# Patent

## Title
Schizochytrium fatty acid synthase (FAS) and products and methods related thereto

## Abstract

Disclosed are a fatty acid synthase (FAS) from Schizochytrium, biologically active fragments and homologues thereof, a nucleic acid sequence encoding such FAS, fragments and homologues thereof, the gene encoding Schizochytrium FAS, host cells and organisms that recombinantly express the FAS, host cells and organisms in which the expression and/or activity of the endogenous FAS has been attenuated, and various methods for making and using any of these proteins, nucleic acid molecules, genes, host cells or organisms.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a Divisional application of U.S. application Ser. No. 11/058,046, filed on Feb. 14, 2005, now US Publication No. 2005/0191679, which claims priority under 35 U.S.C. §119(e) from U.S. Provisional Application Ser. No. 60/544,692, filed Feb. 13, 2004. The entire disclosures of each of the foregoing applications are incorporated herein by reference.


FIELD OF THE INVENTION
This invention generally relates to a fatty acid synthase (FAS) from Schizochytrium and to products and methods related thereto.
BACKGROUND OF THE INVENTION
The de novo synthesis of short and medium chain saturated fatty acids from acetyl-CoA and malonyl-CoA is a complex process catalyzed by several enzyme activities. In most bacteria and in plants these activities are associated with discrete monofunctional polypeptides. In fungi and animals however, these activities are integrated into one or two multifunctional polypeptide chains. Fatty acids, in particular in the form of oils and fats, which are glycerol esters, play a major role in human nutrition because of their high energy content. Additionally, specific types of fatty acids (e.g., polyunsaturated fatty acids such as DHA) have a wide range of physiological effects. There is great interest in being able to manipulate specific types of fatty acids made in organisms that produce these fatty acids (e.g., in the form of oils and/or phospholipids).
SUMMARY OF THE INVENTION
One embodiment of the present invention relates to an isolated protein comprising an amino acid sequence selected from: (a) an amino acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, an amino acid sequence consisting of positions 1-500 of SEQ ID NO:2, an amino acid sequence consisting of positions 450-1300 of SEQ ID NO:2, an amino acid sequence consisting of positions 1250-1700 of SEQ ID NO:2, an amino acid sequence consisting of positions 1575-2100 of SEQ ID NO:2, an amino acid sequence consisting of positions 2025-2850 of SEQ ID NO:2, an amino acid sequence consisting of positions 2800-3350 of SEQ ID NO:2, an amino acid sequence consisting of positions 3300-3900 of SEQ ID NO:2, an amino acid sequence consisting of positions 3900-4136 of SEQ ID NO:2, and a biologically active fragment thereof; and (b) an amino acid sequence that is at least about 45% identical to any of the amino acid sequences of (a) and having the biological activity of the amino acid sequence of (a). In other aspects of this embodiment, the isolated protein comprises an amino acid sequence that is at least about 60% identical, at least about 80% identical, or at least about 95% identical to any of the amino acid sequences of (a). In a preferred aspect of this embodiment, the protein comprises an amino acid sequence selected from: an amino acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, an amino acid sequence consisting of positions 1-500 of SEQ ID NO:2, an amino acid sequence consisting of positions 450-1300 of SEQ ID NO:2, an amino acid sequence consisting of positions 1250-1700 of SEQ ID NO:2, an amino acid sequence consisting of positions 1575-2100 of SEQ ID NO:2, an amino acid sequence consisting of positions 2025-2850 of SEQ ID NO:2, an amino acid sequence consisting of positions 2800-3350 of SEQ ID NO:2, an amino acid sequence consisting of positions 3300-3900 of SEQ ID NO:2, an amino acid sequence consisting of positions 3900-4136 of SEQ ID NO:2, or biologically active fragments of any of these sequences. In an even more preferred embodiment, the protein comprises an amino acid sequence selected from: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:1, SEQ ID NO:12, and SEQ ID NO:13.
In one aspect of this embodiment, the protein comprises any two or more amino acid sequences selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13. In another aspect, the protein comprises SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13. In yet another aspect, the isolated protein is from a Thraustochytriales microorganism and in a preferred embodiment, from a Schizochytrium microorganism.
Yet another embodiment of the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding any of the above-identified proteins, or a nucleic acid sequence that is fully complementary thereto. Another embodiment of the present invention relates to a recombinant nucleic acid molecule comprising such an isolated nucleic acid molecule, operatively linked to a transcription control sequence. In one aspect, the transcription control sequence is a tissue-specific transcription control sequence. In another aspect, the recombinant nucleic acid molecule further comprises a targeting sequence. Yet another embodiment of the present invention relates to a recombinant cell that has been transformed with such a recombinant nucleic acid molecule.
Another embodiment of the present invention relates to a genetically modified microorganism for producing short chain fatty acids by a biosynthetic process, the microorganism being transformed with a recombinant nucleic acid molecule as described above.
Another embodiment of the present invention relates to a genetically modified plant for producing short chain fatty acids by a biosynthetic process, the plant being transformed with a recombinant nucleic acid molecule as described above.
Yet another embodiment of the present invention relates to a genetically modified microorganism for producing short chain fatty acids by a biosynthetic process. The microorganism comprises a nucleic acid molecule encoding a fatty acid synthase, wherein the nucleic acid molecule has been modified to increase the expression or biological activity of the fatty acid synthase. The fatty acid synthase comprises an amino acid sequence selected from: (a) an amino acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, an amino acid sequence consisting of positions 1-500 of SEQ ID NO:2, an amino acid sequence consisting of positions 450-1300 of SEQ ID NO:2, an amino acid sequence consisting of positions 1250-1700 of SEQ ID NO:2, an amino acid sequence consisting of positions 1575-2100 of SEQ ID NO:2, an amino acid sequence consisting of positions 2025-2850 of SEQ ID NO:2, an amino acid sequence consisting of positions 2800-3350 of SEQ ID NO:2, an amino acid sequence consisting of positions 3300-3900 of SEQ ID NO:2, an amino acid sequence consisting of positions 3900-4136 of SEQ ID NO:2, and a biologically active fragment thereof; or (b) an amino acid sequence that is at least about 45% identical to any of the amino acid sequences of (a) and having the biological activity of the amino acid sequence of (a). In one embodiment, the nucleic acid molecule encoding a fatty acid synthase is an endogenous gene in the microorganism. In another embodiment, the microorganism has been transformed with a nucleic acid molecule encoding the fatty acid synthase. In yet another embodiment, the microorganism comprises an endogenous gene encoding the fatty acid synthase and has been transformed with a recombinant nucleic acid molecule encoding a fatty acid synthase. In this embodiment, one or both of the gene and the recombinant nucleic acid molecule has been modified to increase the expression or biological activity of the fatty acid synthase. Such genetically modified microorganisms can include Thraustochytriales microorganism, and in one aspect, a Schizochytrium microorganism.
Another embodiment of the invention relates to a biomass comprising the genetically modified microorganism described above, to a food product comprising such a biomass, or to a pharmaceutical product comprising such a biomass.
Another embodiment of the present invention relates to a method to produce short chain fatty acids by a biosynthetic process, comprising culturing in a fermentation medium a genetically modified microorganism as described above. Another embodiment of the present invention relates to a method to produce short chain fatty acids by a biosynthetic process, comprising growing a genetically modified plant that has been transformed with a recombinant nucleic acid molecule as described above.
Yet another embodiment of the present invention relates to an oligonucleotide, comprising at least 12 contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:4, or a nucleic acid sequence fully complementary thereto.
Another embodiment of the present invention relates to a genetically modified microorganism with reduced production of short chain fatty acids, wherein the microorganism has been genetically modified to selectively attenuate a fatty acid synthase gene or portion thereof encoding a functional domain. The fatty acid synthase gene comprises a nucleic acid sequence selected from: (a) a nucleic acid sequence encoding SEQ ID NO:2; and (b) a nucleic acid sequence encoding an amino acid sequence that is at least about 45% identical to SEQ ID NO:2, wherein the protein having the amino acid sequence has a biological activity selected from the group consisting of acetyl-transferase (AT) activity; enoyl ACP reductase (ER) activity; dehydrase (DH) activity; malonyl/palmitoyl acyltransferase (M/PAT) activity; a first acyl carrier protein (ACP) activity; a second acyl carrier protein (ACP) activity; keto-acyl ACP reductase (KR) activity; keto-acyl ACP synthase (KS) activity; and phosphopantetheinyl transferase (PPT) activity. In one aspect, the fatty acid synthase gene comprises a nucleic acid sequence represented by SEQ ID NO:1. In another aspect, the microorganism has increased production of at least one polyunsaturated fatty acid (PUFA). The microorganism can include, but is not limited to, a Thraustochytriales microorganism, and particularly, a Schizochytrium. In one aspect, the fatty acid synthase gene has been modified in a regulatory region to reduce expression of the gene. In another aspect, the fatty acid synthase gene has been modified in the coding region to reduce the biological activity of one or more functional domains of the fatty acid synthase. In yet another aspect, the fatty acid synthase gene has been mutated by targeted homologous recombination with a nucleic acid sequence that hybridizes to the fatty acid synthase gene and includes a heterologous nucleic acid sequence that modifies the coding region of the fatty acid synthase gene to reduce the expression or activity of the fatty acid synthase encoded thereby.
Another embodiment of the invention relates to a biomass comprising the genetically modified microorganisms described directly above, wherein the microorganisms have reduced production of short chain fatty acids as compared to a wild-type microorganism of the same species. Also included in the invention are food products and pharmaceutical products comprising such a biomass.
Yet another embodiment of the present invention relates to a method for increasing the production of polyunsaturated fatty acids (PUFAs) in a biosynthetic process. The method includes the step of culturing under conditions effective to produce lipids comprising the PUFAs, genetically modified microorganisms as set forth directly above. Products comprising the lipids produced by such a method, including food products and pharmaceutical products, are also encompassed by the present invention.



BRIEF DESCRIPTION OF THE FIGURES OF THE INVENTION
FIG. 1 is a schematic representation of the putative enzymatic domains present in the Schizochytrium FAS protein.
FIG. 2A is a schematic representation of the construction of a plasmid used for targeted inactivation of the Schizochytrium FAS gene.
FIG. 2B is a schematic representation of the events that are believed to occur and result in the stable inactivation of the FAS gene in Schizochytrium. 
FIG. 3 is a digitized image of the synthesis of fatty acids from [1-14C]-malonyl-CoA in cell free homogenates from a cell wall deficient strain of Schizochytrium (AC66) and mutants of that strain in which either the PUFA polyketide synthase orfC gene (PUFA-KO) or the FAS gene (FAS-KO) have been inactivated.



DETAILED DESCRIPTION OF THE INVENTION
The present invention generally relates to a fatty acid synthase (FAS) from Schizochytrium, to biologically active fragments and homologues thereof, to a nucleic acid sequence encoding such FAS, fragments and homologues thereof, to the gene encoding Schizochytrium FAS, to host cells and organisms that recombinantly express the FAS, to host cells and organisms in which the expression and/or activity of the endogenous FAS has been attenuated, and to various methods for making and using any of these proteins, nucleic acid molecules, genes, host cells or organisms. The FAS protein of the present invention is a protein with multiple enzymatic domains that are homologous, in terms of both amino acid sequences and in linear domain organization, to the enzymatic domains encompassed by two proteins in fungi. The FAS domains in mammals are also found on one large protein, but the organization and the specific types of enzymatic activities of the domains are significantly different than the Schizochytrium and fungal FAS. Additionally, the amino acid sequence homology between the Schizochytrium FAS domains and those found in mammalian FAS is significantly less than between Schizochytrium and fungal FAS.
This discovery of a multi-functional protein provides a novel approach for the economic production of short chain fatty acids. For example, it is now possible to clone and express one gene with key sequential enzymatic functions that are found in at least two genes in other (non-mammalian) organisms, which will greatly facilitate the genetic modification of production organisms. In addition, it is possible to use the enzymatic domains of the Schizochytrium FAS gene individually or in various combinations to construct various recombinant/synthetic genes expressing, one or more of the domains.
More specifically, the present inventors have cloned a region of genomic DNA from Schizochytrium sp. ATCC 20888 that contains a single orf (open reading frame) that encodes a fatty acid synthase (FAS gene). The putative function of the enzyme, (i.e., synthesis of short chain saturated fatty acids such as C14:0 and C16:0), was verified by showing that strains in which the gene had been disrupted require supplementation with short chain fatty acids for survival. The FAS encoded by the Schizochytrium gene has some novel features. The organization of the domains in the protein is similar to that found in many fungi (e.g., baker's yeast). However, in all of the fungal enzymes characterized to date, the FAS is encoded in two subunits (i.e., two proteins), while the Schizochytrium FAS is one large protein. Based on homology to the fungal systems, it is likely that the Schizochytrium FAS uses acetyl-CoA and malonyl-CoA along with NADH and NADPH as substrates. The product is probably released as an acyl-CoA ester. As in yeast, the protein contains a phosphopantetheinyl transferase domain that is believed to activate an embedded acyl carrier protein domain. The Schizochytrium FAS is an ‘all-in-one’protein for short chain saturated fatty acid synthesis.
As used herein, a short chain fatty acid is defined as a fatty acid having 18 or fewer carbons. The FAS system of the invention produces short chain fatty acids, which can include any short chain fatty acid, and typically fatty acids having 16, 14, or 12 carbons, and include saturated and monounsaturated fatty acids. For example, short chain fatty acids produced by a FAS system include, but are not limited to, short chain saturated fatty acids such as C14:0 and C16:0. The present invention encompasses the production of any product of the FAS system.
Schizochytrium is a marine microalga that has been developed as a commercial source of oil enriched in DHA. The DHA in Schizochytrium is the product of a highly specialized PUFA synthase (described in PCT Publication No. WO 00/42195 and PCT Publication No. WO 02/083870). Here the present inventors describe the FAS that is responsible for producing the other major fatty acids (C14:0 and C16:0) found in Schizochytrium oil. The products of the FAS of the present invention are of interest by themselves and as a major component of the DHA-enriched oil. Alteration of the activity of the FAS in Schizochytrium may influence the relative amounts of DHA and medium chain saturated fatty acids that accumulate in that oil, thereby altering its commercial value as a DHA-enriched oil.
Accordingly, the Schizochytrium FAS gene and its encoded product as described herein have several uses. First, subclones of the FAS genomic region can be used to make a knockout plasmid construct and to create mutants of Schizochytrium in which the FAS gene has been inactivated. Such constructs have already been produced by the inventors and are described herein (see Examples 2 and 3). These mutants may have utility in a variety of biochemical and genetic studies. In addition, attenuation of the expression and/or activity of the FAS gene in Thraustochytrids such as Schizochytrium is a particularly preferred embodiment of the invention, because reduction of FAS activity in Thraustochytrids is predicted to increase the accumulation of highly desirable long chain fatty acids in the organism. For example, one embodiment of the invention relates to an organism of the order, Thraustochytriales, in which the FAS gene expression is attenuated (resulting in reduced FAS activity), thereby increasing the accumulation of long chain fatty acids and particularly, polyunsaturated fatty acids (PUFAs), by the organism. According to the present invention, reference to an attenuated gene or protein is to a gene or protein that is not deleted or completely activated, but for which the expression and/or biological activity has been reduced (inhibited, down-regulated, decreased) as compared to the expression and/or biological activity of the wild-type gene or protein under normal conditions. Therefore, a FAS having attenuated expression or activity is still expressed and still has some biological activity (e.g., so that an organism expressing the FAS is viable), but the expression or biological activity is reduced as compared to the wild-type FAS.
In another embodiment, expression of this gene in heterologous systems (such as in the cytoplasm of plant cells) is expected to lead to the production and accumulation of short chain saturated fatty acids in those cells. This will provide a means to produce oils enriched in short chain fatty acids in commercial oil-seed crops. This would be an alternative method to the current use of chain-length specific thioesterases targeted to the plastids of plant cells. The end product of the Schizochytrium FAS is likely to be an ester of CoA, and therefore it would be compatible with oil and phospholipid synthesis in the plant cell cytoplasm.
Accordingly, one embodiment of the present invention relates to an isolated fatty acid synthase (FAS). As used herein, reference to an isolated protein, including an isolated FAS, is to a protein (including a polypeptide or peptide) that has been removed from its natural milieu (i.e., that has been subject to human manipulation) and can include purified proteins, partially purified proteins, recombinantly produced proteins, and synthetically produced proteins, for example. As such, “isolated” does not reflect the extent to which the protein has been purified. Preferably, an isolated FAS of the present invention is produced recombinantly. In addition, and by way of example, a “Schizochytrium FAS” refers to a FAS (generally including a homologue of a naturally occurring FAS) from a Schizochytrium or to a FAS protein that has been otherwise produced from the knowledge of the structure (e.g., sequence) and perhaps the function of a naturally occurring FAS from Schizochytrium. In other words, a Schizochytrium FAS includes any FAS that has substantially similar structure and function of a naturally occurring FAS from Schizochytrium or that is a biologically active (i.e., has biological activity) homologue of a naturally occurring FAS from Schizochytrium as described in detail herein. As such, a Schizochytrium FAS protein can include purified, partially purified, recombinant, mutated/modified and synthetic proteins. According to the present invention, the terms “modification” and “mutation” can be used interchangeably, particularly with regard to the modifications/mutations to the amino acid sequences of FAS (or nucleic acid sequences) described herein.
According to the present invention, a homologue of a FAS protein (i.e., a FAS homologue) includes FAS proteins in which at least one or a few, but not limited to one or a few, amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide or fragment), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, famasylation, amidation and/or addition of glycosylphosphatidyl inositol). In a preferred embodiment, a FAS homologue has measurable or detectable FAS enzymatic activity (i.e., has biological activity). Measurable or detectable FAS enzymatic activity can include the enzymatic activity of just one, two, three, etc., up to all ten of the functional domains in the FAS protein of the present invention (discussed in detail below). In another embodiment, a FAS homologue may or may not have measurable FAS functional (biological) activity, but is used for the preparation of antibodies or the development of oligonucleotides useful for identifying other FAS proteins. For example, the production of an antibody against FAS and production of probes and primers useful in the cloning of a FAS are useful for tracking the presence of FAS nucleic acids or proteins in genetically modified organisms or for identifying naturally occurring FAS homologues in other organisms (e.g., in other members of Thraustochytriales).
FAS homologues can be the result of natural allelic variation or natural mutation. FAS homologues of the present invention can also be produced using techniques known in the art including, but not limited to, direct modifications to the protein or modifications to the gene encoding the protein using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis. A naturally occurring allelic variant of a nucleic acid encoding a FAS protein is a gene that occurs at essentially the same locus (or loci) in the genome as the gene which encodes the FAS protein of the present invention (e.g., SEQ ID NO:2), but which, due to natural variations caused by, for example, mutation or recombination, has a similar but not identical sequence. Natural allelic variants typically encode proteins having similar activity to that of the protein encoded by the gene to which they are being compared. One class of allelic variants can encode the same protein but have different nucleic acid sequences due to the degeneracy of the genetic code. Allelic variants can also comprise alterations in the 5′ or 3′ untranslated regions of the gene (e.g., in regulatory control regions). Allelic variants are well known to those skilled in the art. Homologues can be produced using techniques known in the art for the production of proteins including, but not limited to, direct modifications to the isolated, naturally occurring protein, direct protein synthesis, or modifications to the nucleic acid sequence encoding the protein using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis.
Modifications in FAS homologues, as compared to the wild-type protein, increase, decrease, or do not substantially change, the basic biological activity of the FAS homologue as compared to the naturally occurring protein, FAS. In general, the biological activity or biological action of a protein refers to any function(s) exhibited or performed by the protein that is ascribed to the naturally occurring form of the protein as measured or observed in vivo (i.e., in the natural physiological environment of the protein) or in vitro (i.e., under laboratory conditions). Modifications of a protein, such as in a homologue or mimetic (discussed below), may result in proteins having the same biological activity as the naturally occurring protein, or in proteins having decreased or increased biological activity as compared to the naturally occurring protein. Modifications which result in a decrease in protein expression or a decrease in the activity of the protein, can be referred to as inactivation (complete or partial), down-regulation, or decreased action of a protein. Similarly, modifications which result in an increase in protein expression or an increase in the activity of the protein, can be referred to as amplification, overproduction, activation, enhancement, up-regulation or increased action of a protein.
According to one embodiment of the present invention, a biologically active FAS, including a biologically active homologue or fragment thereof, has at least one characteristic of biological activity of activity a wild-type, or naturally occurring, FAS protein described herein. A FAS biological activity includes the ability to catalyze the synthesis of short chain fatty acids, including by using acetyl-CoA and malonyl-CoA along with NADH and NADPH as substrates to produce a short chain fatty acid. More particularly, a FAS biological activity can include any one or more of the biological activities of the nine domains of FAS described herein. According to the present invention, a FAS protein of the present invention has at least one, and preferably two, and more preferably three, and more preferably four, and more preferably five, and more preferably six, and more preferably seven, and more preferably eight, and most preferably nine, biological activities. These biological activities are: (1) acetyl-transferase (AT) activity; (2) enoyl ACP reductase (ER) activity; (3) dehydratase (DH) activity; (4) malonyl/palmitoyl acyltransferase (M/PAT) activity; (5) a first acyl carrier protein (ACP) activity; (6) a second acyl carrier protein (ACP) activity; (7) keto-acyl ACP reductase (KR) activity; (8) keto-acyl ACP synthase (KS) activity; and (9) phosphopantetheinyl transferase (PPT) activity. General reference to FAS biological activity typically refers to all biological activities, but does not exclude reference to only one, two, three, four, five, six, seven or eight of the biological activities. Methods for measuring these various activities are known in the art.
Methods to measure protein expression levels according to this invention, include, but are not limited to: Western blot, immunoblot, enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, surface plasmon resonance, chemiluminescence, fluorescent polarization, phosphorescence, immunohistochemical analysis, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, microcytometry, microarray, microscopy, fluorescence activated cell sorting (FACS), and flow cytometry, as well as assays based on a property of the protein including but not limited to substrate binding. Binding assays are also well known in the art. For example, a BIAcore machine can be used to determine the binding constant of a complex between two proteins. The dissociation constant for the complex can be determined by monitoring changes in the refractive index with respect to time as buffer is passed over the chip (O'Shannessy et al. Anal. Biochem. 212:457-468 (1993); Schuster et al., Nature 365:343-347 (1993)). Other suitable assays for measuring the binding of one protein to another include, for example, immunoassays such as enzyme linked immunoabsorbent assays (ELISA) and radioimmunoassays (RIA), or determination of binding by monitoring the change in the spectroscopic or optical properties of the proteins through fluorescence, UV absorption, circular dichrosim, or nuclear magnetic resonance (NMR).
FIG. 1 is a schematic drawing showing the domain organization of the Schizochytrium FAS protein of the present invention as compared to the domain organization of the individual α and β subunit proteins of yeast FAS systems.
The complete Schizochytrium FAS-encoding sequence is a 12,408 nucleotide sequence (not including the stop codon), represented herein by SEQ ID NO:1, which encodes a 4136 amino acid sequence, represented herein as SEQ ID NO:2. Within the Schizochytrium FAS protein are nine domains as described above: (a) one acetyl-transferase (AT) domain; (b) one enoyl ACP reductase (ER) domain; (c) one dehydrase (DH) domain; (d) one malonyl/palmitoyl acyltransferase (M/PAT) domain; (e) two acyl carrier protein (ACP) domains; (f) one keto-acyl ACP reductase (KR) domain; (g) one keto-acyl ACP synthase (KS) domain; and (h) one phosphopantetheinyl transferase (PPT) domain.
SEQ ID NO:2 was compared with known sequences in a standard protein BLAST search (BLAST 2.0 Basic BLAST homology search using blastp for amino acid searches, wherein the query sequence is filtered for low complexity regions by default (described in Altschul, S. F., Madden, T. L., Schääffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997) “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.” Nucleic Acids Res. 25:3389-3402, incorporated herein by reference in its entirety)). The BLAST search used the following parameters: low complexity filter Off, matrix=BLOSUM62, gap penalties are: Existence: 11, Extension 1. BLAST results of entire FAS protein revealed that the Schizochytrium FAS can be viewed in general, as a protein with homology to a head to tail fusion of fungal α and βFAS subunits. All of the domains currently identified in the two yeast proteins have counterparts in the Schizochytrium FAS. In addition, the Schizochytrium FAS has a second ACP domain. In all cases, the best matches in the BLAST results are fungi such as: Saccharomyces cerevisiae, Candida albicans, Neurospora crassa, Saccharomyces kluyveri, Aspergillus nidulans and Yarrowia lipolytica. At the amino acid level, the sequences with the greatest degree of homology to SEQ ID NO:2 were: (1) the Candida albicans FAS β-subunit (GenBank Accession No. P34731), which was 34% identical to amino acids 1-2100 of SEQ ID NO:2 (identity was actually over 2038 amino acids of this region of SEQ ID NO:2); and (2) Candida albicans FAS α-subunit (GenBank Accession No. P43098), which was 41% identical to amino acids 2101-4136 of SEQ ID NO:2 (identity was actually over 1864 amino acids of this region of SEQ ID NO:2). Several other yeast strains showed similar homology to this portion of Schizochytrium FAS. Since yeast FAS systems have only one ACP domain, whereas the Schizochytrium FAS described herein has two ACP domains, the homology to the yeast α-subunit is found from the second of the two ACPs in Schizochytrium through the end of the protein.
The first domain in the Schizochytrium FAS protein is an acetyl-transferase (AT) domain, also referred to herein as FAS-AT. This domain is contained within a region of SEQ ID NO:2 spanning from about position 1 to about position 500 of SEQ ID NO:2. The amino acid sequence containing the FAS-AT domain is represented herein as SEQ ID NO:5 (positions 45-415 of SEQ ID NO:2). BLAST results show that this domain has high homology to the first domain of several fungal FAS β-subunits. The fungal protein having the closest identity to this domain of the FAS protein of the present invention is a portion of a Yarrowia lipolytica FAS β-subunit (GenBank Accession No. P34229), which is 30% identical over 392 amino acids when compared to SEQ ID NO:5 (i.e., containing the AT domain of the FAS of the present invention). An AT generally refers to a class of enzymes that can carry out a number of distinct acyl transfer reactions. The fungal FAS β-subunit has been shown to have specifically acetyltransacylase activity, which is related to malonyl acyltransferase, except that it transfers an acetyl group which serves as the primer for fatty acid synthesis. In the AT domain, the active site motif is identified as GHS*XG, which in SEQ ID NO:2 is GHS*QG (positions 156-160 of SEQ ID NO:2), where S* (position 158 of SEQ ID NO:2) is the acyl group binding site.
The second domain in the Schizochytrium FAS protein is an enoyl reductase (ER) domain, also referred to herein as FAS-ER. This domain is contained within a region of SEQ ID NO:2 spanning from about position 450 to about position 1300 of SEQ ID NO:2. The amino acid sequence containing the FAS-ER domain is represented herein as SEQ ID NO:6 (positions 460-1210 of SEQ ID NO:2). BLAST results show that this domain has high homology to the second domain of several fungal FAS β-subunits. The fungal protein having the closest identity to this domain of the FAS protein of the present invention is a portion of a Emericella nidulans FAS β-subunit (GenBank Accession No. AAB41494.1), which is 46% identical over 561 amino acids when compared to SEQ ID NO:6 (i.e., containing the ER domain of the FAS of the present invention). An ER enzyme reduces the trans-double bond (introduced by the DH activity) in the fatty acyl-ACP, resulting in fully saturating those carbons.
The third domain in the Schizochytrium FAS protein is a dehydratase (DH) domain, also referred to herein as FAS-DH. This domain is contained within a region of SEQ ID NO:2 spanning from about position 1300 to about position 1700 of SEQ ID NO:2. The amino acid sequence containing the FAS-DH domain is represented herein as SEQ ID NO:7 (positions 1450-1575 of SEQ ID NO:2). BLAST results show that this domain has high homology to the third domain of several fungal FAS β-subunits. The fungal protein having the closest identity to this domain of the FAS protein of the present invention is the Magnaporthe grisea monoamine oxidase C (MaoC) protein (GenBank Accession No. EAA50359.1), which is 39% identical over 121 amino acids when compared to SEQ ID NO:7 (i.e., containing the DH domain of the FAS of the present invention). MaoC shares similarity with a family of proteins with a variety of functions, including the FAS β-keto-acyl dehydratase (DH) activity. This class of enzymes removes HOH from a β-keto acyl-ACP and leaves a trans double bond in the carbon chain.
The fourth domain in the Schizochytrium FAS protein is a malonyl/palmitoyl acyltransferase (M/PAT) domain, also referred to herein as FAS-M/PAT. This domain is contained within a region of SEQ ID NO:2 spanning from about position 1575 to about position 2100 of SEQ ID NO:2. The amino acid sequence containing the FAS-M/PAT domain is represented herein as SEQ ID NO:8 (positions 1575-2025 of SEQ ID NO:2). BLAST results show that this domain has high homology to the fourth domain of several fungal FAS β-subunits. The fungal protein having the closest identity to this domain of the FAS protein of the present invention is a portion of a Neurospora crassa protein (GenBank Accession No. EAA33229.1), which is 47% identical over 397 amino acids when compared to SEQ ID NO:8 (i.e., containing the M/PAT domain of the FAS of the present invention). In yeast FAS, FabD (malonyl-CoA:ACP acyltransferase) has been shown to have the dual functions of transferring the malonyl group from CoA to the FAS ACP domain, and also of transferring the fatty acid product of FAS (a palmitoyl group) from the FAS ACP to CoA. The active site motif of this protein is GHS*XG, which in the Schizochytrium FAS-M/PAT is GHS*LG (positions 1723-1727 of SEQ ID NO:2), where the S* (position 1725 of SEQ ID NO:2) is the position where the acyl group binds.
The fifth and sixth domains in the Schizochytrium FAS protein are acyl carrier protein (ACP) domains, also referred to herein as FAS-ACP. These domains are contained within a region of SEQ ID NO:2 spanning from about position 2025 to about position 2850 of SEQ ID NO:2. The amino acid sequence containing the first FAS-ACP domain (FAS-ACP1) is represented herein as SEQ ID NO:9 (positions 2140-2290 of SEQ ID NO:2). The amino acid sequence containing the second FAS-ACP domain (FAS-ACP2) is represented herein as SEQ ID NO:10 (positions 2325-2585 of SEQ ID NO:2). BLAST results show that these domains have high homology to the N-terminus of several yeast FAS α-subunits which have been designated in the literature as ACP domains. However, all fungal FAS proteins appear to have only one ACP domain, whereas the Schizochytrium FAS protein of the present invention has two ACP domains. The fungal protein having the closest identity to the first ACP domain of the FAS protein of the present invention is a portion of a Neurospora crassa protein (GenBank Accession No. EAA33230.1), which is 44% identical over 149 amino acids when compared to SEQ ID NO:9 (i.e., containing the first ACP domain of the FAS of the present invention). The fungal protein having the closest identity to the second ACP domain of the FAS protein of the present invention is a portion of a Neurospora crassa protein (GenBank Accession No. EAA33230.1), which is 36% identical over 262 amino acids when compared to SEQ ID NO:10 (i.e., containing the second ACP domain of the FAS of the present invention). By alignment with the yeast sequences, two putative phosphopanthelyation sites are identified in the ACP domains: the serine residues at positions 2160 (in FAS-ACP1) and 2353 (in FAS-ACP-2) of SEQ ID NO:2. A domain or protein having acyl carrier protein (ACP) biological activity (function) is characterized as being a small polypeptide (typically, 80 to 100 amino acids long), that functions as a carrier for growing fatty acyl chains via a thioester linkage to a covalently bound co-factor of the protein. They occur as separate units or as domains within larger proteins. ACPs are converted from inactive apo-forms to functional holo-forms by transfer of the phosphopantetheinyl moeity of CoA to a highly conserved serine residue of the ACP. Acyl groups are attached to ACP by a thioester linkage at the free terminus of the phosphopantetheinyl moiety.
The seventh domain in the Schizochytrium FAS protein is a keto-acyl ACP reductase (KR) domain, also referred to herein as FAS-KR. This domain is contained within a region of SEQ ID NO:2 spanning from about position 2800 to about position 3350 of SEQ ID NO:2. The amino acid sequence containing the FAS-KR domain is represented herein as SEQ ID NO:11 (positions 2900-3100 of SEQ ID NO:2). BLAST results show that this domain has high homology to the second domain of several fungal FAS α-subunits (FabG or β-keto-acyl ACP reductase). The fungal protein having the closest identity to this domain of the FAS protein of the present invention is a portion of a Neurospora crassa protein (GenBank Accession No. EAA33220.1), which is 50% identical over 205 amino acids when compared to SEQ ID NO:11 (i.e., containing the KR domain of the FAS of the present invention). As in the yeast FAS, a domain or protein having keto-acyl ACP reductase (KR) biological activity (function), is characterized as an enzyme that catalyzes the pyridine-nucleotide-dependent reduction of β-keto acyl forms of ACP. It is the first reductive step in the de novo fatty acid biosynthesis elongation cycle.
The eighth domain in the Schizochytrium FAS protein is a keto-acyl ACP synthase (KS) domain, also referred to herein as FAS-KS. This domain is contained within a region of SEQ ID NO:2 spanning from about position 3300 to about position 3900 of SEQ ID NO:2. The amino acid sequence containing the FAS-KS domain is represented herein as SEQ ID NO:12 (positions 3350-3875 of SEQ ID NO:2). BLAST results show that this domain has high homology to the third domain of several fungal FAS α-subunits, and particularly, FabB (β-keto-acyl ACP synthase). The fungal protein having the closest identity to this domain of the FAS protein of the present invention is a portion of a Candida albicans FAS α-subunit protein (GenBank Accession No. P43098), which is 55% identical over 548 amino acids when compared to SEQ ID NO:12 (i.e., containing the KS domain of the FAS of the present invention). The active site cysteine of this domain has been identified at position 3530 of SEQ ID NO:2. According to the present invention, a domain or protein having (KS) biological activity (function) is characterized as an enzyme that carries out the initial step of the FAS elongation reaction cycle. The acyl group destined for elongation is linked to a cysteine residue at the active site of the enzyme by a thioester bond. In the multi-step reaction, the acyl-enzyme undergoes condensation with malonyl-ACP to form -keto-acyl-ACP, CO2 and free enzyme. The KS plays a key role in the elongation cycle and in many systems has been shown to possess greater substrate specificity than other enzymes of the reaction cycle.
The ninth domain in the Schizochytrium FAS protein is a phosphopantetheinyl transferase (PPT domain), also referred to herein as FAS-PPT. This domain is contained within a region of SEQ ID NO:2 spanning from about position 3900 to about position 4136 of SEQ ID NO:2. The amino acid sequence containing the FAS-PPT domain is represented herein as SEQ ID NO:13 (positions 4025-4136 of SEQ ID NO:2). BLAST results show that this domain has high homology to the C-terminal domain of several fungal FAS α-subunits, and particularly, to AcpS (holo-ACP synthase, also known as 4-phosphopantetheinyl transferase). The fungal protein having the closest identity to this domain of the FAS protein of the present invention is a portion of a Schizosaccharomyces pombe FAS α-subunit protein (GenBank Accession No. BAB62031.1), which is 46% identical over 110 amino acids when compared to SEQ ID NO:13 (i.e., containing the PPT domain of the FAS of the present invention). A PPT domain is required for the attachment of a phosphopantetheine cofactor to produce the active, holo-ACP.
In one embodiment, a FAS protein (e.g., including homologues of the FAS isolated from Schizochytrium and described in detail herein) includes proteins that have at least one of: (a) acetyl-transferase (AT) activity; (b) enoyl ACP reductase (ER) activity; (c) dehydratase (DH) activity; (d) malonyl/palmitoyl acyltransferase (M/PAT) activity; (e) acyl carrier protein (ACP) activity; (f) keto-acyl ACP reductase (KR) activity; (g) keto-acyl ACP synthase (KS) activity; and (h) phosphopantetheinyl transferase (PPT) activity. In one embodiment of the invention, an isolated FAS comprises an amino acid sequence selected from: (a) an amino acid sequence selected from: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:13, an amino acid sequence consisting of positions 1-500 of SEQ ID NO:2, an amino acid sequence consisting of positions 450-1300 of SEQ ID NO:2, an amino acid sequence consisting of positions 1250-1700 of SEQ ID NO:2, an amino acid sequence consisting of positions 1575-2100 of SEQ ID NO:2, an amino acid sequence consisting of positions 2025-2850 of SEQ ID NO:2, an amino acid sequence consisting of positions 2800-3350 of SEQ ID NO:2, an amino acid sequence consisting of positions 3300-3900 of SEQ ID NO:2, an amino acid sequence consisting of positions 3900-4136 of SEQ ID NO:2, or biologically active fragments of any of these sequences or any combinations of these sequences; or, (b) an amino acid sequence that is at least about 45% identical to any of these sequences, wherein the amino acid sequence has the biological activity of the reference sequence (biological activities of these sequences are described above).
In one aspect of the invention, as discussed above a FAS protein comprises an amino acid sequence that is at least about 45% identical to any of the above-described amino acid sequences representing the full-length FAS of the invention (i.e., SEQ ID NO:2), a region containing a biologically active domain of the FAS of the invention (i.e., a region of SEQ ID NO:2 defined by positions numbers above), or a biologically active domain of the FAS of the invention (i.e., any one of SEQ ID NOs:5-13), over the full length of that protein, region, or domain. In another aspect, a FAS protein of the invention comprises an amino acid sequence that is at least 50% identical to any of the above-identified protein, regions or domains, and in another aspect at least about 55%, and in another aspect at least about 60%, and in another aspect at least about 65%, and in another aspect at least about 70%, and in another aspect at least about 75%, and in another aspect at least about 80%, and in another aspect at least about 85%, and in another aspect at least about 90%, and in another aspect at least about 95% identical, and in another aspect at least about 96% identical, and in another aspect at least about 97% identical, and in another aspect at least about 98% identical, and in another aspect at least about 99% identical, to the amino acid sequence defining any of the above-identified protein, regions or domains. Preferably, a FAS protein of the present invention comprises at least one, two, three, four, five, six, seven, eight, or all nine biological activities of a FAS protein of the invention selected from: (a) acetyl-transferase (AT) activity; (b) enoyl ACP reductase (ER) activity; (c) dehydratase (DH) activity; (d) malonyl/palmitoyl acyltransferase (M/PAT) activity; (e) acyl carrier protein (ACP) activity; (f) keto-acyl ACP reductase (KR) activity; (g) keto-acyl ACP synthase (KS) activity; and (h) phosphopantetheinyl transferase (PPT) activity.
In one embodiment of the present invention, a FAS homologue according to the present invention has an amino acid sequence that is less than about 100% identical to any of the above-identified amino acid sequences for a full-length FAS protein, or a region or domain thereof according to the present invention. In another aspect of the invention, a FAS homologue according to the present invention has an amino acid sequence that is less than about 99% identical to any of the above-identified amino acid sequences, and in another embodiment, is less than is less than 98% identical to any of the above-identified amino acid sequences, and in another embodiment, is less than 97% identical to any of the above-identified amino acid sequences, and in another embodiment, is less than 96% identical to any of the above-identified amino acid sequences, and in another embodiment, is less than 95% identical to any of the above-identified amino acid sequences, and in another embodiment, is less than 94% identical to any of the above-identified amino acid sequences, and in another embodiment, is less than 93% identical to any of the above-identified amino acid sequences, and in another embodiment, is less than 92% identical to any of the above-identified amino acid sequences, and in another embodiment, is less than 91% identical to any of the above-identified amino acid sequences, and in another embodiment, is less than 90% identical to any of the above-identified amino acid sequences, and so on, in increments of whole integers.
As used herein, unless otherwise specified, reference to a percent (%) identity refers to an evaluation of homology which is performed using: (1) a BLAST 2.0 Basic BLAST homology search using blastp for amino acid searches and blastn for nucleic acid searches with standard default parameters, wherein the query sequence is filtered for low complexity regions by default (described in Altschul, S. F., Madden, T. L., Schääffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997) “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.” Nucleic Acids Res. 25:3389-3402, incorporated herein by reference in its entirety); (2) a BLAST 2 alignment (using the parameters described below); (3) and/or PSI-BLAST with the standard default parameters (Position-Specific Iterated BLAST. It is noted that due to some differences in the standard parameters between BLAST 2.0 Basic BLAST and BLAST 2, two specific sequences might be recognized as having significant homology using the BLAST 2 program, whereas a search performed in BLAST 2.0 Basic BLAST using one of the sequences as the query sequence may not identify the second sequence in the top matches. In addition, PSI-BLAST provides an automated, easy-to-use version of a “profile” search, which is a sensitive way to look for sequence homologues. The program first performs a gapped BLAST database search. The PSI-BLAST program uses the information from any significant alignments returned to construct a position-specific score matrix, which replaces the query sequence for the next round of database searching. Therefore, it is to be understood that percent identity can be determined by using any one of these programs.
Two specific sequences can be aligned to one another using BLAST 2 sequence as described in Tatusova and Madden, (1999), “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250, incorporated herein by reference in its entirety. BLAST 2 sequence alignment is performed in blastp or blastn using the BLAST 2.0 algorithm to perform a Gapped BLAST search (BLAST 2.0) between the two sequences allowing for the introduction of gaps (deletions and insertions) in the resulting alignment. For purposes of clarity herein, a BLAST 2 sequence alignment is performed using the standard default parameters as follows.
For blastn, using 0 BLOSUM62 matrix:
Reward for match=1
Penalty for mismatch=−2
Open gap (5) and extension gap (2) penalties
gap x_dropoff (50) expect (10) word size (11) filter (on)
For blastp, using 0 BLOSUM62 matrix:
Open gap (11) and extension gap (1) penalties
gap x_dropoff (50) expect (10) word size (3) filter (on).
A FAS protein can also include proteins having an amino acid sequence comprising at least 10 contiguous amino acid residues of any of the above-identified amino acid sequences (i.e., 10 contiguous amino acid residues having 100% identity with 10 contiguous amino acids of the reference amino acid sequence). In another aspect, a homologue of a FAS amino acid sequence includes amino acid sequences comprising at least 20, or at least about 30, or at least about 40, or at least about 50, or at least about 75, or at least about 100, or at least about 115, or at least about 130, or at least about 150, or at least about 200, or at least about 250, or at least about 300, or at least about 350, or at least about 400, or at least about 500, or at least about 600, or at least about 700, or at least about 800, or at least about 900, or at least about 1000, or at least about 1100, or at least about 1200, and so on, in increments of 10 amino acids, up to at least about 4130 contiguous amino acid residues of the amino acid sequence represented by SEQ ID NO:2. A FAS homologue can include proteins encoded by a nucleic acid sequence comprising at least about 30, or at least about 60, or at least about 90, or at least about 150, or at least about 225, or at least about 300, or at least about 750, or at least about 900, or at least about 1050, or at least about 1200, or at least about 1500, or at least about 1800, or at least about 2100, or at least about 2400, or at least about 2700, or at least about 3000, and so on, in increments of 30 nucleotides, up to at least about 12,400 contiguous nucleotides of the nucleic acid sequence represented by SEQ ID NO:1. In a preferred embodiment, a FAS homologue has measurable FAS biological activity (i.e., has biological activity), as described above, including any one or more of the biological activities described for a FAS of the present invention.
According to the present invention, the term “contiguous” or “consecutive”, with regard to nucleic acid or amino acid sequences described herein, means to be connected in an unbroken sequence. For example, for a first sequence to comprise 30 contiguous (or consecutive) amino acids of a second sequence, means that the first sequence includes an unbroken sequence of 30 amino acid residues that is 100% identical to an unbroken sequence of 30 amino acid residues in the second sequence. Similarly, for a first sequence to have “100% identity” with a second sequence means that the first sequence exactly matches the second sequence with no gaps between nucleotides or amino acids.
In another embodiment, a FAS protein, including a FAS homologue, includes a protein having an amino acid sequence that is sufficiently similar to a natural FAS amino acid sequence that a nucleic acid sequence encoding the homologue is capable of hybridizing under moderate, high or very high stringency conditions (described below) to (i.e., with) a nucleic acid molecule encoding the natural FAS (i.e., to the complement of the nucleic acid strand encoding the natural FAS amino acid sequence). Preferably, a homologue of a FAS protein is encoded by a nucleic acid molecule comprising a nucleic acid sequence that hybridizes under moderate, high or very high stringency conditions to the complement of a nucleic acid sequence that encodes a protein comprising SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13. Even more preferably, a homologue of a FAS protein is encoded by a nucleic acid molecule comprising a nucleic acid sequence that hybridizes under moderate, high or very high stringency conditions to the complement of the nucleic acid sequence represented by SEQ ID NO:1.
A nucleic acid sequence complement of nucleic acid sequence encoding a FAS of the present invention refers to the nucleic acid sequence of the nucleic acid strand that is complementary to the strand which encodes FAS. It will be appreciated that a double stranded DNA which encodes a given amino acid sequence comprises a single strand DNA and its complementary strand having a sequence that is a complement to the single strand DNA. As such, nucleic acid molecules of the present invention can be either double-stranded or single-stranded, and include those nucleic acid molecules that form stable hybrids under stringent hybridization conditions with a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:2, for example, and/or with the complement of the nucleic acid sequence that encodes an amino acid sequence of SEQ ID NO:2. Methods to deduce a complementary sequence are known to those skilled in the art. It should be noted that since nucleic acid sequencing technologies are not entirely error-free, the sequences presented herein, at best, represent apparent sequences of a FAS protein of the present invention.
As used herein, reference to hybridization conditions refers to standard hybridization conditions under which nucleic acid molecules are used to identify similar nucleic acid molecules. Such standard conditions are disclosed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989. Sambrook et al., ibid., is incorporated by reference herein in its entirety (see specifically, pages 9.31-9.62). In addition, formulae to calculate the appropriate hybridization and wash conditions to achieve hybridization permitting varying degrees of mismatch of nucleotides are disclosed, for example, in Meinkoth et al., 1984, Anal. Biochem. 138, 267-284; Meinkoth et al., ibid., is incorporated by reference herein in its entirety.
More particularly, moderate stringency hybridization and washing conditions, as referred to herein, refer to conditions which permit isolation of nucleic acid molecules having at least about 70% nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction (i.e., conditions permitting about 30% or less mismatch of nucleotides). High stringency hybridization and washing conditions, as referred to herein, refer to conditions which permit isolation of nucleic acid molecules having at least about 80% nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction (i.e., conditions permitting about 20% or less mismatch of nucleotides). Very high stringency hybridization and washing conditions, as referred to herein, refer to conditions which permit isolation of nucleic acid molecules having at least about 90% nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction (i.e., conditions permitting about 10% or less mismatch of nucleotides). As discussed above, one of skill in the art can use the formulae in Meinkoth et al., ibid. to calculate the appropriate hybridization and wash conditions to achieve these particular levels of nucleotide mismatch. Such conditions will vary, depending on whether DNA:RNA or DNA:DNA hybrids are being formed. Calculated melting temperatures for DNA:DNA hybrids are 10° C. less than for DNA:RNA hybrids. In particular embodiments, stringent hybridization conditions for DNA:DNA hybrids include hybridization at an ionic strength of 6×SSC (0.9 M Na+) at a temperature of between about 20° C. and about 35° C. (lower stringency), more preferably, between about 28° C. and about 40° C. (more stringent), and even more preferably, between about 35° C. and about 45° C. (even more stringent), with appropriate wash conditions. In particular embodiments, stringent hybridization conditions for DNA:RNA hybrids include hybridization at an ionic strength of 6×SSC (0.9 M Na+) at a temperature of between about 30° C. and about 45° C., more preferably, between about 38° C. and about 50° C., and even more preferably, between about 45° C. and about 55° C., with similarly stringent wash conditions. These values are based on calculations of a melting temperature for molecules larger than about 100 nucleotides, 0% formamide and a G+C content of about 40%. Alternatively, Tm can be calculated empirically as set forth in Sambrook et al., supra, pages 9.31 to 9.62. In general, the wash conditions should be as stringent as possible, and should be appropriate for the chosen hybridization conditions. For example, hybridization conditions can include a combination of salt and temperature conditions that are approximately 20-25° C. below the calculated Tm of a particular hybrid, and wash conditions typically include a combination of salt and temperature conditions that are approximately 12-20° C. below the calculated Tm of the particular hybrid. One example of hybridization conditions suitable for use with DNA:DNA hybrids includes a 2-24 hour hybridization in 6×SSC (50% formamide) at about 42° C., followed by washing steps that include one or more washes at room temperature in about 2×SSC, followed by additional washes at higher temperatures and lower ionic strength (e.g., at least one wash as about 37° C. in about 0.1×-0.5×SSC, followed by at least one wash at about 68° C. in about 0.1×-0.5×SSC).
FAS proteins also include expression products of fusions (e.g., fusion proteins, for example, used to overexpress soluble, active forms of the recombinant protein), of mutagenized genes (such as genes having codon modifications to enhance gene transcription and translation), and of truncated genes (such as genes having membrane binding domains removed to generate soluble forms of a membrane protein, or genes having signal sequences removed which are poorly tolerated in a particular recombinant host). It is noted that FAS and protein homologues of the present invention include proteins which do not have any FAS activity or more preferably, that have attenuated FAS activity. Such proteins are useful, for example, for the production of antibodies or for production of genetically modified organisms that lack the ability to produce one or more short chain fatty acids.
The minimum size of a protein and/or homologue of the present invention is a size sufficient to have FAS biological activity or, when the protein is not required to have such activity, sufficient to be useful for another purpose associated with a FAS protein of the present invention, such as for the production of antibodies that bind to a naturally occurring FAS. As such, the minimum size of a FAS or homologue of the present invention is a size suitable to form at least one epitope that can be recognized by an antibody, and is typically at least 8 amino acids in length, and preferably 10, and more preferably 15, and more preferably 20, and more preferably 25, and even more preferably 30 amino acids in length, and up to 4136 amino acids in length, in increments of any whole integer from 1 to 4136, with preferred sizes depending on whether full-length, multivalent (i.e., fusion protein having more than one domain, each of which has a function), or functional portions of such proteins are desired. There is no limit, other than a practical limit, on the maximum size of such a protein in that the protein can include a portion of a FAS protein (including FAS homologues) or a full-length FAS.
Similarly, the minimum size of a nucleic acid molecule of the present invention is a size sufficient to encode a protein having FAS activity (including the activity of one or more domains of a FAS of the present invention), sufficient to encode a protein comprising at least one epitope which binds to an antibody, or sufficient to form a probe or oligonucleotide primer that is capable of forming a stable hybrid with the complementary sequence of a nucleic acid molecule encoding a natural FAS (e.g., under low, moderate or high stringency conditions). As such, the size of the nucleic acid molecule encoding such a protein can be dependent on nucleic acid composition and percent homology or identity between the nucleic acid molecule and complementary sequence as well as upon hybridization conditions per se (e.g., temperature, salt concentration, and formamide concentration). The minimal size of a nucleic acid molecule that is used as an oligonucleotide primer or as a probe is typically at least about 12 to about 15 nucleotides in length if the nucleic acid molecules are GC-rich and at least about 15 to about 18 bases in length if they are AT-rich. There is no limit, other than a practical limit, on the maximal size of a nucleic acid molecule of the present invention, in that the nucleic acid molecule can include a portion of a FAS encoding sequence, a nucleic acid sequence encoding a full-length FAS (including a FAS gene), or multiple genes, or portions thereof.
The present invention also includes a fusion protein that includes a FAS-containing domain (including a homologue or functional domain of a FAS) attached to one or more fusion segments. Suitable fusion segments for use with the present invention include, but are not limited to, segments that can: enhance a protein's stability; provide other desirable biological activity; and/or assist with the purification of a FAS protein (e.g., by affinity chromatography). A suitable fusion segment can be a domain of any size that has the desired function (e.g., imparts increased stability, solubility, action or biological activity; and/or simplifies purification of a protein). Fusion segments can be joined to amino and/or carboxyl termini of the FAS-containing domain of the protein and can be susceptible to cleavage in order to enable straight-forward recovery of a FAS protein. Fusion proteins are preferably produced by culturing a recombinant cell transformed with a fusion nucleic acid molecule that encodes a protein including the fusion segment attached to either the carboxyl and/or amino terminal end of a FAS-containing domain.
In one embodiment of the present invention, any of the amino acid sequences described herein can be produced with from at least one, and up to about 20, additional heterologous amino acids flanking each of the C- and/or N-terminal ends of the specified amino acid sequence. The resulting protein or polypeptide can be referred to as “consisting essentially of” the specified amino acid sequence. According to the present invention, the heterologous amino acids are a sequence of amino acids that are not naturally found (i.e., not found in nature, in vivo) flanking the specified amino acid sequence, or that are not related to the function of the specified amino acid sequence, or that would not be encoded by the nucleotides that flank the naturally occurring nucleic acid sequence encoding the specified amino acid sequence as it occurs in the gene, if such nucleotides in the naturally occurring sequence were translated using standard codon usage for the organism from which the given amino acid sequence is derived. Similarly, the phrase “consisting essentially of”, when used with reference to a nucleic acid sequence herein, refers to a nucleic acid sequence encoding a specified amino acid sequence that can be flanked by from at least one, and up to as many as about 60, additional heterologous nucleotides at each of the 5′ and/or the 3′ end of the nucleic acid sequence encoding the specified amino acid sequence. The heterologous nucleotides are not naturally found (i.e., not found in nature, in vivo) flanking the nucleic acid sequence encoding the specified amino acid sequence as it occurs in the natural gene or do not encode a protein that imparts any additional function to the protein or changes the function of the protein having the specified amino acid sequence.
FAS proteins as described herein can be isolated from a various microorganisms including members of the order Thraustochytriales. For example, preferred microorganisms from which a FAS protein of the present invention may be derived include microorganisms from a genus including, but not limited to: Thraustochytrium, Labyrinthuloides, Japonochytrium, and Schizochytrium. Preferred species within these genera include, but are not limited to: any Schizochytrium species, including Schizochytrium aggregatum, Schizochytrium limacinum, Schizochytrium minutum; any Thraustochytrium species (including former Ulkenia species such as U. visurgensis, U. amoeboida, U. sarkariana, U. profunda, U. radiata, U. minuta and Ulkenia sp. BP-5601), and including Thraustochytrium striatum, Thraustochytrium aureum, Thraustochytrium roseum; and any Japonochytrium species. Particularly preferred strains of Thraustochytriales include, but are not limited to: Schizochytrium sp. (S31)(ATCC 20888); Schizochytrium sp. (S8)(ATCC 20889); Schizochytrium sp. (LC-RM)(ATCC 18915); Schizochytrium sp. (SR21); Schizochytrium aggregatum (Goldstein et Belsky)(ATCC 28209); Schizochytrium limacinum (Honda et Yokochi)(IFO 32693); Thraustochytrium sp. (23B)(ATCC 20891); Thraustochytrium striatum (Schneider)(ATCC 24473); Thraustochytrium aureum (Goldstein)(ATCC 34304); Thraustochytrium roseum (Goldstein)(ATCC 28210); and Japonochytrium sp. (L1)(ATCC 28207).
According to the present invention, the terms/phrases “Thraustochytrid”, “Thraustochytriales microorganism” and “microorganism of the order Thraustochytriales” can be used interchangeably and refer to any members of the order Thraustochytriales, which includes both the family Thraustochytriaceae and the family Labyrinthulaceae. The terms “Labyrinthulid” and “Labyrinthulaceae” are used herein to specifically refer to members of the family Labyrinthulaceae. To specifically reference Thraustochytrids that are members of the family Thraustochytriaceae, the term “Thraustochytriaceae” is used herein. Thus, for the present invention, members of the Labyrinthulids are considered to be included in the Thraustochytrids.
Developments have resulted in frequent revision of the taxonomy of the Thraustochytrids. Taxonomic theorists generally place Thraustochytrids with the algae or algae-like protists. However, because of taxonomic uncertainty, it would be best for the purposes of the present invention to consider the strains described in the present invention as Thraustochytrids to include the following organisms: Order: Thraustochytriales; Family: Thraustochytriaceae (Genera: Thraustochytrium, Schizochytrium, Japonochytrium, Aplanochytrium, or Elina) or Labyrinthulaceae (Genera Labyrinthula, Labyrinthuloides, or Labyrinthomyxa). Also, the following genera are sometimes included in either family Thraustochytriaceae or Labyrinthulaceae: Althornia, Corallochytrium, Diplophyrys, and Pyrrhosorus), and for the purposes of this invention are encompassed by reference to a Thraustochytrid or a member of the order Thraustochytriales. It is recognized that at the time of this invention, revision in the taxonomy of Thraustochytrids places the genus Labyrinthuloides in the family of Labyrinthulaceae and confirms the placement of the two families Thraustochytriaceae and Labyrinthulaceae within the Stramenopile lineage. It is noted that the Labyrinthulaceae are sometimes commonly called labyrinthulids or labyrinthula, or labyrinthuloides and the Thraustochytriaceae are commonly called thraustochytrids, although, as discussed above, for the purposes of clarity of this invention, reference to Thraustochytrids encompasses any member of the order Thraustochytriales and/or includes members of both Thraustochytriaceae and Labyrinthulaceae. Recent taxonomic changes are summarized below.
Strains of certain unicellular microorganisms disclosed herein are members of the order Thraustochytriales. Thraustochytrids are marine eukaryotes with an evolving taxonomic history. Problems with the taxonomic placement of the Thraustochytrids have been reviewed by Moss (in “The Biology of Marine Fungi”, Cambridge University Press p. 105 (1986)), Bahnweb and Jackle (ibid. p. 131) and Chamberlain and Moss (BioSystems 21:341 (1988)).
For convenience purposes, the Thraustochytrids were first placed by taxonomists with other colorless zoosporic eukaryotes in the Phycomycetes (algae-like fungi). The name Phycomycetes, however, was eventually dropped from taxonomic status, and the Thraustochytrids were retained in the Oomycetes (the biflagellate zoosporic fungi). It was initially assumed that the Oomycetes were related to the heterokont algae, and eventually a wide range of ultrastructural and biochemical studies, summarized by Barr (Barr. Biosystems 14:359 (1981)) supported this assumption. The Oomycetes were in fact accepted by Leedale (Leedale. Taxon 23:261 (1974)) and other phycologists as part of the heterokont algae. However, as a matter of convenience resulting from their heterotrophic nature, the Oomycetes and Thraustochytrids have been largely studied by mycologists (scientists who study fungi) rather than phycologists (scientists who study algae).
From another taxonomic perspective, evolutionary biologists have developed two general schools of thought as to how eukaryotes evolved. One theory proposes an exogenous origin of membrane-bound organelles through a series of endosymbioses (Margulis, 1970, Origin of Eukaryotic Cells. Yale University Press, New Haven); e.g., mitochondria were derived from bacterial endosymbionts, chloroplasts from cyanophytes, and flagella from spirochaetes. The other theory suggests a gradual evolution of the membrane-bound organelles from the non-membrane-bounded systems of the prokaryote ancestor via an autogenous process (Cavalier-Smith, 1975, Nature (Lond.) 256:462-468). Both groups of evolutionary biologists however, have removed the Oomycetes and Thraustochytrids from the fungi and place them either with the chromophyte algae in the kingdom Chromophyta (Cavalier-Smith BioSystems 14:461 (1981)) (this kingdom has been more recently expanded to include other protists and members of this kingdom are now called Stramenopiles) or with all algae in the kingdom Protoctista (Margulis and Sagen. Biosystems 18:141 (1985)).
With the development of electron microscopy, studies on the ultrastructure of the zoospores of two genera of Thraustochytrids, Thraustochytrium and Schizochytrium, (Perkins, 1976, pp. 279-312 in “Recent Advances in Aquatic Mycology” (ed. E. B. G. Jones), John Wiley & Sons, New York; Kazama. Can. J. Bot. 58:2434 (1980); Barr, 1981, Biosystems 14:359-370) have provided good evidence that the Thraustochytriaceae are only distantly related to the Oomycetes. Additionally, genetic data representing a correspondence analysis (a form of multivariate statistics) of 5-S ribosomal RNA sequences indicate that Thraustochytriales are clearly a unique group of eukaryotes, completely separate from the fungi, and most closely related to the red and brown algae, and to members of the Oomycetes (Mannella et al. Mol. Evol. 24:228 (1987)). Most taxonomists have agreed to remove the Thraustochytrids from the Oomycetes (Bartnicki-Garcia. p. 389 in “Evolutionary Biology of the Fungi” (eds. Rayner, A. D. M., Brasier, C. M. & Moore, D.), Cambridge University Press, Cambridge).
In summary, employing the taxonomic system of Cavalier-Smith (Cavalier-Smith. BioSystems 14:461 (1981); Cavalier-Smith. Microbiol. Rev. 57:953 (1993)), the Thraustochytrids are classified with the chromophyte algae in the kingdom Chromophyta (Stramenopiles). This taxonomic placement has been more recently reaffirmed by Cavalier-Smith et al. using the 18s rRNA signatures of the Heterokonta to demonstrate that Thraustochytrids are chromists not Fungi (Cavalier-Smith et al. Phil. Tran. Roy. Soc. London Series BioSciences 346:387 (1994)). This places the Thraustochytrids in a completely different kingdom from the fungi, which are all placed in the kingdom Eufungi.
Currently, there are 71 distinct groups of eukaryotic organisms (Patterson. Am. Nat. 154:S96 (1999)) and within these groups four major lineages have been identified with some confidence: (1) Alveolates, (2) Stramenopiles, (3) a Land Plant-green algae-Rhodophyte_Glaucophyte (“plant”) lade and (4) an Opisthokont lade (Fungi and Animals). Formerly these four major lineages would have been labeled Kingdoms but use of the “kingdom” concept is no longer considered useful by some researchers.
As noted by Armstrong, Stramenopile refers to three-parted tubular hairs, and most members of this lineage have flagella bearing such hairs. Motile cells of the Stramenopiles (unicellular organisms, sperm, zoospores) are asymmetrical having two laterally inserted flagella, one long, bearing three-parted tubular hairs that reverse the thrust of the flagellum, and one short and smooth. Formerly, when the group was less broad, the Stramenopiles were called Kingdom Chromista or the heterokont (=different flagella) algae because those groups consisted of the Brown Algae or Phaeophytes, along with the yellow-green Algae, Golden-brown Algae, Eustigmatophytes and Diatoms. Subsequently some heterotrophic, fungal-like organisms, the water molds, and labyrinthulids (slime net amoebas), were found to possess similar motile cells, so a group name referring to photosynthetic pigments or algae became inappropriate. Currently, two of the families within the Stramenopile lineage are the Labyrinthulaceae and the Thraustochytriaceae. Historically, there have been numerous classification strategies for these unique microorganisms and they are often classified under the same order (i.e., Thraustochytriales). Relationships of the members in these groups are still developing. Porter and Leander have developed data based on 18S small subunit ribosomal DNA indicating the thraustochytrid-labyrinthulid lade in monophyletic. However, the lade is supported by two branches; the first contains three species of Thraustochytrium and Ulkenia profunda, and the second includes three species of Labyrinthula, two species of Labyrinthuloides and Schizochytrium aggregatum. 
The taxonomic placement of the Thraustochytrids as used in the present invention is therefore summarized below:
Kingdom: Chromophyta (Stramenopiles)
Phylum: Heterokonta
Order: Thraustochytriales (Thraustochytrids)
Family: Thraustochytriaceae or Labyrinthulaceae
Genera: Thraustochytrium, Schizochytrium, Japonochytrium, Aplanochytrium, Elina, Labyrinthula, Labyrinthuloides, or Labyrinthulomyxa 
Some early taxonomists separated a few original members of the genus Thraustochytrium (those with an amoeboid life stage) into a separate genus called Ulkenia. However it is now known that most, if not all, Thraustochytrids (including Thraustochytrium and Schizochytrium), exhibit amoeboid stages and as such, Ulkenia is not considered by some to be a valid genus. As used herein, the genus Thraustochytrium will include Ulkenia. 
Despite the uncertainty of taxonomic placement within higher classifications of Phylum and Kingdom, the Thraustochytrids remain a distinctive and characteristic grouping whose members remain classifiable within the order Thraustochytriales.
Further embodiments of the present invention include nucleic acid molecules that encode a FAS protein. An isolated nucleic acid molecule of the present invention includes a nucleic acid molecule comprising a nucleic acid sequence encoding any of the isolated FAS proteins, including a FAS homologue or fragment, described above.
In one embodiment, such nucleic acid molecules include isolated nucleic acid molecules that hybridize under moderate stringency conditions, and even more preferably under high stringency conditions, and even more preferably under very high stringency conditions with the complement of a nucleic acid sequence encoding a naturally occurring FAS protein (i.e., including naturally occurring allelic variants encoding a FAS protein). Preferably, an isolated nucleic acid molecule encoding a FAS protein of the present invention comprises a nucleic acid sequence that hybridizes under moderate, high, or very high stringency conditions to the complement of a nucleic acid sequence that encodes any of the proteins described above. In one embodiment, an isolated nucleic acid molecule comprises a nucleic acid sequence that hybridizes under moderate, high or very high stringency conditions to the complement of a nucleic acid sequence represented by SEQ ID NO:1. Such conditions have been described in detail above.
In accordance with the present invention, an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subject to human manipulation) and can include DNA, RNA, or derivatives of either DNA or RNA, including cDNA. As such, “isolated” does not reflect the extent to which the nucleic acid molecule has been purified. An isolated FAS nucleic acid molecule of the present invention can be isolated from its natural source or produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. Isolated FAS nucleic acid molecules can include, for example, FAS genes, natural allelic variants of FAS genes, FAS coding regions or portions thereof, and FAS coding and/or regulatory regions modified by nucleotide insertions, deletions, substitutions, and/or inversions in a manner such that the modifications do not substantially interfere with the nucleic acid molecule's ability to encode a FAS protein of the present invention or to form stable hybrids under stringent conditions with natural gene isolates. An isolated FAS nucleic acid molecule can include degeneracies. As used herein, nucleotide degeneracies refers to the phenomenon that one amino acid can be encoded by different nucleotide codons. Thus, the nucleic acid sequence of a nucleic acid molecule that encodes a FAS protein of the present invention can vary due to degeneracies. It is noted that an isolated FAS nucleic acid molecule of the present invention is not required to encode a protein having FAS activity. A FAS nucleic acid molecule can encode a truncated, mutated or inactive protein, for example. Such nucleic acid molecules and the proteins encoded by such nucleic acid molecules are useful in as probes and primers for the identification of other FAS proteins.
According to the present invention, reference to a FAS gene includes all nucleic acid sequences related to a natural (i.e. wild-type) FAS gene, such as regulatory regions that control production of the FAS encoded by that gene (such as, but not limited to, transcription, translation or post-translation control regions) as well as the coding region itself. The present inventors provide herein the 5′ untranslated region of the FAS gene of the present invention (represented herein by SEQ ID NO:4), as well as the 3′ untranslated region (represented herein by SEQ ID NO:3). The use of these regions of the FAS gene to regulate the expression and/or biological activity of a FAS-encoding nucleic acid molecule, such as an endogenous FAS gene, or to regulate the expression or activity of a heterologous nucleic acid molecule (i.e., a molecule encoding a different protein) is encompassed by the present invention. In addition, the regulation of the expression or activity of a FAS gene or protein according to the present invention also encompasses the use of any regulatory sequence or protein, including heterologous regulatory sequences and proteins (i.e., those that are not naturally associated with the FAS gene or protein) to regulate the expression and/or biological activity of FAS. Such uses will be described below.
In another embodiment, a FAS gene can be a naturally occurring allelic variant that includes a similar but not identical sequence to the nucleic acid sequence encoding a given FAS protein. Allelic variants have been previously described above. The phrases “nucleic acid molecule” and “gene” can be used interchangeably when the nucleic acid molecule comprises a gene as described above.
A FAS nucleic acid molecule homologue (i.e., encoding a FAS homologue) can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al.). For example, nucleic acid molecules can be modified using a variety of techniques including, but not limited to, by classic mutagenesis and recombinant DNA techniques (e.g., site-directed mutagenesis, chemical treatment, restriction enzyme cleavage, ligation of nucleic acid fragments and/or PCR amplification), or synthesis of oligonucleotide mixtures and ligation of mixture groups to “build” a mixture of nucleic acid molecules and combinations thereof. Another method for modifying a recombinant nucleic acid molecule encoding a FAS is gene shuffling (i.e., molecular breeding) (See, for example, U.S. Pat. No. 5,605,793 to Stemmer; Minshull and Stemmer; 1999, Curr. Opin. Chem. Biol. 3:284-290; Stemmer, 1994, P.N.A.S. USA 91:10747-10751, all of which are incorporated herein by reference in their entirety). This technique can be used to efficiently introduce multiple simultaneous changes in the FAS activity. Nucleic acid molecule homologues can be selected by hybridization with a FAS gene or by screening the function of a protein encoded by a nucleic acid molecule (e.g., enzymatic activity).
One embodiment of the present invention relates to an oligonucleotide, comprising at least 12 contiguous nucleotides of SEQ ID NO:1, and a nucleic acid sequence fully complementary thereto. The minimal size of a nucleic acid molecule that is used as an oligonucleotide primer or as a probe is typically at least about 12 to about 15 nucleotides in length if the nucleic acid molecules are GC-rich and at least about 15 to about 18 bases in length if they are AT-rich. There is no limit, other than a practical limit, on the maximal size of an oligonucleotide probe or primer of the present invention, in that the probe or primer can include any portion of a FAS gene of the invention that is suitable for the intended use, with probes typically being larger than primers. As such, an oligonucleotide of the invention can include any length fragment between about 12 and about 12,408 nucleotides or even larger probes, in whole integers (e.g., 12, 13, 14, 15, 16 . . . 12,407, 12,408).
One embodiment of the present invention includes a recombinant nucleic acid molecule, which includes at least one isolated nucleic acid molecule of the present invention inserted into any nucleic acid vector (e.g., a recombinant vector) which is suitable for cloning, sequencing, and/or otherwise manipulating the nucleic acid molecule, such as expressing and/or delivering the nucleic acid molecule into a host cell to form a recombinant cell. Such a vector contains heterologous nucleic acid sequences, that is nucleic acid sequences that are not naturally found adjacent to nucleic acid molecules of the present invention, although the vector can also contain regulatory nucleic acid sequences (e.g., promoters, untranslated regions) which are naturally found adjacent to nucleic acid molecules of the present invention (discussed in detail below). The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid. The vector can be maintained as an extrachromosomal element (e.g., a plasmid) or it can be integrated into the chromosome. The entire vector can remain in place within a host cell, or under certain conditions, the plasmid DNA can be deleted, leaving behind the nucleic acid molecule of the present invention. The integrated nucleic acid molecule can be under chromosomal promoter control, under native or plasmid promoter control, or under a combination of several promoter controls. Single or multiple copies of the nucleic acid molecule can be integrated into the chromosome. The vector can be designed for tissue-specific expression in the host cell, such as by using tissue-specific promoters. Several recombinant nucleic acid molecules useful in the present invention, including several recombinant vectors, are described in detail in the Examples.
Typically, a recombinant molecule includes a nucleic acid molecule of the present invention operatively linked to one or more transcription control sequences (e.g., promoters, operators, repressors, enhancers, terminators). As used herein, the phrase “recombinant molecule” or “recombinant nucleic acid molecule” primarily refers to a nucleic acid molecule or nucleic acid sequence operatively linked to a transcription control sequence, but can be used interchangeably with the phrase “nucleic acid molecule”, when such nucleic acid molecule is a recombinant molecule as discussed herein. According to the present invention, the phrase “operatively linked” refers to linking a nucleic acid molecule to a transcription control sequence in a manner such that the molecule is able to be expressed when transformed (i.e., transformed, transduced, transfected, or conjugated) into a host cell. Transcription control sequences are sequences which control the initiation, elongation, or termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells useful for expressing a FAS protein of the present invention. A variety of such transcription control sequences are known to those skilled in the art. Preferred transcription control sequences include those which function in Thraustochytriales microorganisms, bacterial, fungal (e.g., yeast), or plant cells. Other preferred transcription control sequences are for plants and include those that promote gene expression in specific tissues (e.g., leaves, stems, roots, flowers, seeds) and can be referred to herein as tissue-specific transcription control sequences. Such sequences are well known in the art.
In one embodiment of the invention, a suitable transcription control sequence includes the regulatory sequences that are naturally found in the FAS gene of the present invention. For example, regulatory sequences of a Schizochytrium FAS, which include a FAS promoter, are found in nucleotides represented herein by SEQ ID NO:3 (3′ untranslated region) or in nucleotides represented herein by SEQ ID NO:4 (5′ untranslated region). In another embodiment, any regulatory sequence can be used which increases (enhances, upregulates), allows/maintains or decreases (attenuates, downregulates, reduces) the expression and/or biological activity of an endogenous and/or recombinant FAS gene or protein of the present invention. Regulatory sequences, including promoter sequences, for a variety of host organisms are known in the art and all are encompassed for use in the present invention.
Recombinant molecules of the present invention, which can be either DNA or RNA, can also contain additional regulatory sequences, such as transcription regulatory sequences, translation regulatory sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell. In one embodiment, a recombinant molecule of the present invention, including those which are integrated into the host cell chromosome, also contains signal (targeting) (i.e., signal segment nucleic acid sequences) to enable an expressed FAS to be secreted from the cell that produces the protein or targeted to a particular organelle or membrane. For example, in one embodiment, suitable signal segments include a signal segment that is naturally associated with a FAS of the present invention or any heterologous signal segment capable of directing the secretion of a FAS protein according to the present invention. In another embodiment, a recombinant molecule of the present invention comprises a signal sequence to enable an expressed FAS protein to be delivered to and inserted into the membrane of a host cell. In another embodiment, a recombinant molecule of the present invention comprises a signal sequence which specifically targets the delivery of a FAS to specific sub-cellular organelles or compartments, such as the endoplasmic reticulum, the chloroplast, the chromoplast, other plastids, or the cytoplasm.
One or more recombinant molecules of the present invention can be used to produce an encoded product (e.g., a FAS protein) of the present invention. In one embodiment, an encoded product is produced by expressing a nucleic acid molecule as described herein under conditions effective to produce the protein. A preferred method to produce an encoded protein is by transforming a host cell with one or more recombinant molecules to form a recombinant cell. Suitable host cells to transform include, but are not limited to, any microalgal cell, including a Thraustochytriales microorganism, or any bacterial cell, fungal (e.g., yeast) cell, other microbial cell, or plant cell that can be transformed. Host cells can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule.
Preferred host cells for use in the present invention include any microorganism cell or plant cell which is suitable for expression of a FAS protein of the present invention, including, but not limited to: (1) plants, including, but not limited to, crop plants (e.g., canola—Brassica napus, rice, corn, flax, safflower, soy, sunflower, rapeseed, linseed); (2) fungi, including, but not limited to, Phycomyces sp., Neurospora sp., Mucor sp. (e.g., Mucor circinelloides), Blakeslea sp., Mortierella sp. (e.g., Mortierella alpina), Rhodotorula sp., Lipomyces sp. (e.g., Lipomyces starkeyi), Cryptococcus sp. (e.g., Cryptococcus curvatus, aka Apiotricum curvatum), Cunninghamella sp. (e.g., Cunninghamella echinulata), Yarrowia sp. (e.g., Yarrowia lipolytica) and yeast (e.g., Saccharomyces sp. (e.g., Saccharomyces cerevisiae), Phaffia rhodozyma, Xanthophyllomyces dendrohous, Candida sp. (e.g., Candida utilus); (3) algae, including but not limited to, green algae (e.g., Haematococcus pluvialus, Chlorococcum, Spongiococcum, Neospongiococcum, Dunaliella), Crypthecodinium cohnii, Porphyridium cruentum, Phaeodactylum tricornicum, Nannochloropsis oculata, Isochrysis galbana, Chlorella sp.; (4) bacteria, including, but not limited to, blue-green (e.g., Spirulina, Synechococcus, Synechocystis), Escherichia coli, Flavobacterium, Paracoccus, Erwinia, Agrobacterium, Rhodococcus, Mycobacterium, Streptomyces, Thodococcus, Nocardia, Pseudomonas; and (5) members of the order, Thraustochytriales, including but not limited to: Thraustochytrium sp. (e.g., including former Ulkenia species such as U. visurgensis, U. amoeboida, U. sarkariana, U. profunda, U. radiata, U. minuta and Ulkenia sp. BP-5601, and including Thraustochytrium striatum, Thraustochytrium aureum, and Thraustochytrium roseum); Labyrinthuloides, Japonochytrium (e.g., Japonochytrium sp.), and Schizochytrium (e.g., Schizochytrium sp., Schizochytrium aggregatum, Schizochytrium limacinum, Schizochytrium minutum).
According to the present invention, the term “transformed” or “transformation” is used to refer to any method by which an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid molecule) can be inserted into the cell. In microbial systems, the term “transformation” is used to describe an inherited change due to the acquisition of exogenous nucleic acids by the microorganism and can be essentially synonymous with the term “transfection”, which is more commonly used in reference to the similar process in animal cells. The term “transformation” is preferably used herein to refer to the introduction of nucleic acid molecules into microbial cells, such as bacteria and yeast, or into plant cells. Therefore, transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, biolistic methods (particle bombardment), adsorption, Agrobacterium-mediated transformation, infection and protoplast fusion. Methods of transforming prokaryotic and eukaryotic host cells are well known in the art. See, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (1982), Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (1989), incorporated herein by reference in its entirety. A preferred method for transforming members of the order Thraustochytriales is described in U.S. patent application Ser. No. 10/124,807, filed Apr. 16, 2002, incorporated by reference in its entirety.
Numerous methods for plant transformation have been developed, including biological and physical transformation protocols. See, for example, Miki et al., “Procedures for Introducing Foreign DNA into Plants” in Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pp. 67-88. In addition, vectors and in vitro culture methods for plant cell or tissue transformation and regeneration of plants are available. See, for example, Gruber et al., “Vectors for Plant Transformation” in Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pp. 89-119.
The most widely utilized method for introducing an expression vector into plants is based on the natural transformation system of Agrobacterium. See, for example, Horsch et al., Science 227:1229 (1985). A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteria which genetically transform plant cells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carry genes responsible for genetic transformation of the plant. See, for example, Kado, C. I., Crit. Rev. Plant. Sci. 10:1 (1991). Descriptions of Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer are provided by numerous references, including Gruber et al., supra, Miki et al., supra, Moloney et al., Plant Cell Reports 8:238 (1989), and U.S. Pat. Nos. 4,940,838 and 5,464,763, each of which is incorporated herein by reference in its entirety.
A generally applicable method of plant transformation is microprojectile-mediated transformation wherein DNA is carried on the surface of microprojectiles. The expression vector is introduced into plant tissues with a biolistic device that accelerates the microprojectiles to speeds sufficient to penetrate plant cell walls and membranes. Sanford et al., Part. Sci. Technol. 5:27 (1987), Sanford, J. C., Trends Biotech. 6:299 (1988), Sanford, J. C., Physiol. Plant 79:206 (1990), Klein et al., Biotechnology 10:268 (1992), each of which is incorporated herein by reference in its entirety.
Another method for physical delivery of DNA to plants is sonication of target cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively, liposome or spheroplast fusion have been used to introduce expression vectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christou et al., Proc Natl. Acad. Sci. USA 84:3962 (1987), each of which is incorporated herein by reference in its entirety. Direct uptake of DNA into protoplasts using CaCl2 precipitation, polyvinyl alcohol or poly-L-ornithine have also been reported. Hain et al., Mol. Gen. Genet. 199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982), each of which is incorporated herein by reference in its entirety. Electroporation of protoplasts and whole cells and tissues have also been described. Donn et al., In Abstracts of VIIth International Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p. 53 (1990); D'Halluin et al., Plant Cell 4:1495-1505 (1992) and Spencer et al., Plant Mol. Biol. 24:51-61 (1994), each of which is incorporated herein by reference in its entirety.
In one embodiment, an isolated FAS protein of the present invention is produced by culturing a cell that expresses the protein under conditions effective to produce the protein, and recovering the protein. A preferred cell to culture is a recombinant cell of the present invention. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production. An effective medium refers to any medium in which a cell is cultured to produce a FAS protein of the present invention. Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and Petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.
Depending on the vector and host system used for production, resultant proteins of the present invention may either remain within the recombinant host cell; be secreted into the culture medium; be secreted into a space between two cellular membranes, such as the periplasmic space in E. coli; or be retained on the outer surface of a cell membrane. The phrase “recovering the protein” refers to collecting the whole culture medium containing the protein and need not imply additional steps of separation or purification. Proteins of the present invention can be purified, if desired, using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization. If proteins of the present invention are purified, they are preferably retrieved in “substantially pure” form. As used herein, “substantially pure” refers to a purity that allows for the effective use of the protein as a biocatalyst or other reagent.
To produce significantly high yields of short chain fatty acids by the methods of the present invention, a microorganism or plant (or part of a plant, e.g., seeds, pollen, embryos, flowers, fruits, shoots, leaves, roots, stems, explants, etc.) can be genetically modified to increase the action of the FAS protein of the present invention, and preferably, to enhance production of the FAS protein, and thereby, a short chain fatty acid endproduct.
In a preferred embodiment of the invention, a microorganism that contains an endogenous FAS protein of the invention (e.g., Schizochytrium) is genetically modified to increase or reduce the expression and activity of the FAS protein. Without being bound by theory, the present inventors believe that attenuation of the expression or activity of the endogenous FAS protein in Thraustochytrids such as Schizochytrium will increase the accumulation of the highly desirable polyunsaturated fatty acids (PUFAs) by the organism, the synthesis of which proceeds using the PUFA polyketide synthase (PKS) system, which shares some of the same substrates with the FAS system (this PUFA PKS system is described in detail in PCT Publication No. WO 02/083870, published Oct. 24, 2002, which is incorporated herein by reference in its entirety). Therefore, decreasing the expression or activity of the FAS in the microorganism will increase the production of PUFAs.
As used herein, a genetically modified microorganism, such as a genetically modified bacterium, protist, microalga, fungus, or other microbe, and particularly, any member of the genera of the order Thraustochytriales (e.g., a Thraustochytrid) described herein (e.g., Schizochytrium, Thraustochytrium, Japonochytrium, Labyrinthuloides), has a genome which is modified (i.e., mutated or changed) from its normal (i.e., wild-type or naturally occurring) form such that the desired result is achieved (i.e., increased or modified FAS expression and/or activity, production of a desired product using the FAS protein, or decreased or modified FAS expression and/or activity). Genetic modification of a microorganism can be accomplished using classical strain development and/or molecular genetic techniques. Such techniques are generally disclosed for microorganisms, for example, in Sambrook et al., 1989, supra, incorporated by reference herein in its entirety. A genetically modified microorganism can include a microorganism in which nucleic acid molecules have been inserted, deleted or modified (i.e., mutated; e.g., by insertion, deletion, substitution, and/or inversion of nucleotides), in such a manner that such modifications provide the desired effect within the microorganism.
Preferred microorganism host cells to modify according to the present invention include, but are not limited to, any bacteria, protist, microalga, fungus, or protozoa. In one aspect, preferred microorganisms to genetically modify include, but are not limited to, any microorganism of the order Thraustochytriales. Particularly preferred host cells for use in the present invention could include microorganisms from a genus including, but not limited to: Thraustochytrium, Labyrinthuloides, Japonochytrium, and Schizochytrium. Preferred species within these genera include, but are not limited to: any Schizochytrium species, including Schizochytrium aggregatum, Schizochytrium limacinum, Schizochytrium minutum; any Thraustochytrium species (including former Ulkenia species such as U. visurgensis, U. amoeboida, U. sarkariana, U. profunda, U. radiata, U. minuta and Ulkenia sp. BP-5601), and including Thraustochytrium striatum, Thraustochytrium aureum, Thraustochytrium roseum; and any Japonochytrium species. Particularly preferred strains of Thraustochytriales include, but are not limited to: Schizochytrium sp. (S31)(ATCC 20888); Schizochytrium sp. (S8)(ATCC 20889); Schizochytrium sp. (LC-RM)(ATCC 18915); Schizochytrium sp. (SR21); Schizochytrium aggregatum (Goldstein et Belsky)(ATCC 28209); Schizochytrium limacinum (Honda et Yokochi)(IFO 32693); Thraustochytrium sp. (23B)(ATCC 20891); Thraustochytrium striatum (Schneider)(ATCC 24473); Thraustochytrium aureum (Goldstein)(ATCC 34304); Thraustochytrium roseum (Goldstein)(ATCC 28210); and Japonochytrium sp. (L1)(ATCC 28207). Other examples of suitable host microorganisms for genetic modification include, but are not limited to, yeast including Saccharomyces cerevisiae, Saccharomyces carlsbergensis, or other yeast such as Candida, Kluyveromyces, or other fungi, for example, filamentous fungi such as Aspergillus, Neurospora, Penicillium, etc. Bacterial cells also may be used as hosts. This includes Escherichia coli, which can be useful in fermentation processes. Alternatively, a host such as a Lactobacillus species or Bacillus species can be used as a host.
As used herein, a genetically modified plant can include any genetically modified plant including higher plants and particularly, any consumable plants or plants useful for producing a desired product of the present invention (e.g., short chain fatty acids or any other lipid product). Such a genetically modified plant has a genome which is modified (i.e., mutated or changed) from its normal (i.e., wild-type or naturally occurring) form such that the desired result is achieved (i.e., increased or modified FAS expression and/or activity and/or production of a desired product using the FAS protein). Genetic modification of a plant can be accomplished using classical strain development and/or molecular genetic techniques. Methods for producing a transgenic plant, wherein a recombinant nucleic acid molecule encoding a desired amino acid sequence is incorporated into the genome of the plant, are known in the art and have been described briefly above. A preferred plant to genetically modify according to the present invention is preferably a plant suitable for consumption by animals, including humans.
Preferred plants to genetically modify according to the present invention (i.e., plant host cells) include, but are not limited to any higher plants, and particularly consumable plants, including crop plants and especially plants used for their oils. Such plants can include, for example: canola, soybeans, rapeseed, linseed, corn, safflowers, flax, sunflowers, tobacco, rice, tomatoes and carrots. Other preferred plants include those plants that are known to produce compounds used as pharmaceutical agents, flavoring agents, neutraceutical agents, functional food ingredients or cosmetically active agents or plants that are genetically engineered to produce these compounds/agents.
According to the present invention, a genetically modified microorganism or plant includes a microorganism or plant that has been modified using recombinant technology. As used herein, genetic modifications that result in a decrease in gene expression, in the function of the gene, or in the function of the gene product (i.e., the protein encoded by the gene) can be referred to as inactivation (complete or partial), attenuation, deletion, interruption, blockage or down-regulation of a gene. For example, a genetic modification in a gene which results in a decrease in the function of the protein encoded by such gene, can be the result of a complete deletion of the gene (i.e., the gene does not exist, and therefore the protein does not exist), a mutation in the gene which results in incomplete or no translation of the protein (e.g., the protein is not expressed), or a mutation in the gene or introduction of a regulatory sequence or protein into the host which decreases, attenuates or abolishes the expression and/or the natural function of the protein (e.g., a protein is expressed which has decreased or no enzymatic activity or action). In the present invention, genetic modifications that decrease the expression or activity of a FAS are preferably not complete inactivations, as such mutations are lethal in microorganisms. Preferably, such modifications reduce or attenuate, but do not entirely delete, the expression or function of a FAS protein. Genetic modifications that result in an increase in gene expression or function can be referred to as amplification, overproduction, overexpression, activation, enhancement, addition, or up-regulation of a gene.
In one embodiment of the present invention, a genetic modification of a microorganism or plant increases or decreases the expression and/or activity of a FAS protein of the present invention. Such a genetic modification includes any type of modification and specifically includes modifications made by recombinant technology and/or by classical mutagenesis. It should be noted that reference to increasing the action (activity) of FAS refers to any genetic modification in the microorganism or plant in question and/or in the recombinant nucleic acids containing the FAS-encoding DNA with which the organism is transformed that results in increased functionality of the protein and can include higher activity of the protein (e.g., specific activity or in vivo enzymatic activity), reduced inhibition or degradation of the protein, and overexpression of the protein. For example, gene copy number can be increased, expression levels can be increased by use of a promoter that gives higher levels of expression than that of the native promoter, or a gene can be altered by genetic engineering or classical mutagenesis to increase the action of an enzyme. In one aspect, FAS activity or expression can be modified by modifying a nucleic acid or protein that interacts with a FAS gene or protein and normally modulates the expression or activity of the FAS gene or protein. Such a modification can be achieved by recombinant or classical mutational techniques.
Similarly, reference to decreasing the action (activity) of a FAS protein refers to any genetic modification in the microorganism or plant in question and/or in the recombinant nucleic acids containing the FAS-encoding DNA (including FAS regulatory regions or inhibitors thereof) with which the organism is transformed that results in decreased functionality of the enzymes and includes decreased activity of the enzymes (e.g., specific activity), increased inhibition or degradation of the enzymes and a reduction or elimination of expression of the enzyme. For example, the action of FAS of the present invention can be decreased by blocking or reducing the production of the protein, “knocking out” all or a portion of the gene encoding the protein, reducing FAS activity, or inhibiting the activity of FAS (any one, two or more of the biological activities of a FAS of the invention). Blocking or reducing the production of an enzyme can include placing the gene encoding the protein under the control of a heterologous promoter that requires the presence of an inducing compound in the growth medium. By establishing conditions such that the inducer becomes depleted from the medium, the expression of the gene encoding the protein (and therefore, of protein synthesis) could be turned off or on as desired. Blocking or reducing the activity of a protein could also include using an excision technology approach similar to that described in U.S. Pat. No. 4,743,546, incorporated herein by reference in its entirety. To use this approach, the gene encoding the protein of interest is cloned between specific genetic sequences that allow specific, controlled excision of the gene from the genome. Excision could be prompted by, for example, a shift in the cultivation temperature of the culture, as in U.S. Pat. No. 4,743,546, or by some other physical or nutritional signal. PCT Publication No. WO 02/083869, published Oct. 24, 2002, describes methods for inactivation of genes in Thraustochytriales, and is incorporated herein by reference in its entirety.
In one embodiment of the present invention, it is contemplated that a mutagenesis program could be combined with a selective screening process to obtain microorganisms of interest. The mutagenesis methods could include, but are not limited to: chemical mutagenesis, gene shuffling, switching regions of the genes encoding specific enzymatic domains, or mutagenesis restricted to specific regions of those genes, as well as other methods. For example, high throughput mutagenesis methods could be used to influence or optimize production of the desired fatty acids or other lipid products. Such methods could be combined with selective (i.e., targeted or directed) modification of the FAS by molecular biology techniques. For example, one could use selective modification techniques to modify a microorganism, for example, by introduction of a recombinant nucleic acid molecule encoding the FAS protein of the invention into any suitable host cell, including host cells comprising an endogenous FAS, and then use mutagenesis technologies to optimize fatty acid production and to create strains having improved fatty acid synthesis activity or to select for microorganisms with other improved or desired qualities. Screening methods are also useful for identifying other organisms having homologous FAS genes to the FAS of Schizochytrium. Homologous FAS genes identified in such organisms can be used in methods similar to those described herein.
In one embodiment of the present invention, a genetically modified microorganism or plant includes a microorganism or plant that has an enhanced ability to synthesize fatty acids in general or an enhanced ability to synthesize specific short chain fatty acids. According to the present invention, “an enhanced ability to synthesize” a product refers to any enhancement, or up-regulation, in a pathway related to the synthesis of the product such that the microorganism or plant produces an increased amount of the product compared to the wild-type microorganism or plant, cultured or grown, under the same conditions. In one embodiment of the present invention, enhancement of the ability of a microorganism or plant to synthesize short chain fatty acids is accomplished by amplification of the expression of the FAS gene. Amplification of the expression of FAS can be accomplished in any suitable host cell (e.g., a Thraustochytriales cell, a bacterial cell, a yeast cell, a plant cell), for example, by introduction of a recombinant nucleic acid molecule encoding the FAS gene, or by modifying regulatory control over a native FAS gene, in the case of Thraustochytriales.
According to the present invention, “selective modification” of an organism or nucleic acid molecule refers to a targeted, or directed, modification, where the modification to be made is predetermined and designed, for example, by knowledge of the gene structure of the FAS of the present invention. For example, selective modification of an organism can be achieved by introduction (e.g., overexpression) of a recombinant nucleic acid molecule encoding a FAS protein, or by targeted modification of an endogenous gene, such as by homologous recombination. Selective modification is distinguished from random mutagenesis techniques, where in the latter process, the mutation is randomly generated by a non-target-specific method and the desired phenotype is subsequently selected through screening of mutants for the phenotype. Selective modification techniques and classical random mutagenesis and screening techniques can be combined in the present invention to produce a variety of genetically modified organisms.
Therefore, it is an embodiment of the present invention to provide a microorganism or plant that is transformed with a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding FAS of the present invention. Preferred recombinant nucleic acid molecules comprising such a nucleic acid sequence include recombinant nucleic acid molecules comprising any of the FAS nucleic acid sequences previously described herein. It is one embodiment of the present invention to provide a microorganism or plant which is transformed with a genetically modified recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a mutant, or homologue, FAS. Protein homologues have been described in detail herein.
It is another embodiment of the present invention to provide a genetically modified microorganism for producing a fatty acid by a biosynthetic process, wherein the microorganism comprises a nucleic acid molecule encoding a FAS protein of the present invention and wherein the nucleic acid molecule encoding the FAS protein has been modified to increase the expression or biological activity of the FAS. The FAS can be any FAS described herein, including homologues and biologically active fragments as described herein. In one aspect of the invention, the microorganism has an endogenous FAS (e.g., a member of Thraustochytriales), and the endogenous gene is modified to increase the expression or activity of the FAS (e.g., by introducing a promoter that gives higher levels of expression than that of the native promoter, by genetically mutating the endogenous gene to increase the activity of the enzyme, etc.). In another embodiment, the microorganism is genetically modified by transformation with a recombinant nucleic acid molecule encoding a FAS of the invention. Such a microorganism can be any suitable host microorganism and in one embodiment, is a Thraustochytriales microorganism (e.g., a Schizochytrium), such that the microorganism comprises both an endogenous FAS and a recombinant FAS. The FAS proteins in this scenario need not be identical, since one or both of the endogenous and recombinant FAS proteins can be modified as compared to a wild-type Schizochytrium FAS disclosed herein to produce a FAS homologue. For example, one or both of the endogenous or recombinant FAS-encoding nucleic acid molecules can be modified to increase the expression or activity of the FAS proteins.
Accordingly, one embodiment of the invention is a biomass comprising any of the microorganisms described herein comprising a nucleic acid molecule encoding a FAS of the present invention that has been modified to increase the expression or biological activity of the FAS as described above. As used herein, a biomass refers to a population of microbial cells that have been harvested from a fermentation or culture process. Various fermentation parameters for inoculating, growing and recovering microfloral biomasses are discussed in detail in U.S. Pat. No. 5,130,242, incorporated herein by reference in its entirety. The biomass harvested from a fermentation run can be dried (e.g., spray drying, tunnel drying, vacuum drying, or a similar process) and used in any food, pharmaceutical or other desired product. Alternatively, the harvested and washed biomass can be used directly (without drying) in various products. To extend its shelf life, the wet biomass can be acidified (approximate pH=3.5-4.5) and/or pasteurized or flash heated to inactivate enzymes and then canned, bottled or packaged under a vacuum or non-oxidizing atmosphere (e.g., N2 or CO2).
One embodiment of the present invention is a method to produce a short chain fatty acid by a biosynthetic process, comprising culturing in a fermentation medium a genetically modified microorganism that has increased expression or biological activity of a FAS protein as described above. For example, the microorganism can have increased expression or biological activity of any FAS proteins described herein, including homologues and enzymatically active portions thereof. The FAS protein can be an endogenous FAS protein and/or a recombinant FAS protein according to the invention. The microorganism is cultured or grown in a suitable medium, under conditions effective to produce the desired fatty acid or other lipid product. An appropriate, or effective, medium refers to any medium in which a genetically modified microorganism of the present invention, when cultured, is capable of producing the desired product. Such a medium is typically an aqueous medium comprising assimilable carbon, nitrogen and phosphate sources. Such a medium can also include appropriate salts, minerals, metals and other nutrients. Microorganisms of the present invention can be cultured in conventional fermentation bioreactors. The microorganisms can be cultured by any fermentation process which includes, but is not limited to, batch, fed-batch, cell recycle, and continuous fermentation. Preferred growth conditions for potential host microorganisms according to the present invention are well known in the art. The desired products produced by the genetically modified microorganism can be recovered from the fermentation medium using conventional separation and purification techniques. For example, the fermentation medium can be filtered or centrifuged to remove microorganisms, cell debris and other particulate matter, and the product can be recovered from the cell-free supernatant by conventional methods, such as, for example, ion exchange, chromatography, extraction, solvent extraction, membrane separation, electrodialysis, reverse osmosis, distillation, chemical derivatization and crystallization. Alternatively, microorganisms producing the desired product, or extracts and various fractions thereof, can be used without removal of the microorganism components from the product, such as in a biomass of the invention.
One embodiment of the present invention is a method to produce short chain fatty acids by growing or culturing a genetically modified plant of the present invention as previously described herein. Such a method includes the step of culturing in a fermentation medium or growing in a suitable environment, such as soil, a plant having a genetic modification to increase the action of FAS. Preferably, the genetic modification includes transformation or transfection of the plant with a recombinant nucleic acid molecule that expresses a protein having FAS biological activity. Such a protein can include any of the FAS proteins described herein, including any homologue of a naturally occurring FAS having biological activity.
In the method for production of short chain fatty acids of the present invention, a plant that has a genetic modification to increase the action of FAS is cultured in a fermentation medium or grown in a suitable medium such as soil for production of the FAS. An appropriate, or effective, fermentation medium has been discussed in detail above. A suitable growth medium for higher plants includes any growth medium for plants, including, but not limited to, soil, sand, any other particulate media that support root growth (e.g. vermiculite, perlite, etc.) or Hydroponic culture, as well as suitable light, water and nutritional supplements which optimize the growth of the higher plant. The genetically modified plants of the present invention are engineered to produce significant quantities of short chain fatty acids through increased action of the FAS protein of the present invention. The short chain fatty acids can be recovered through purification processes which extract these products from the plant. In a preferred embodiment, the fatty acids are recovered by harvesting the plant or plant fraction (e.g., oils). In this embodiment, the plant or plant fraction can be consumed in its natural state or further processed into consumable products.
Another embodiment of the invention relates to a genetically modified microorganism with a reduced ability to produce short chain fatty acids (resulting in reduced amounts of short chain fatty acid production in the microorganism), wherein the microorganism has been genetically modified to selectively delete, but more preferably attenuate (inhibit, reduce the expression or activity of, etc., without actually deleting or completely inactivating the gene or its protein product) a FAS gene or portion thereof encoding a functional domain. In a preferred embodiment, the microorganism is a microalga, and in a more preferred embodiment, is a Thraustochytriales microorganism (e.g., a Schizochytrium). The FAS gene can be modified by modification to the coding region of the FAS gene or to a regulatory region of the FAS gene, such that expression and/or biological activity of the FAS gene is reduced, and preferably inhibited so that the microorganism has reduced production of short chain fatty acids and more preferably, increased production of long chain fatty acids, and particularly, of polyunsaturated fatty acids (PUFAs). In one embodiment the FAS gene is partially or completely deleted or inactivated, including by replacing the gene with a non-FAS nucleic acid sequence, such as by gene disruption through homologous recombination. In this aspect, the FAS gene is mutated or inactivated (or deleted) by targeted homologous recombination with a nucleic acid sequence that hybridizes to the FAS gene that includes a heterologous nucleic acid sequence that disrupts the coding region of the FAS gene.
Production of a microorganism that has reduced FAS expression or activity has commercial benefits, as described above. Microorganisms that contain the FAS of the present invention include members of Thraustochytriales, which are known to be valuable organisms for the production of lipids containing high levels of polyunsaturated fatty acids (PUFAs), including highly unsaturated fatty acids such as omega-3 fatty acids. Polyunsaturated fatty acids (PUFAs) are critical components of membrane lipids in most eukaryotes (Lauritzen et al., Prog. Lipid Res. 40 1 (2001); McConn et al., Plant J. 15, 521 (1998)) and are precursors of certain hormones and signaling molecules (Heller et al., Drugs 55, 487 (1998); Creelman et al., Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 355 (1997)). According to the present invention, a preferred PUFA is a long chain PUFA, which is defined as a PUFA having eighteen carbons or more. PUFAs include any omega-3 or omega-6 polyunsaturated fatty acids with three or more double bonds. Omega-3 PUFAs are polyethylenic fatty acids in which the ultimate ethylenic bond is three carbons from and including the terminal methyl group of the fatty acid and include, for example, docosahexaenoic acid C22:6(n-3) (DHA) and omega-3 docosapentaenoic acid C22:5(n-3) (DPAn-3). Omega-6 PUFAs are polyethylenic fatty acids in which the ultimate ethylenic bond is six carbons from and including the terminal methyl group of the fatty acid and include, for example, arachidonic acid C20:4(n-6) (ARA), C22:4(n-6), omega-6 docosapentaenoic acid C22:5(n-6) (DPAn-6) and dihomogammalinolenic acid C20:3(n-6)(dihomo GLA). Members of Thraustochytriales, such as Schizochytrium, accumulate large quantities of triacylglycerols rich in PUFAs. Since these lipid products are useful in a variety of food and other commercial products, it would be useful to enhance the ability of microorganisms to preferentially produce the PUFAs. The present invention provides one method by which this goal can be achieved (i.e., by attenuation of the competing FAS system).
Accordingly, another embodiment of the invention relates to a biomass comprising genetically modified microorganism (e.g., a microorganism of the order Thraustochytriales (e.g., Schizochytrium, Thraustochytrium)) that have reduced short chain fatty acid synthesis and more preferably, increased PUFA synthesis, as compared to a wild-type microorganism of the same species, as described above. It is to be understood that organisms other than Thraustochytriales may be discovered which contain a FAS protein having homology and most or all of the biological activities of the full length FAS described herein. Such microorganisms can also be modified to reduce the expression or activity of the FAS system, particularly if such microorganisms are also useful for producing PUFAs.
Fatty acids produced in accordance with the methods of the present invention are typically produced as lipids. As used herein, the term “lipid” includes phospholipids (PL); free fatty acids; esters of fatty acids; triacylglycerols (TAG); diacylglycerides; phosphatides; sterols and sterol esters; carotenoids; xanthophylls (e.g., oxycarotenoids); hydrocarbons; and other lipids known to one of ordinary skill in the art. The term “fatty acid” (including as the term is used in “polyunsaturated fatty acid” and “PUFA”) includes not only the free fatty acid form, but other forms as well, such as the triacyglycerol (TAG) form and the phospholipids (PL) form.
A food product, as used herein, includes any food ingredient (e.g., a food product that is part of another food product, such as an oil), and also includes, but is not limited to: fine bakery wares, bread and rolls, breakfast cereals, processed and unprocessed cheese, condiments (ketchup, mayonnaise, etc.), dairy products (milk, yoghurt), puddings and gelatine desserts, carbonated drinks, teas, powdered beverage mixes, processed fish products, fruit-based drinks, chewing gum, hard confectionery, frozen dairy products, processed meat products, nut and nut-based spreads, pasta, processed poultry products, gravies and sauces, potato chips and other chips or crisps, chocolate and other confectionery, soups and soup mixes, soya based products (milks, drinks, creams, whiteners), vegetable oil-based spreads, and vegetable-based drinks. Other products include dietary supplements, a pharmaceutical formulation (e.g., a pharmaceutical product), humanized animal milk, and infant formulas. Suitable pharmaceutical formulations or products include, but are not limited to, an anti-inflammatory formulation, a chemotherapeutic agent, an active excipient, an osteoporosis drug, an anti-depressant, an anti-convulsant, an anti-Heliobactor pylori drug, a drug for treatment of neurodegenerative disease, a drug for treatment of degenerative liver disease, an antibiotic, a cholesterol lowering formulation, and products used to treat a condition selected from the group consisting of: chronic inflammation, acute inflammation, gastrointestinal disorder, cancer, cachexia, cardiac restenosis, neurodegenerative disorder, degenerative disorder of the liver, blood lipid disorder, osteoporosis, osteoarthritis, autoimmune disease, preeclampsia, preterm birth, age related maculopathy, pulmonary disorder, and peroxisomal disorder.
Therefore, another embodiment of the present invention relates to a method for producing lipids, and preferably PUFAs, from a biosynthetic process, comprising culturing under conditions effective to produce the lipids genetically modified microorganisms (e.g., of the order Thraustochytriales) as previously described herein, wherein the microorganisms have been genetically modified to selectively increase or decrease (depending on the goal) a FAS gene as described above. The lipids can be recovered using any one of a variety of recovery techniques known in the art or the entire microorganism or extracts thereof can be recovered. One aspect of the invention relates to a method for recovering lipids from a biosynthetic process, comprising recovering lipids from a culture of genetically modified microorganisms (e.g., of the order Thraustochytriales), wherein the microorganisms have been genetically modified to selectively increase or decrease a FAS gene as described above. Techniques for recovery of lipids from the culture are known in the art and include, but are not limited to, ion exchange, chromatography, extraction, solvent extraction, phase separation, membrane separation, electrodialysis, reverse osmosis, distillation, chemical derivatization and crystallization.
Another embodiment of the present invention is a method for producing short chain fatty acids using an isolated FAS, including a homologue of a FAS as described herein. The method can be operated in batch or continuous mode using a stirred tank, a plug-flow column reactor or other apparatus known to those skilled in the art.
In one embodiment, the FAS is bound to a solid support, i.e., an immobilized enzyme. As used herein, a FAS bound to a solid support (i.e., an immobilized FAS) includes immobilized isolated FAS, immobilized cells which contain a FAS (including immobilized and genetically modified Thraustochytriales, bacterial, fungal (e.g., yeast), microalgal, or plant cells), stabilized intact cells and stabilized cell/membrane homogenates. Stabilized intact cells and stabilized cell/membrane homogenates include cells and homogenates from naturally occurring microorganisms expressing FAS or from genetically modified microorganisms or plants as disclosed elsewhere herein. Thus, although methods for immobilizing FAS are discussed below, it will be appreciated that such methods are equally applicable to immobilizing cells and in such an embodiment, the cells can be lysed.
A variety of methods for immobilizing an enzyme are disclosed in Industrial Enzymology 2nd Ed., Godfrey, T. and West, S. Eds., Stockton Press, New York, N.Y., 1996, pp. 267-272; Immobilized Enzymes, Chibata, I. Ed., Halsted Press, New York, N.Y., 1978; Enzymes and Immobilized Cells in Biotechnology, Laskin, A. Ed., Benjamin/Cummings Publishing Co., Inc., Menlo Park, Calif., 1985; and Applied Biochemistry and Bioengineering, Vol. 4, Chibata, I. and Wingard, Jr., L. Eds, Academic Press, New York, N.Y., 1983, which are incorporated herein in their entirety.
Briefly, a solid support refers to any solid organic supports, artificial membranes, biopolymer supports, or inorganic supports that can form a bond with FAS (or cell) without significantly affecting the activity of isolated FAS. Exemplary organic solid supports include polymers such as polystyrene, nylon, phenol-formaldehyde resins, acrylic copolymers (e.g., polyacrylamide), stabilized intact whole cells, and stabilized crude whole cell/membrane homogenates. Exemplary biopolymer supports include cellulose, polydextrans (e.g., Sephadex®), agarose, collagen and chitin. Exemplary inorganic supports include glass beads (porous and nonporous), stainless steel, metal oxides (e.g., porous ceramics such as ZrO2, TiO2, Al2O3, and NiO) and sand. Preferably, the solid support is selected from the group consisting of stabilized intact cells and/or crude cell homogenates. Preparation of such supports requires a minimum of handling and cost. Additionally, such supports provide excellent stability of the enzyme.
Stabilized intact cells and/or cell/membrane homogenates can be produced, for example, by using bifunctional crosslinkers (e.g., glutaraldehyde) to stabilize cells and cell homogenates. In both the intact cells and the cell membranes, the cell wall and membranes act as immobilizing supports. In such a system, integral membrane proteins are in the “best” lipid membrane environment. Whether starting with intact cells or homogenates, in this system the cells are either no longer “alive” or “metabolizing”, or alternatively, are “resting” (i.e., the cells maintain metabolic potential and active FAS, but under the culture conditions are not growing); in either case, the immobilized cells or membranes serve as biocatalysts.
FAS can be bound to a solid support by a variety of methods including adsorption, cross-linking (including covalent bonding), and entrapment. Adsorption can be through van der Waal's forces, hydrogen bonding, ionic bonding, or hydrophobic binding. Exemplary solid supports for adsorption immobilization include polymeric adsorbents and ion-exchange resins. Solid supports in a bead form are particularly well-suited. The particle size of an adsorption solid support can be selected such that the immobilized enzyme is retained in the reactor by a mesh filter while the substrate (e.g., the precursor or substrate used as a starting material to produce the desired fatty acid) is allowed to flow through the reactor at a desired rate. With porous particulate supports it is possible to control the adsorption process to allow FAS or genetically modified microorganism cells to be embedded within the cavity of the particle, thus providing protection without an unacceptable loss of activity.
Cross-linking of a FAS to a solid support involves forming a chemical bond between a solid support and a FAS. It will be appreciated that although cross-linking generally involves linking a FAS to a solid support using an intermediary compound, it is also possible to achieve a covalent bonding between the enzyme and the solid support directly without the use of an intermediary compound. Cross-linking commonly uses a bifunctional or multifunctional reagent to activate and attach a carboxyl group, amino group, sulfur group, hydroxy group or other functional group of the enzyme to the solid support. The term “activate” refers to a chemical transformation of a functional group which allows a formation of a bond at the functional group. Exemplary amino group activating reagents include water-soluble carbodiimides, glutaraldehyde, cyanogen bromide, N-hydroxysuccinimide esters, triazines, cyanuric chloride, and carbonyl diimidazole. Exemplary carboxyl group activating reagents include water-soluble carbodiimides, and N-ethyl-5-phenylisoxazolium-3-sulfonate. Exemplary tyrosyl group activating reagents include diazonium compounds. And exemplary sulfhydryl group activating reagents include dithiobis-5,5′-(2-nitrobenzoic acid), and glutathione-2-pyridyl disulfide. Systems for covalently linking an enzyme directly to a solid support include Eupergit®, a polymethacrylate bead support available from Rohm Pharma (Darmstadt, Germany), kieselguhl (Macrosorbs), available from Sterling Organics, kaolinite available from English China Clay as “Biofix” supports, silica gels which can be activated by silanization, available from W. R. Grace, and high-density alumina, available from UOP (Des Plaines, Ill.).
Entrapment can also be used to immobilize FAS. Entrapment of FAS involves formation of, inter alia, gels (using organic or biological polymers), vesicles (including microencapsulation), semipermeable membranes or other matrices. Exemplary materials used for entrapment of an enzyme include collagen, gelatin, agar, cellulose triacetate, alginate, polyacrylamide, polystyrene, polyurethane, epoxy resins, carrageenan, and egg albumin. Some of the polymers, in particular cellulose triacetate, can be used to entrap the enzyme as they are spun into a fiber. Other materials such as polyacrylamide gels can be polymerized in solution to entrap the enzyme. Still other materials such as polyglycol oligomers that are functionalized with polymerizable vinyl end groups can entrap enzymes by forming a cross-linked polymer with UV light illumination in the presence of a photosensitizer.
The present invention also includes isolated (i.e., removed from their natural milieu) antibodies, or antigen binding fragments thereof, that are capable of selectively binding to a FAS protein of the present invention (e.g., FAS antibodies). The phrase “selectively binds” refers to the specific binding of one protein to another (e.g., an antibody, fragment thereof, or binding partner to an antigen), wherein the level of binding, as measured by any standard assay (e.g., an immunoassay), is statistically significantly higher than the background control for the assay. For example, when performing an immunoassay, controls typically include a reaction well/tube that contain antibody or antigen binding fragment alone (i.e., in the absence of antigen), wherein an amount of reactivity (e.g., non-specific binding to the well) by the antibody or antigen binding fragment thereof in the absence of the antigen is considered to be background. Binding can be measured using a variety of methods standard in the art including enzyme immunoassays (e.g., ELISA), immunoblot assays, etc.). Antibodies of the present invention can be polyclonal or monoclonal, functional equivalents such as antibody fragments and genetically-engineered antibodies, including single chain antibodies or chimeric antibodies, including bi-specific antibodies that can bind to more than one epitope.
Generally, in the production of an antibody, a suitable experimental animal, such as, for example, but not limited to, a rabbit, a sheep, a hamster, a guinea pig, a mouse, a rat, or a chicken, is exposed to an antigen against which an antibody is desired. Typically, an animal is immunized with an effective amount of antigen that is injected into the animal. An effective amount of antigen refers to an amount needed to induce antibody production by the animal. The animal's immune system is then allowed to respond over a pre-determined period of time. The immunization process can be repeated until the immune system is found to be producing antibodies to the antigen. In order to obtain polyclonal antibodies specific for the antigen, serum is collected from the animal that contains the desired antibodies (or in the case of a chicken, antibody can be collected from the eggs). Such serum is useful as a reagent. Polyclonal antibodies can be further purified from the serum (or eggs) by, for example, treating the serum with ammonium sulfate.
Monoclonal antibodies may be produced according to the methodology of Kohler and Milstein (Nature 256:495-497, 1975). For example, B lymphocytes are recovered from the spleen (or any suitable tissue) of an immunized animal and then fused with myeloma cells to obtain a population of hybridoma cells capable of continual growth in suitable culture medium. Hybridomas producing the desired antibody are selected by testing the ability of the antibody produced by the hybridoma to bind to the desired antigen.
Genetically engineered antibodies of the invention include those produced by standard recombinant DNA techniques involving the manipulation and re-expression of DNA encoding antibody variable and/or constant regions. Particular examples include, chimeric antibodies, where the VH and/or VL domains of the antibody come from a different source to the remainder of the antibody, and CDR grafted antibodies (and antigen binding fragments thereof), in which at least one CDR sequence and optionally at least one variable region framework amino acid is (are) derived from one source and the remaining portions of the variable and the constant regions (as appropriate) are derived from a different source. Construction of chimeric and CDR-grafted antibodies are described, for example, in European Patent Applications: EP-A 0194276, EP-A 0239400, EP-A 0451216 and EP-A 0460617.
The following examples are provided for the purpose of illustration and are not intended to limit the scope of the present invention.
EXAMPLES
Example 1
The following example describes the cloning and characterization of the Schizochytrium FAS proteins of the invention.
Briefly, a cDNA library was prepared from mRNA isolated from Schizochytrium cells as described in Metz et al., Science 293, pp 290-292 (2001). Approximately 8,500 clones were randomly chosen and sequenced from the 5′ end using a vector-derived primer. A search of this database using various FAS proteins as queries revealed the presence of many homologues. When the database was queried using the yeast FAS subunits (a and b), over 40 sequences were found with significant homology. Most of these Schizochytrium sequences could be assembled into a single large ( ̃4000 bp) contig. When individual cDNA sequences were used as queries in a BLAST search (tblastn) the best matches were, in most cases, to the FAS subunits of fungal organisms. In these organisms the Type I FAS is composed of two subunits (a and b), each carrying a distinct set of enzymatic domains. One anomaly of the cDNA sequence data was that all but one of the matches were to the “a” subunit. Using the sequence tags as a guide, the inventors cloned the corresponding regions of genomic DNA encoding those cDNA-derived sequences. As the project proceeded, the reason for the anomalous representation of the subunits in the cDNA library became clear. The FAS in Schizochytrium is encoded by a single large gene. The FAS Orf contains 12,408 bp and encodes a protein with a deduced molecular mass of 444,884 Daltons. No evidence for introns could be found, either by analysis of the genomic sequence itself or by comparison to available cDNA sequences. Blast and Pfam results plus motif analyses were used to establish a preliminary structure and functional identification of the domains of this Type I protein. Both in terms of amino acid sequence and in the sequential organization, the Schizochytrium FAS resembles a fusion of the head (N-terminus) of the fungal FAS a subunit to the tail (C-terminus) of the b subunit.
The DNA fragments containing the Schizochytrium FAS open reading frame were cloned from a lambda library of genomic DNA. Standard methods were used to produce that library as well as PCR derived probes (based on sequences obtained from cDNA clones) for isolation of the respective DNA fragments. The Orf encoding the FAS gene was sequenced using a combination of subcloning and primer walking.
The complete Schizochytrium FAS-encoding sequence is a 12,408 nucleotide sequence (not including the stop codon), represented herein by SEQ ID NO:1, which encodes a 4136 amino acid sequence, represented herein as SEQ ID NO:2. Within the Schizochytrium FAS protein are nine domains as described above: (a) one acetyl-transferase (AT) domain (SEQ ID NO:5); (b) one enoyl ACP reductase (ER) domain (SEQ ID NO:6); (c) one dehydrase (DH) domain (SEQ ID NO:7); (d) one malonyl/palmitoyl acyltransferase (M/PAT) domain (SEQ ID NO:8); (e) two acyl carrier protein (ACP) domains (SEQ ID NO:9 and (SEQ ID NO:10); (f) one keto-acyl ACP reductase (KR) domain (SEQ ID NO:11); (g) one keto-acyl ACP synthase (KS) domain (SEQ ID NO:12); and (h) one phosphopantetheinyl transferase (PPT) domain (SEQ ID NO:13).
FIG. 1 is a schematic representation of the putative enzymatic domains present in the Schizochytrium FAS protein based on the identification, cloning and sequencing of this protein by the present inventors, followed by analysis of the sequences. The size and positions of the putative domains relative to the entire protein, and the identification of the functions of those domains, are based on Pfam analyses results and on homology to the well-characterized FAS (α and β subunits) of baker's yeast (Saccharomyces cerevisiae) (references 1, 2 & 3). The domain organization and sizes relative to the overall protein sizes of the yeast FAS (α and β subunits) are shown for comparison.
Abbreviations: (AT) acetyl-transferase; (ER) enoyl ACP reductase; (DH) dehydrase (DH); (M/PAT) malonyl/palmitoyl acyltransferase; (ACP #1) the first acyl carrier protein; (ACP #2) the second acyl carrier protein; (R) keto-acyl ACP reductase; (KS) keto-acyl ACP synthase; and (PPT) phosphopantetheinyl transferase.
REFERENCES
1. Mohamed et al., J Biol Chem. 1988, Sep. 5; 263(25):12315-25.
2. Chirala et al., J Biol Chem. 1987, Mar. 25; 262(9):4231-40.
3. Fichtlscherer et al., Eur J Biochem. 2000, May; 267(9):2666-71.
Example 2
The following example describes the targeted inactivation of the Schizochytrium FAS gene.
The Schizochytrium FAS gene was inactivated by targeted inactivation, using technology that is described in PCT Publication No. WO 02/083869, published Oct. 24, 2002. PCT Publication No. WO 02/083869 is incorporated herein by reference in its entirety.
FIG. 2A diagrams the construction of a plasmid used for targeted inactivation of the Schizochytrium FAS gene. The straight line represents the region of the Schizochytrium chromosome containing the FAS gene. The arrow above the line indicates the position of the open reading frame (Orf) that encodes the FAS protein. The boxed area on the line (both striped and solid) represents the portion of the chromosome cloned into the plasmid; JK870. Digestion of JK870 with the restriction enzyme BglII, followed by dilution and ligation, resulted in recovery of plasmid JK876, in which the BglII fragment (solid box) has been deleted. A fragment of DNA containing the ble gene (encoding a protein that confers resistance to ZeocinTM) along with the α-tubulin promoter region and a SV40 terminator region (see below), isolated from the plasmid pTUBZEO11-2, by digestion with BamHI, is then cloned into the BglII site of JK876 to generate the plasmid JK874.
FIG. 2B diagrams the events that are believed to occur and result in the stable inactivation of the FAS gene in Schizochytrium. The plasmid JK874 is introduced into whole cells of Schizochytrium (either the 20888 strain, or the cell wall deficient strain—AC66) via particle bombardment (as described in PCT Publication No. WO 02/083869, ibid.). The initial selection for transformed cells (i.e., having the ZeocinTM resistance gene from plasmid JK874 introduced into the chromosome in a functional manner) was made on agar plates with growth medium containing ZeocinTM and supplemented with free saturated fatty acids (i.e., C16:0) solubilized with β-cyclodextrin. Colonies that grow under these selective conditions were transferred to a new plate in a grid-like array. The transformants were then tested for their ability to grow without fatty acid supplementation by transferring cells from the colonies to a plate containing ZeocinTM but lacking the fatty acid. The colonies that are ZeocinTM resistant and require supplementation with 16:0 fatty acids for growth were presumed to have had the FAS gene inactivated via a homologous recombination event. Those colonies that did not show reversion to 16:0 prototrophy were presumed to have integrated the ZeocinTM resistance gene cassette into the FAS region via a double crossover event as diagramed. The stability of the phenotype (requirement for supplementation with 16:0 fatty acid for growth) indicates a portion of the FAS Orf has been replaced with the DNA conferring ZeocinTM resistance. The integration into the FAS portion of the chromosome was then confirmed via a PCR reaction as diagramed. In this manner, FAS knock-outs in both Schizochytrium strains ATCC 20888 and AC66 were obtained.
Example 3
The following example describes in vitro fatty acid synthesis assays in strains in which the Schizochytrium FAS system of the invention has been inactivated.
FIG. 3 shows the synthesis of fatty acids from [1-14C]-malonyl-CoA in cell free homogenates from a cell wall deficient strain of Schizochytrium (AC66) and mutants of that strain in which either the PUFA polyketide synthase orfC gene (PUFA-KO) or the FAS gene (FAS-KO) (see FIGS. 2A and 2B above) have been inactivated. Cells were disrupted by sonication in 100 mM phosphate buffer, pH 7.2 containing 2 mM DTT, 1 mM EDTA and 10% glycerol. Aliquots of the homogenates were supplemented with 10 μM acetyl-CoA, 100 μM [1-14C]-malonyl-CoA, 2 mM NADH and 2 mM NADPH and incubated for 30 min at 30° C. Fatty acids in the reaction mixture were converted to methyl esters, extracted into hexane and separated by AgNO3 thin layer chromatography (TLC) (solvent: hexane/diethyl ether/HOAc 70/30/2 by vol). Radioactivity on the TLC plates was detected using a Molecular Dynamics phosphorimaging system. Migration distances of fatty acid methyl ester standards (16:0, 18:1, DPA n-6, and DHA) are indicated to the right side of the diagram.
Assays of the homogenate of the AC66 parent strain reveal the presence of radiolabeled bands that co-migrate with the 16:0-, 18:1- and DHA-methyl ester standards. In the PUFA-KO mutant strain, the radiolabeled band that co-migrates with the DHA methyl ester standard is greatly reduced (or lacking). In the FAS-KO strain, radiolabeled bands that co-migrate with the 16:0- and 18:1-methyl ester standards (as well as another band) are greatly reduced (or lacking). These data indicate that inactivation of the FAS gene results in the loss of the ability to synthesize short chain saturated fatty acids and suggest that 18:1 is derived from products of the FAS. The data also show that synthesis of DHA and DPA in Schizochytrium is not dependent on products of the FAS system.
Each publication cited herein is incorporated herein by reference in its entirety.
While various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art. It is to be expressly understood, however, that such modifications and adaptations are within the scope of the present invention, as set forth in the following claims.

## Claims

1. A genetically modified Thraustochytriales microorganism for producing short chain fatty acids by a biosynthetic process, wherein the microorganism comprises a nucleic acid molecule encoding a fatty acid synthase and wherein the nucleic acid molecule encoding the fatty acid synthase has been modified to increase the expression of the fatty acid synthase, the fatty acid synthase comprising an amino acid sequence selected from the group consisting of:
a) an amino acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, an amino acid sequence consisting of positions 1-500 of SEQ ID NO:2, an amino acid sequence consisting of positions 450-1300 of SEQ ID NO:2, an amino acid sequence consisting of positions 1250-1700 of SEQ ID NO:2, an amino acid sequence consisting of positions 1575-2100 of SEQ ID NO:2, an amino acid sequence consisting of positions 2025-2850 of SEQ ID NO:2, an amino acid sequence consisting of positions 2800-3350 of SEQ ID NO:2, an amino acid sequence consisting of positions 3300-3900 of SEQ ID NO:2, an amino acid sequence consisting of positions 3900-4136 of SEQ ID NO:2, and a biologically active fragment thereof wherein the biologically active fragment is selected from the group of consisting of acetyl-transferase (AT) activity; enoyl ACP reductase (ER) activity; dehydrase (DH) activity; malonyl/palmitoyl acyltransferase (M/PAT) activity; a first acyl carrier protein (ACP) activity; a second acyl carrier protein (ACP) activity; keto-acyl ACP reductase (KR) activity; keto-acyl ACP synthase (KS) activity; and phosphopantetheinyl transferase (PPT) activity; and
b) an amino acid sequence that is at least 85% identical to any of the amino acid sequences of (a) and having the biological activity of the amino acid sequence of (a).

2. The genetically modified microorganism of claim 1, wherein the nucleic acid molecule encoding the fatty acid synthase is an endogenous gene in the microorganism.

3. The genetically modified microorganism of claim 1, wherein the microorganism has been transformed with the nucleic acid molecule encoding the fatty acid synthase.

4. The genetically modified microorganism of claim 1, wherein the microorganism comprises an endogenous gene encoding the fatty acid synthase and has been transformed with a recombinant nucleic acid molecule encoding the fatty acid synthase, wherein the gene, and the recombinant nucleic acid molecule, or both have been modified to increase the expression of the fatty acid synthase.

5. A biomass comprising the genetically modified microorganism of claim 1.

6. A food product or a pharmaceutical product comprising the biomass of claim 5.

7. A method to produce short chain fatty acids by a biosynthetic process, comprising culturing in a fermentation medium the genetically modified microorganism according to claim 1.

8. A genetically modified Thraustochytriales microorganism for producing short chain fatty acids by a biosynthetic process, wherein the microorganism comprises multiple copies of a nucleic acid molecule encoding a fatty acid synthase, wherein the nucleic acid molecule encoding the fatty acid synthase has been modified to increase the expression of the fatty acid synthase, and wherein the fatty acid synthase comprises an amino acid sequence selected from the group consisting of:
a) an amino acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, an amino acid sequence consisting of positions 1-500 of SEQ ID NO:2, an amino acid sequence consisting of positions 450-1300 of SEQ ID NO:2, an amino acid sequence consisting of positions 1250-1700 of SEQ ID NO:2, an amino acid sequence consisting of positions 1575-2100 of SEQ ID NO:2, an amino acid sequence consisting of positions 2025-2850 of SEQ ID NO:2, an amino acid sequence consisting of positions 2800-3350 of SEQ ID NO:2, an amino acid sequence consisting of positions 3300-3900 of SEQ ID NO:2, an amino acid sequence consisting of positions 3900-4136 of SEQ ID NO:2, and a biologically active fragment thereof wherein the biologically active fragment is selected from the group of consisting of acetyl-transferase (AT) activity; enoyl ACP reductase (ER) activity; dehydrase (DH) activity; malonyl/palmitoyl acyltransferase (M/PAT) activity; a first acyl carrier protein (ACP) activity; a second acyl carrier protein (ACP) activity; keto-acyl ACP reductase (KR) activity; keto-acyl ACP synthase (KS) activity; and phosphopantetheinyl transferase (PPT) activity; and
b) an amino acid sequence that is at least 85% identical to any of the amino acid sequences of (a) and having the biological activity of the amino acid sequence of (a).

9. The genetically modified Thraustochytriales microorganism of claim 1, wherein the amino acid sequence is at least 90% identical to any of the amino acid sequences of (a) and having the biological activity of the amino acid sequence of (a).

10. The genetically modified Thraustochytriales microorganism of claim 1, wherein the amino acid sequence is at least 95% identical to any of the amino acid sequences of (a) and having the biological activity of the amino acid sequence of (a).

11. The genetically modified Thraustochytriales microorganism of claim 1, wherein the amino acid sequence is at least 96% identical to any of the amino acid sequences of (a) and having the biological activity of the amino acid sequence of (a).

12. The genetically modified Thraustochytriales microorganism of claim 1, wherein the amino acid sequence is at least 97% identical to any of the amino acid sequences of (a) and having the biological activity of the amino acid sequence of (a).

13. The genetically modified Thraustochytriales microorganism of claim 1, wherein the amino acid sequence is at least 98% identical to any of the amino acid sequences of (a) and having the biological activity of the amino acid sequence of (a).

14. The genetically modified Thraustochytriales microorganism of claim 1, wherein the amino acid sequence is at least 99% identical to any of the amino acid sequences of (a) and having the biological activity of the amino acid sequence of (a).

15. The genetically modified Thraustochytriales microorganism of claim 8, wherein the amino acid sequence is at least 90% identical to any of the amino acid sequences of (a) and having the biological activity of the amino acid sequence of (a).

16. The genetically modified Thraustochytriales microorganism of claim 8, wherein the amino acid sequence is at least 95% identical to any of the amino acid sequences of (a) and having the biological activity of the amino acid sequence of (a).

17. The genetically modified Thraustochytriales microorganism of claim 8, wherein the amino acid sequence is at least 96% identical to any of the amino acid sequences of (a) and having the biological activity of the amino acid sequence of (a).

18. The genetically modified Thraustochytriales microorganism of claim 8, wherein the amino acid sequence is at least 97% identical to any of the amino acid sequences of (a) and having the biological activity of the amino acid sequence of (a).

19. The genetically modified Thraustochytriales microorganism of claim 8, wherein the amino acid sequence is at least 98% identical to any of the amino acid sequences of (a) and having the biological activity of the amino acid sequence of (a).

20. The genetically modified Thraustochytriales microorganism of claim 8, wherein the amino acid sequence is at least 99% identical to any of the amino acid sequences of (a) and having the biological activity of the amino acid sequence of (a).