step can be based on reduced production of lactic acid or lactate, acetic acid or acetate or formic acid or formate, etc... In a further aspect, methods for producing a fatty acid or a product derived therefrom at high yield, especially under anaerobic or quasi anaerobic conditions are provided. Indeed, high yield production of fatty acids or products derived therefrom is of interest in view of its numerous industrial applications, such as for instance but not limited to diesel fuel, jet fuel, lubricants, polyoleifins (e.g. polyethylenes, polypropylene), elastomers, polyesters, polyamides, plastic processing additives, coating resins, surfactants. In particular embodiments, the methods of the present invention comprise the steps of obtaining a micro-organism which is can produce high concentrations of fatty acid by genetically modifying it to increase yield of the fatty acid and culturing the thus obtained microorganism in the presence of specific substrates or chemical building blocks.

In particular embodiments, the methods of the present invention comprise the steps of (i) modifying a strain such that it is capable of producing the fatty acid of interest, preferably at high yield, from a substrate such as pentose or hexose sugars; and (ii) culturing said strain in the presence of said suitable substrate.

In particular embodiments of the methods for the production of fatty acids, the produced fatty acid is a carboxylic acid, even more particularly such as but not limited to butanoic acid, hexanoic acid, caprylic acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid. In further particular embodiments compositions comprising a specific amount of one or more specified fatty acids are envisaged. Indeed, it has been found that the fatty acids produced by the micro-organism may vary, depending on the genes that have been introduced. Methods for producing a micro-organism and methods of modifying said organism to increase fatty acid production yield and/or fatty acid composition are described hereinabove and illustrated in the Examples section.

In particular embodiments of the process envisaged herein, the micro-organism or strain are cultivated in a medium that includes a sugar that is fermentable by the transformed strain. The sugar may comprise monosaccharides, such as but not limited to glucose, xylose, arabinose, galactose, disaccharides, such as but not limited to maltose, sucrose, lactose, cellobiose, polyalcohols, such as but not limited to mannitol, sorbitol, glycerol, and mixtures thereof.

In particular embodiments, the micro-organism is modified to ensure the ability to use a specific substrate in the production of fatty acids. Additionally or alternatively, enzymes can be added to the cultivation medium to ensure degradation of another substrate into fermentable hexose sugars.

In particular embodiments of the invention, the medium contains at least (the equivalent of) 5 g/L, at least 10 g/L, at least 20 g/L, at least 30g/L, more particularly at least 40g/L, and even more particularly at least 50 g/L glucose. In further particular embodiments, the medium comprises at least 100 g/L, more particularly between 100 and 500 g/L glucose. The medium may optionally contain further nutrients as required by the particular strain, including inorganic nitrogen sources such as ammonia or ammonium salts, and the like, and minerals and the like. However, in more particular embodiments, the medium is a complete mineral medium comprising a hexose sugar as the only carbon source. The ability of the strains of the present invention to grow on this simple medium greatly reduces cost of cultivation and simplifies purification of the fatty acid produced.

Other growth conditions, such as temperature, cell density, and the like are not considered to be critical to obtain the effects envisaged herein and are generally selected to provide an economical process. Temperatures during each of the growth phase and the production phase may range from above the freezing temperature of the medium to about 50°C. A preferred temperature, particularly during the production phase, is from about 28-45°C. The culturing step of the methods of the invention may be conducted aerobically, microaerobically or anaerobically. Quasi-anaerobic conditions or oxygen limited conditions, in which no oxygen is added during the process but dissolved oxygen is present in the medium at the start of the production process, can also be used.

In particular embodiments, the methods as envisaged herein comprise cultivation of micro-organisms (strains) which exhibit the ability to convert sugars to fatty acid (or another product derived therefrom of interest) under anaerobic or oxygen-limited conditions.

The cultivation step of the methods according to this aspect can be conducted continuously, batch-wise, or some combination thereof.

The yield of fatty acid obtained by the tools and methods envisaged herein will depend on the cultivation conditions used. In certain embodiments, the methods of producing fatty acids or products derived therefrom according to the present invention result in a yield of fatty acid (or derived product) that is at least 0.5 g/L, particularly at least 2 g/L, more particularly at least 5 g/L, particularly at least 50 g/L, and most particularly between 50-100 g/L. In further particular embodiments the productivity of about 2-3 g/L/hour.

In particular embodiments the micro-organisms envisaged herein are capable of converting at least 50%, more particularly at least 60%, even more particularly 75%, most particularly at least 95%, and up to 100% of the substrate consumed. In practice, the yield obtained in particular embodiments of the methods of the present invention is at least 0.5g/g sugar, more particularly at least 0.6g/g sugar, but may be up to 0.95g/g sugar.

In further embodiments, methods are provided for producing a fatty acid or a product derived therefrom which, in addition to the steps detailed above further comprise the step of recovering the fatty acid of interest. In particular embodiments, recovery of the fatty acid from cultivation medium in the methods of the present invention is greatly simplified in view of the fact that the organisms can be grown on a mineral medium containing only sugars as a carbon source. Suitable purification can be carried out by methods known to the person skilled in the art such as by using extraction, ion exchange resins, electrodialysis, nanofiltration, etc... In further particular embodiments, where a product derived from a fatty acid, such as an ester is envisaged, the methods may comprise the step of recovering said product of interest.

In a further aspect, the compositions obtainable by the methods for producing fatty acids or products derived therefrom described herein are provided, which compositions comprise one or more fatty acids and/or one or more products derived therefrom, such as for instance but not limited to butanoic acid, hexanoic acid, caprylic acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, or products such as alcohols or esters obtained therefrom. The present invention will now be further illustrated by means of the following non-limiting examples. EXAMPLES

Example 1 : Fatty acid production in E. coli

A genetically modified E. coli was generated by ensuring overexpression of enzymes involved in the beta-oxidation pathway and expression of an NADH dependent enoyl-CoA reductase (ter).

Thus, in total four genes were selected for expression in E. coli. These four genes code for:

• FadB, exhibiting enoyl-CoA hydratase (EC 4.2.1.17) and 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) activity

• FadA, exhibiting 3-ketoacyl-CoA thiolase (EC 2.3.1.16) activity. FadB and FadA form the multienzyme complex (FadA ₂ FadB ₂ ).

· FadM which is a thioesterase (EC 3.1.2.-)

• TER (from Euglena gracilis) which is a frans-enoyl-CoA reductase.

The nucleotide sequence was codon optimized.

Individual cloning of the genes into an expression vector

With the aim of obtaining good expression levels, the four genes fadA, fadB, fadM and ter were cloned step by step into the pMA expression vector. Therefore, suitable oligonucleotides were generated and the genes were amplified by PCR using pMA::4 as template.

· The fragment fadBA was cloned with into the MCS2 of the vector (Figure 4a)

• The ter gene was cloned into the MCS1 of the plasmid (Figure 4b)

• The combination of fadAB in MCS2 and ter in MCS1 was also made (Figure 4c)

• The full construct fadAB in MCS2 and ter+fadM in MCS1 is illustrated in Figure 4d. In a next step IPTG inducible expression vectors were constructed as listed in Table 1 Table 1. IPTG inducible expression vectors

Transformation was checked by streaking transformants on agar plate containing X-Gal and IPTG. After incubation at 37 °C over night, the streak of LacZ-containing cells is blue, whereas the streak of the control strain is not. Expression of genes was further tested by Western blot. Increase of fatty acid synthesis by reduction of production of side products

In order to further increase fatty acid production, the genes for fatty acid production were transferred to E. coli and derivate deletion strains with a disrupted lactate dehydrogenase gene (AldhA, EC 1.1.1.28), with a disrupted phosphotransacetylase gene (Apta, EC 2.3.1.8), and with the double knockout.

Prior to transformation, these strains were checked to determine substrate and product analysis.

E. coli wild type strain BW251 13 and derivate deletion strains with disrupted lactate dehydrogenase gene (AldhA, EC 1.1.1.28), phosphotransacetylase gene (Apta, EC 2.3.1.8), and double knock out (AldhA+Apta) were cultivated in mineral medium (Evans medium) under anaerobic conditions in serum flasks closed with a butyl rubber stopper. After 24 hours of incubation the substrate and product concentrations were determined. The results are shown in Table 2. Strongly reduced lactate production was observed in AldhA strains and strongly reduced acetate production in Apta strains. This resulted in the strong production of pyruvate by the double knock out mutant.

Table 2. Glucose consumption and product formation by E. coli wild type and Apta, Aldh and AptaAIdh strains after 24 hours incubation.

Compound (mm) Wt Apta Aldh AldhApta

Glucose 45 35 37 22

Lactate 38 74 3 0

Acetate 27 2 32 3

Formate 18 2 36 1 1

Succinate 6 5 7 7

Pyruvate 1 3 7 17

Ethanol 17 3 23 10

Transformation

For expression the constructs were transferred to E. coli and derived deletion strains described above with disrupted lactate dehydrogenase gene (AldhA, EC 1.1.1.28), with disrupted phosphotransacetylase gene (Apta, EC 2.3.1.8), and a double knockout with disrupted lactate dehydrogenase gene (AldhA, EC 1.1.1.28) and disrupted phosphotransacetylase gene (Apta, EC 2.3.1.8)

The control strain for expression was E. coli BL21 (DE3). As the IPTG inducible vectors need a T7-RNA polymerase to be expressed in the E. coli strain used, this gene was introduced into the E. coli BW251 13 Apta AldhA strain resulting in E. coli BW251 13 Apta AldhA+T7pol. Expression analysis of double transformant strains E. coli BL21 and E. coli BW25113 Apta AldhA (+T7 pol.) containing the pACYCDuet and the pCOLADuet vectors showed expression of three genes fadA, fadB, fadM and ter (data not shown).

Clear expression of the fadA, fadB and the ter gene was observed in the control strain (E. coli BL21 ) (data not shown).

Cell free extracts of E. coli cells potentially expressing fadM were also analyzed for FadM enzyme activity (using C18:0 acyl-CoA as a substrate and Ellman's reagent for detecting the release of CoASH, Nie et al. 2008). These assays clearly showed that IPTG induced E. coli cells have a strongly enhanced FadM activity (Figure 5).

Thus, the genes fadA, fadB, fadM and ter were found to be well expressed in both the E. coli BL21 and E. coli BW25113 Apta AldhA (+T7 pol.). Fatty acid analysis

The following protocol for the analysis of methylated fatty acids was developed for freeze- dried wild-type E. coli cells.

The fatty acids are extracted from the dry cells using chloroform or chloroform/methanol. The fatty acids are methylated using TMSH (W. Butte, J. Chromatogr. 261. 1983) and analysed by GC. An internal standard is used to quantify the peaks and a standard series of fatty acids is used to identify the individual fatty acids.

• A Chloroform/methanol (1 :2) mixture was chosen as solvent for extraction of fatty acids from dry cells, as more types of fatty acids, and a higher concentration of fatty acids was detected after GC analysis than after use of chloroform alone as solvent.

• An extraction time of 16 - 20 h gave maximal peak areas and is thus the extraction time to be used.

Samples from cultures of transformant and wild type strains double are analysed using this procedure. Table 3 shows the amount of fatty acids (C16:0 palmitic acid, C16: 1 palmitoleic acid, C18:1 oleic acid) accumulated by the wild type E. coli strain and the double deletion strain after growth in LB or Evans medium under aerobic conditions at 37 °C.

Table 3. Determined amount of fatty acids in control strains of E. coli BW25113 (wt) and E. coli BW251 13 Mdh Apia

Strain Medium C16:0 C16:0 C16:1 C16:1 C18:1 C18:1 mg/ml nmol/ml Mg/ml nmol/ml mg/ml nmol/ml

Wt LB 0.40 17.8 0.01 2.6 1.13 0.2

Wt Evans 0.72 31 .9 0.25 98.7 10.78 1.8

AldhApta LB 0.27 1 1 .9 0.03 1 1.1 1.03 0.2

AldhApta Evans 0.85 37.6 0.36 141.0 1.18 0.2

The strains E. coli BL21 and E. coli BW25113 Apta AldhA (+T7 pol.) containing the genes fadA, fadB, Ter and fadM were analysed for fatty acid production. No extracellular fatty acids were found (results not shown). The double knockout induced with IPTG showed a 2-3 fold higher intracellular fatty acid content with modified fatty acid composition (enriched in C14:0, C16:0 and C18:0), compared to the non-induced strain, suggesting that the synthetic pathway is active.

The fatty acid compositions of the biomass showed some differentiation. All transformants induced with IPTG showed lower C14:0, C17:0D and C19:0D contents and increased C16: 1 and C18:1 contents, as compared to the non-induced cultures. All transformants contained the FadM gene, such that it appears that this gene caused the observed differences. When all four enzymes of the reversed beta-oxidation pathway were present (i.e. the combination of FadABM+TER) a much larger impact on fatty acid composition was realized: it was the only transformant producing C12:0, and showed clearly increased levels of C14:0 and C18:0. These compounds were produced at the expense of C16:0, C17:0D and C18: 1. Moreover, the FadABM + TER transformant produced as only strain trace amounts of nine, sofar unknown, fatty acids. This strain also shows the largest increase in fatty acids content of the biomass (g/g) and in fatty acid concentration (g/g) upon induction with IPTG.

Table 4. Dry weight (g/l), fatty acid content of biomass (%, wt/wt) and fatty acid concentration of transformants, with and without IPTG

AldhApta + FadABM +FadABM + FadM +FadABM + FadM +FadABM +FadABM

1 2 +Ter +Ter +YdiO +YdiO +YdiO

+YdbK

IPTG - + - + - + - + - + - + - + +

Fatty acid % % % % % % % % % % % % % % % %

DW (g/l) 0,8 0,8 0,8 0,7 0,9 0,9 0,8 0,8 0,8 0.9 1.0 0,9 0,8 0,7 0,6 0,5

FA % 5,4 5,8 4,3 4,6 5,6 5,2 7,2 6,6 5,2 7.0 7,5 8,4 5,6 5,4 5,1 5,7

FA (mg/l) 44 47 36 34 50 46 60 50 43 61 73 78 43 37 31 30

41

?=unknown peaks

Example 2: Fatty acid production in yeast Identification of relevant genes The yeast C. curvatus is used as a model organism. The genes corresponding to DGAT (DGAT1 , DGAT2),PDAT, and FAS involved in the production of triacyl-glycerols were identified from the draft genome sequence for C. curvatus that is available at FBR. Also the GPD, pyrG, Ku70 and the gene corresponding to AOX involved in fatty acid degradation was identified.

Codon optimized synthetic genes encoding FadA, FadB, FadM and TER were ordered.

Using PCR, genes, gene fragments and the necessary marker genes which are necessary to build the vectors were cloned and sequenced.

Vector construction

Vectors were prepared by joining restriction fragments from the cloned PCR fragments or from the synthetic genes. Transformation vectors were built from a combination of a GPD promoter fragment, a gene to be expressed and a GPD terminator fragment. The following vectors were made:

GPD promoter-Hygromycin resistance gene- GPD terminator (GHG)

GPD promoter-Nourseothricin resistance gene- GPD terminator (GNG)

GPD promoter-FadA gene- GPD terminator (GFadAG)

GPD promoter-FadB gene- GPD terminator (GFadBG)

GPD promoter-FadM gene- GPD terminator (GFadMG)

GPD promoter-TER- GPD terminator (GTERG)

Suppressing endogenous fatty acid consumption

Knock out vectors aimed at insertion in a specific endogenous gene in order to inactivate this gene are prepared by selecting a 500 base pair fragment homologous to the 5'-part of the gene to be inactivated and cloning it in front of the first GPD promoter and a 500 base pair fragment homologous to the 3'-part of the gene to be inactivated cloned after of the last GPD terminator. The following vectors were generated for the C. curvatus dgatl and dgat2 genes: 5 GAT1 -GPD promoter-Hygromycin gene-GPD terminator-3'DGAT1 (DGAT1 -GHG) 5 GAT2-GPD promoter-Nourseothricine gene-GPD terminator-3'DGAT2 (DGAT2-GNG) 5'pyrG-GPD promoter-Nourseothricine gene-GPD terminator-3'pyrG (PyrG-GNG) 5'Ku70-GPD promoter-Nourseothricine gene-GPD terminator-3'Ku70 (Ku70-GNG)

The DGAT1 , DGAT2 and pyrG knockout vectors contained 500 bp overlap regions.

Similarly, knock out vectors aimed at insertion within the FAS gene and thereby inactivating this gene are prepared by selecting a 500 base pair fragment homologous to the 5'-part of the gene to be inactivated and cloning it in front of the first GPD promoter and a 500 base pair fragment homologous to the 3'-part of the gene to be inactivated cloned after of the last GPD terminator. The following vector was generated:

5'FAS-2-GPD promoter-Hygromycin gene-GPD terminator-3'FAS-2 (FAS-2-GHG) After transformation of a FadA, FadB, FadM, TER containing C. curvatus strain colonies were plated on oleic acid/tween80 containing plates and selected for oleic acid auxotrophy.

Reducing endogenous fatty acid degradation

Knocking out the AOX gene reduces the fatty acid degradation in C. curvatus and thus enhances the effect of the reversed beta-oxidation pathway.

Knock out vectors aimed at insertion in the AOX gene and thereby inactivating this gene are prepared by selecting a 500 base pair fragment homologous to the 5'-part of the gene to be inactivated and cloning it in front of the first GPD promoter and a 500 base pair fragment homologous to the 3'-part of the gene to be inactivated cloned after of the last GPD terminator. The following vector was generated:

5'AOX-GPD promoter-Hygromycin gene-GPD terminator-3'AOX (AOX-GHG)

After transformation of a FadA, FadB, FadM, TER containing C. curvatus strain, with the AOX knock out vector colonies were plated antibiotic containing plates and selected for AOX knock out by means of PCR. Increasing homologous recombination efficiency

In order to further enhance homologous recombination efficiency, the genome of C. curvatus was analysed for the presence of Ku70 or Ku80 genes, as knocking out Ku70 and/or Ku80 was shown to enhance homologous recombination in a number of yeasts and fungi.

Analysis of the genome of C. curvatus showed that the genome contains has a Ku70 homologue.

The following vector was constructed containing 800 bp flanking region of the Ku70 gene and a NAT marker gene in between:

5'Ku70-GPD promoter-Nourseothricine gene-GPD terminator-3'Ku70 (Ku70-GNG)

Transformation of Cryptococcus curvatus A TCC20509.

Preparation of competent cells

10 ml YPD (in a 100 ml Erlenmeyer flask) is inoculated with a colony or with part of a glycerol stock and grow overnight (o/n) at 30 ^° C and 250 rpm. 1 ml from the o/n culture is used to inoculate 50 ml of YPD medium in a 250 ml Erlenmeyer flask and is grown for 3-4 hours at 30 ^° C and 250 rpm. In this time the culture should reach an OD650 of 0.8-1. The culture is then put on ice.

50 ml of culture in is then transferred to a Costar tube at 3500 rpm (4 ^° C) 10 -15 min., and the medium is carefully discarded. Thereafter, 10 ml cold filter sterilised 50 mM SodiumPhosphate buffer pH 7.5 + 25 mM DTT is added. The yeast pellet is carefully re- suspended by swirling with a sterile pipette.

The culture is then incubated approx. 12 minutes at 37 ^° C in a waterbath. The tubes are cooled quickly on ice and centrifuged 15 min. as before. The buffer/DTT is discarded and replaced with 10 ml of cold sterile 270 mM Sucrose, 10 mM Tris/HCI pH 7.5, 1 mM MgCI2. The pellet is carefully re-suspended and centrifuged again in the same way. The washing step is repeated.

The final pellet is re-suspended in a small volume of the Sucrose solution. First approx. 0.5 ml is used and a cell suspension with a high viscosity is made which can just be handled with a normal 1 ml pipette tip.

Electroporation

For electroporation a Biorad Gene Pulser with 2 mm Cuvettes is used.

Pulse settings; 0.8 kVolt, 1000 Ohm and 25

50 μΙ competent cells are mixed with 5 μΙ DNA* (in water) on ice in an Eppendorf tube.

The mix is transferred to a cuvette (precooled on ice) and the mixture is tapped down. The pulse is provided and immediately thereafter 1 ml. of YPD medium (room temp.) is added. The cells are mixed gently.

The cells are then transferred back to the Eppendorf tube and incubated 2-3 hr. at 30 ^° C.

DNA*

The DNA vector is used linearized or circular (standard plasmid isolates).

Inserting genes together with the selection marker is done by making one vector construct or by means of co-transformation. In the latter case depending on the gene used 1 -3 out of 10 co-transformants are obtained.

The amount of vector used is 100 - 1000 ng. of DNA per transformation resulting in 100 - 500 transformants / μg DNA.

Plating of transformants

After incubations the cells are plated on YPD/ 1.5% agar + the proper antibiotic. Antibiotics to be used are hygromycin, kanamycin, zeocin or nourseothricin. Optimal concentration of nourseothricin for selection was determined to be at 75μg/ml.

After 3-4 days resistant colonies appear on the plates.

Cultivation of transformants Colonies are used to inoculate 3-5 ml cultures YPD + antibiotic.

Fatty acid analysis

The following protocol for the analysis of methylated fatty acids was used for freeze-dried wild-type C. curvatus cells.

The fatty acids are extracted from the dry cells using chloroform or chloroform/methanol. The fatty acids are methylated using TMSH (W. Butte, J. Chromatogr. 261. 1983) and analysed by GC. An internal standard is used to quantify the peaks and a standard series of fatty acids is used to identify the individual fatty acids.

· A Chloroform/methanol (1 :2) mixture was chosen as solvent for extraction of fatty acids from dry cells, as more types of fatty acids, and a higher concentration of fatty acids was detected after GC analysis than after use of chloroform alone as solvent.* An extraction time of 16 - 20 h gave maximal peak areas and is thus the extraction time to be used.

Samples from cultures of transformant and wild type strains double are analysed using this procedure.

Results a) Creating C. curvatus strains containing the alternative fatty acid biosynthesis pathway

Insertion of the alternative fatty acid biosynthesis pathway, fadA + fadB + fadM + TER, was done by co-transformation of a selectable marker gene (either the Hygromycin or Noursethricin resistance gene) with the 4 genes. After growth on selective plates colonies were initially screened for the presence of the heterologous genes by means of colony PCR. Colonies showing the presence of the genes were grown on a streak plate and single colonies were grown in liquid culture for DNA analysis. PCR on isolated DNA was used to confirm the genotype of the transformants. The strains obtained are listed in Table 6. Table 6. Strains and genotypes

* genotype based on colony PCR.

The DNA was used for PCR reactions in order to confirm the genotype. Strains C1-12, C1-14, H01-1 . C1 -7, D1 -2 were selected for fatty acid analysis.

The fermentations with C. curvatus transformants (C1-1 1 and C1-14, carrying the three FAD genes and TER; H01-1 , carrying only the ter gene) and wild type were done in 300ml shake flasks with 50ml minimal medium (Meesters et al) supplemented with 0.27 g/l NH ₄ CI and 16 g/l glucose at pH 5.5. The cultures were inoculated with a 24h old YPD culture. The cultures had an initial optical density of 0.2 (OD446nm) and were cultivated under aerobic conditions at 30°C, 175 rpm. Fatty acid accumulation is stimulated by nitrogen limitation during the experiment. The biomass is harvested after 3 days (without cerulenin) or 5 days (with cerulenin) and freeze dried for fatty acid analysis (Table 7). Table 7: Fatty acid analysis

0,25g/l 1 1 1

No large differences were found in fatty acid composition between wild type strains and transformants. The fatty acid content of the wild type strain was 60%, a value we have found as maximum in comparable experiments in the past. Presence of TER, whether or not with FadA, FadB and FadM resulted in an increase of fatty acid content to 68-72 % of the cell dry weight. These values are exceptionally high for C. curvatus. These results indicate that the reversed beta-oxidation pathway is indeed active in the transformants. The fact that only the presence of ter is sufficient to evoke this, suggests that the homologous beta-oxidation enzymes of C. curvatus are present and active. b) Eliminating endogenous fatty acid consumption pathways

i) Knock-out strategy

From the first experiments using the knock-out vectors, it appeared that the efficiency of recombination was low. Thus a strategy was developed to knock out the endogenous Ku70 gene. Transformation was performed with a vector comprising a sequence containing a flanking region of the Ku70 gene. Transformation with the construct (transformation T) gave 180 colonies.

These knock-outs can be used for further knock-out of the endogenous dgatl, dgat2 and fas genes using the knock-out vectors described above.

Colonies are identified wherein the dgatl , dgat2 and/or fas gene are effectively suppressed.

ii) Reducing endogenous fatty acid synthesis in C. curvatus using Cerulenin.

An alternative way to reduce fatty acid accumulation in C. curvatus is the addition of cerulenin during growth. Cerulenin has been shown to inhibit fatty acid synthesis in a large number of fungi and yeasts. The inhibition occurs at the level of the FAS system. In order to maintain growth the inhibiting effect can partly be complemented by the addition of oleic acid to the growth medium.

Cerulenin was added at a concentration of 25 μg/ml). Oleic acid/tween80 was added at 0.1 g/l, 0.25 g/l. For the

The results of adding cerulenin or cerulenin +oleic acid are provided in Table 7 above.

Addition of cerulenin resulted in low growth, due to an efficient inhibition of fatty acid synthesis. This could partly be overcome by the addition of Tween80/oleic acid.

Also in experiments in which both cerulenin and tween80/oleic acid were added the transformants showed higher fatty acid contents, 7.7-10.2% instead of 5.3%, indicating that the reversed beta-oxidation pathway is indeed active in the transformants. c) Reducing endogenous fatty acid degradation in C. curvatus An FadA, FadB, FadM, and TER containing C. curvatus strain was transformed, with the AOX knock out vector. Colonies are identified wherein the AOX gene is effectively suppressed.

Example 3: The ferredoxin approach in E. coli. Vector construction

The homologous E. coli genes ydiO and ydbK were multiplied by means of PCR and cloned in a plasmid.

Similar to the procedure described in Example 1 , IPTG expression vectors were constructed comprising different combinations of fadA, fadB, fadM, ydiO and ydbK genes.

Transformation For expression the constructs were transferred to E. coli and derived deletion strains described above with disrupted lactate dehydrogenase gene (AldhA, EC 1.1.1.28), with disrupted phosphotransacetylase gene (Apta, EC 2.3.1.8), and a double knockout with disrupted lactate dehydrogenase gene (AldhA, EC 1.1.1.28) and disrupted phosphotransacetylase gene (Apta, EC 2.3.1.8). The control strain for expression was E. coli BL21 (DE3).

As the IPTG inducible vectors need a T7-RNA polymerase to be expressed in the E. coli strain used, this gene is introduced into the E. co// BW25113 Apta AldhA strain resulting in E. coli BW251 13 Apta AldhA+T7pol.

The presence of the constructs was determined by cultivation in media containing the relevant antibiotic resistance markers. Expression analysis of the different strains is performed to check the production of the recombinant genes.

Fatty acid analysis The protocol for the analysis of methylated fatty acids described above was used to analyse samples from cultures of transformant and wild type strains.