Patent Grant
10557114
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 5, 2014, is named SOLAP019AUS_SL.txt and is 579,821 bytes in size.
Certain organisms including plants and some microalgae use a type II fatty acid biosynthetic pathway, characterized by the use of discrete, monofunctional enzymes for fatty acid synthesis. In contrast, mammals and fungi use a single, large, multifunctional protein.
Type II fatty acid biosynthesis typically involves extension of a growing acyl-ACP (acyl-carrier protein) chain by two carbon units followed by cleavage by an acyl-ACP thioesterase. In plants, two main classes of acyl-ACP thioesterases have been identified: (i) those encoded by genes of the FatA class, which tend to hydrolyze oleoyl-ACP into oleate (an 18:1 fatty acid) and ACP, and (ii) those encoded by genes of the FatB class, which liberate C8-C16 fatty acids from corresponding acyl-ACP molecules.
Different FatB genes from various plants have specificities for different acyl chain lengths. As a result, different gene products will produce different fatty acid profiles in plant seeds. See, U.S. Pat. Nos. 5,850,022; 5,723,761; 5,639,790; 5,807,893; 5,455,167; 5,654,495; 5,512,482; 5,298,421; 5,667,997; and 5,344,771; 5,304,481. Recently, FatB genes have been cloned into oleaginous microalgae to produce triglycerides with altered fatty acid profiles. See, WO2010/063032, WO2011/150411, WO2012/106560, and WO2013/158938.
In various aspects, the invention(s) contemplated herein may include, but need not be limited to, any one or more of the following embodiments:
A nucleic acid construct including a regulatory element and a FatB gene expressing an active acyl-ACP thioesterase operable to produce an altered fatty acid profile in an oil produced by a cell expressing the nucleic acid construct, wherein the FatB gene expresses a protein having an amino acid sequence falling within clade 5 of Table 1a, the sequence having at least 94.6% sequence identity with each of SEQ ID NOs: 88, 82, 85, and 103, and optionally wherein the fatty acid of the oil is enriched in C8 and C10 fatty acids.
A nucleic acid construct including a regulatory element and a FatB gene expressing an active acyl-ACP thioesterase operable to produce an altered fatty acid profile in an oil produced by a cell expressing the nucleic acid construct, wherein the FatB gene expresses a protein having an amino acid sequence falling within one of clades 1-12 of Table 1a.
The nucleic acid construct of embodiment 2, wherein the FatB gene expresses a protein having an amino acid sequence falling within clade 1 of Table 1a, the sequence having at least 85.9% sequence identity with each of SEQ ID NOs: 19, 161, 22, and 160, and optionally wherein the fatty acid of the oil is enriched in C14 and C16 fatty acids.
The nucleic acid construct of embodiment 2, wherein the FatB gene expresses a protein having an amino acid sequence falling within clade 2 of Table 1a, the sequence having at least 89.5% sequence identity with each of SEQ ID NOs: 134-136, 132, 133, 137, 124, 122, 123, 125, and optionally wherein the fatty acid of the oil is enriched in C12 and C14 fatty acids.
The nucleic acid construct of embodiment 2, wherein the FatB gene expresses a protein having an amino acid sequence falling within clade 3 of Table 1a, the sequence having at least 92.5% sequence identity with each of SEQ ID NOs: 126 and 127, and optionally wherein the fatty acid of the oil is enriched in C12 and C14 fatty acids.
The nucleic acid construct of embodiment 2, wherein the FatB gene expresses a protein having an amino acid sequence falling within clade 4 of Table 1a, the sequence having at least 83.8% sequence identity with SEQ ID NO: 79, and optionally wherein the fatty acid of the oil is enriched in C12 and C14 fatty acids.
The nucleic acid construct of embodiment 2, wherein the FatB gene expresses a protein having an amino acid sequence falling within clade 6 of Table 1a, the sequence having at least 99.9% sequence identity with each of SEQ ID NOs: 111 and 110, and optionally wherein the fatty acid of the oil is enriched in C10 fatty acids.
The nucleic acid construct of embodiment 2, wherein the FatB gene expresses a protein having an amino acid sequence falling within clade 7 of Table 1a, the sequence having at least 89.5% sequence identity with each of SEQ ID NOs: 73, 106, 185, 172, 171, 173, 174, and optionally wherein the fatty acid of the oil is enriched in C10 and C12 fatty acids.
The nucleic acid construct of embodiment 2, wherein the FatB gene expresses a protein having an amino acid sequence falling within clade 8 of Table 1a, the sequence having at least 85.9% sequence identity with each of SEQ ID NOs: 112, 113, 142, 145, 143, 144, 139, 140, 138, 141, and optionally wherein the fatty acid of the oil is enriched in C12 and C14 fatty acids.
The nucleic acid construct of embodiment 2, wherein the FatB gene expresses a protein having an amino acid sequence falling within clade 9 of Table 1a, the sequence having at least 83.8% sequence identity with each of SEQ ID NOs: 187-189, and optionally wherein the fatty acid of the oil is enriched in C12 and C14 fatty acids.
The nucleic acid construct of embodiment 2, wherein the FatB gene expresses a protein having an amino acid sequence falling within clade 10 of Table 1a, the sequence having at least 95.9% sequence identity with each of SEQ ID NOs: 147, 149, 146, 150, 152, 151, 148, 154, 156, 155, 157, 108, 75, 190, 191, and 192, and optionally wherein the fatty acid of the oil is enriched in C14 and C16 fatty acids.
The nucleic acid construct of embodiment 2, wherein the FatB gene expresses a protein having an amino acid sequence falling within clade 11 of Table 1a, the sequence having at least 88.7% sequence identity with SEQ ID NO: 121, and optionally wherein the fatty acid of the oil is enriched in C14 and C16 fatty acids.
The nucleic acid construct of embodiment 2, wherein the FatB gene expresses a protein having an amino acid sequence falling within clade 12 of Table 1a, the sequence having at least 72.8% sequence identity with each of SEQ ID NOs: 129 and 186, and optionally wherein the fatty acid of the oil is enriched in C16 fatty acids.
An isolated nucleic acid or recombinant DNA construct including a nucleic acid, wherein the nucleic acid has at least 80% sequence identity to any of SEQ ID NOS: 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 65, 66, 68, 69, 71, 72, 74, 76, 78, 80, 81, 83, 84, 86, 87, 89, 90, 92, 93, 95, 96, 98, 99, 101, 102, 104, 105, 107, 109 or any equivalent sequences by virtue of the degeneracy of the genetic code.
An isolated nucleic acid sequence encoding a protein or a host cell expressing a protein having at least 80% sequence identity to any of SEQ ID NOS: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 75, 77, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 108, 110-192 or a fragment thereof having acyl-ACP thioesterase activity.
The isolated nucleic acid of embodiment 15, wherein, the protein has acyl-ACP thioesterase activity operable to alter the fatty acid profile of an oil produced by a recombinant cell including that sequence.
A method of producing a recombinant cell that produces an altered fatty acid profile, the method including transforming the cell with a nucleic acid according to any of embodiments 1-3.
A host cell produced by the method of embodiment 17.
The host cell of embodiment 18, wherein the host cell is selected from a plant cell, a microbial cell, and a microalgal cell.
A method for producing an oil or oil-derived product, the method including cultivating a host cell of embodiment 5 or 6, and extracting oil produced thereby, optionally wherein the cultivation is heterotrophic growth on sugar.
The method of embodiment 20, further including producing a fatty acid, fuel, chemical, or other oil-derived product from the oil.
An oil produced by the method of embodiment 20, optionally having a fatty acid profile including at least 20% C8, C10, C12, C14 or C16 fatty acids.
An oil-derived product produced by the method of embodiment 21.
The oil of embodiment 23, wherein the oil is produced by a microalgae and optionally, lacks C24-alpha sterols.
As used with respect to nucleic acids, the term “isolated” refers to a nucleic acid that is free of at least one other component that is typically present with the naturally occurring nucleic acid. Thus, a naturally occurring nucleic acid is isolated if it has been purified away from at least one other component that occurs naturally with the nucleic acid.
A “natural oil” or “natural fat” shall mean a predominantly triglyceride oil obtained from an organism, where the oil has not undergone blending with another natural or synthetic oil, or fractionation so as to substantially alter the fatty acid profile of the triglyceride. In connection with an oil comprising triglycerides of a particular regiospecificity, the natural oil or natural fat has not been subjected to interesterification or other synthetic process to obtain that regiospecific triglyceride profile, rather the regiospecificity is produced naturally, by a cell or population of cells. In connection with a natural oil or natural fat, and as used generally throughout the present disclosure, the terms oil and fat are used interchangeably, except where otherwise noted. Thus, an “oil” or a “fat” can be liquid, solid, or partially solid at room temperature, depending on the makeup of the substance and other conditions. Here, the term “fractionation” means removing material from the oil in a way that changes its fatty acid profile relative to the profile produced by the organism, however accomplished. The terms “natural oil” and “natural fat” encompass such oils obtained from an organism, where the oil has undergone minimal processing, including refining, bleaching and/or degumming, which does not substantially change its triglyceride profile. A natural oil can also be a “noninteresterified natural oil”, which means that the natural oil has not undergone a process in which fatty acids have been redistributed in their acyl linkages to glycerol and remain essentially in the same configuration as when recovered from the organism.
“Exogenous gene” shall mean a nucleic acid that codes for the expression of an RNA and/or protein that has been introduced into a cell (e.g. by transformation/transfection), and is also referred to as a “transgene”. A cell comprising an exogenous gene may be referred to as a recombinant cell, into which additional exogenous gene(s) may be introduced. The exogenous gene may be from a different species (and so heterologous), or from the same species (and so homologous), relative to the cell being transformed. Thus, an exogenous gene can include a homologous gene that occupies a different location in the genome of the cell or is under different control, relative to the endogenous copy of the gene. An exogenous gene may be present in more than one copy in the cell. An exogenous gene may be maintained in a cell, for example, as an insertion into the genome (nuclear or plastid) or as an episomal molecule.
“Fatty acids” shall mean free fatty acids, fatty acid salts, or fatty acyl moieties in a glycerolipid. It will be understood that fatty acyl groups of glycerolipids can be described in terms of the carboxylic acid or anion of a carboxylic acid that is produced when the triglyceride is hydrolyzed or saponified.
“Microalgae” are microbial organisms that contain a chloroplast or other plastid, and optionally that are capable of performing photosynthesis, or a prokaryotic microbial organism capable of performing photosynthesis. Microalgae include obligate photoautotrophs, which cannot metabolize a fixed carbon source as energy, as well as heterotrophs, which can live solely off of a fixed carbon source. Microalgae include unicellular organisms that separate from sister cells shortly after cell division, such as Chlamydomonas, as well as microbes such as, for example, Volvox, which is a simple multicellular photosynthetic microbe of two distinct cell types. Microalgae include cells such as Chlorella, Dunaliella, and Prototheca. Microalgae also include other microbial photosynthetic organisms that exhibit cell-cell adhesion, such as Agmenellum, Anabaena, and Pyrobotrys. Microalgae also include obligate heterotrophic microorganisms that have lost the ability to perform photosynthesis, such as certain dinoflagellate algae species and species of the genus Prototheca.
An “oleaginous” cell is a cell capable of producing at least 20% lipid by dry cell weight, naturally or through recombinant or classical strain improvement. An “oleaginous microbe” or “oleaginous microorganism” is a microbe, including a microalga that is oleaginous.
The term “percent sequence identity,” in the context of two or more amino acid or nucleic acid sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. For sequence comparison to determine percent nucleotide or amino acid identity, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Optimal alignment of sequences for comparison can be conducted using the NCBI BLAST software (ncbi.nlm.nih.gov/BLAST/) set to default parameters. For example, to compare two nucleic acid sequences, one may use blastn with the “BLAST 2 Sequences” tool Version 2.0.12 (Apr. 21, 2000) set at the following default parameters: Matrix: BLOSUM62; Reward for match: 1; Penalty for mismatch: −2; Open Gap: 5 and Extension Gap: 2 penalties; Gap x drop-off: 50; Expect: 10; Word Size: 11; Filter: on. For a pairwise comparison of two amino acid sequences, one may use the “BLAST 2 Sequences” tool Version 2.0.12 (Apr. 21, 2000) with blastp set, for example, at the following default parameters: Matrix: BLOSUM62; Open Gap: 11 and Extension Gap: 1 penalties; Gap x drop-off 50; Expect: 10; Word Size: 3; Filter: on.
In connection with a natural oil, a “profile” is the distribution of particular species or triglycerides or fatty acyl groups within the oil. A “fatty acid profile” is the distribution of fatty acyl groups in the triglycerides of the oil without reference to attachment to a glycerol backbone. Fatty acid profiles are typically determined by conversion to a fatty acid methyl ester (FAME), followed by gas chromatography (GC) analysis with flame ionization detection (FID). The fatty acid profile can be expressed as one or more percent of a fatty acid in the total fatty acid signal determined from the area under the curve for that fatty acid. FAME-GC-FID measurement approximate weight percentages of the fatty acids.
As used herein, an oil is said to be “enriched” in one or more particular fatty acids if there is at least a 10% increase in the mass of that fatty acid in the oil relative to the non-enriched oil. For example, in the case of a cell expressing a heterologous FatB gene described herein, the oil produced by the cell is said to be enriched in, e.g., C8 and C16 fatty acids if the mass of these fatty acids in the oil is at least 10% greater than in oil produced by a cell of the same type that does not express the heterologous FatB gene (e.g., wild type oil).
“Recombinant” is a cell, nucleic acid, protein or vector that has been modified due to the introduction of an exogenous nucleic acid or the alteration of a native nucleic acid. Thus, e.g., recombinant (host) cells can express genes that are not found within the native (non-recombinant) form of the cell or express native genes differently than those genes are expressed by a non-recombinant cell. Recombinant cells can, without limitation, include recombinant nucleic acids that encode a gene product or suppression elements such as mutations, knockouts, antisense, interfering RNA (RNAi) or dsRNA that reduce the levels of active gene product in a cell. A “recombinant nucleic acid” is a nucleic acid originally formed in vitro, in general, by the manipulation of nucleic acid, e.g., using polymerases, ligases, exonucleases, and endonucleases, using chemical synthesis, or otherwise is in a form not normally found in nature. Recombinant nucleic acids may be produced, for example, to place two or more nucleic acids in operable linkage. Thus, an isolated nucleic acid or an expression vector formed in vitro by nucleic by ligating DNA molecules that are not normally joined in nature, are both considered recombinant for the purposes of this invention. Recombinant nucleic acids can also be produced in other ways; e.g., using chemical DNA synthesis. Once a recombinant nucleic acid is made and introduced into a host cell or organism, it may replicate using the in vivo cellular machinery of the host cell; however, such nucleic acids, once produced recombinantly, although subsequently replicated intracellularly, are still considered recombinant for purposes of this invention. Similarly, a “recombinant protein” is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid.
Embodiments of the present invention relate to the use of FatB genes isolated from plants, which can be expressed in a host cell in order to alter the fatty acid profile of an oil produced by the recombinant cell. Although the microalga, Prototheca moriformis, was used to screen the genes for ability to the alter fatty acid profile, the genes are useful in a wide variety of host cells. For example, the genes can be expressed in bacteria, other microalgae, or higher plants. The genes can be expressed in higher plants according to the methods of U.S. Pat. Nos. 5,850,022; 5,723,761; 5,639,790; 5,807,893; 5,455,167; 5,654,495; 5,512,482; 5,298,421; 5,667,997; 5,344,771; and 5,304,481. The fatty acids can be further converted to triglycerides, fatty aldehydes, fatty alcohols and other oleochemicals either synthetically or biosynthetically.
In specific embodiments, triglycerides are produced by a host cell expressing a novel FatB gene. A triglyceride-containing natural oil can be recovered from the host cell. The natural oil can be refined, degummed, bleached and/or deodorized. The oil, in its natural or processed form, can be used for foods, chemicals, fuels, cosmetics, plastics, and other uses. In other embodiments, the FatB gene may not be novel, but the expression of the gene in a microalga is novel.
The genes can be used in a variety of genetic constructs including plasmids or other vectors for expression or recombination in a host cell. The genes can be codon optimized for expression in a target host cell. The proteins produced by the genes can be used in vivo or in purified form.
For example, the gene can be prepared in an expression vector comprising an operably linked promoter and 5′UTR. Where a plastidic cell is used as the host, a suitably active plastid targeting peptide can be fused to the FATB gene, as in the examples below. Generally, for the newly identified FATB genes, there are roughly 50 amino acids at the N-terminal that constitute a plastid transit peptide, which are responsible for transporting the enzyme to the chloroplast. In the examples below, this transit peptide is replaced with a 38 amino acid sequence that is effective in the Prototheca moriformis host cell for transporting the enzyme to the plastids of those cells. Thus, the invention contemplates deletions and fusion proteins in order to optimize enzyme activity in a given host cell. For example, a transit peptide from the host or related species may be used instead of that of the newly discovered plant genes described here.
A selectable marker gene may be included in the vector to assist in isolating a transformed cell. Examples of selectable markers useful in microlagae include sucrose invertase and antibiotic resistance genes.
The gene sequences disclosed can also be used to prepare antisense, or inhibitory RNA (e.g., RNAi or hairpin RNA) to inhibit complementary genes in a plant or other organism.
FatB genes found to be useful in producing desired fatty acid profiles in a cell are summarized below in Table 1. Nucleic acids or proteins having the sequence of SEQ ID NOS: 1-109 can be used to alter the fatty acid profile of a recombinant cell. Variant nucleic acids can also be used; e.g., variants having at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NOS: 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 65, 66, 68, 69, 71, 72, 74, 76, 78, 80, 81, 83, 84, 86, 87, 89, 90, 92, 93, 95, 96, 98, 99, 101, 102, 104, 105, 107 or 109. Codon optimization of the genes for a variety of host organisms is contemplated, as is the use of gene fragments. Preferred codons for Prototheca strains and for Chlorella protothecoides are shown below in Tables 2 and 3, respectively. Codon usage for Cuphea wrightii is shown in Table 3a. Codon usage for Arabidopsis is shown in Table 3b; for example, the most preferred of codon for each amino acid can be selected. Codon tables for other organisms including microalgae and higher plants are known in the art. In some embodiments, the first and/or second most preferred Prototheca codons are employed for codon optimization. In specific embodiments, the novel amino acid sequences contained in the sequence listings below are converted into nucleic acid sequences according to the most preferred codon usage in Prototheca, Chlorella, Cuphea wrightii, or Arabidopsis as set forth in tables 2 through 3b or nucleic acid sequences having at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to these derived nucleic acid sequences.
In embodiments of the invention, there is protein or a nucleic acid encoding a protein having any of SEQ ID NOS: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 75, 77, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 108, or 110-192. In an embodiment, there is protein or a nucleic acid encoding a protein having at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with any of SEQ ID NOS: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 75, 77, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 108, or 110-192. In certain embodiments, the invention encompasses a fragment any of the above-described proteins or nucleic acids (including fragments of protein or nucleic acid variants), wherein the protein fragment has acyl-ACP thioesterase activity or the nucleic acid fragment encodes such a protein fragment. In other embodiments, the fragment includes a domain of an acyl-ACP thioesterase that mediates a particular function, e.g., a specificity-determining domain. Illustrative fragments can be produced by C-terminal and/or N-terminal truncations and include at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the full-length sequences disclosed herein.
In certain embodiments, percent sequence identity for variants of the nucleic acids or proteins discussed above can be calculated by using the full-length nucleic acid sequence (e.g., one of SEQ ID NOS: 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 65, 66, 68, 69, 71, 72, 74, 76, 78, 80, 81, 83, 84, 86, 87, 89, 90, 92, 93, 95, 96, 98, 99, 101, 102, 104, 105, 107 or 109) or full-length amino acid sequence (e.g., one of SEQ ID NOS: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 75, 77, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 108, or 110-192) as the reference sequence and comparing the full-length test sequence to this reference sequence. In some embodiments relating to fragments, percent sequence identity for variants of nucleic acid or protein fragments can be calculated over the entire length of the fragment.
The nucleic acids can be in isolated form, or part of a vector or other construct, chromosome or host cell. It has been found that is many cases the full length gene (and protein) is not needed; for example, deletion of some or all of the N-terminal hydrophobic domain (typically an 18 amino acid domain starting with LPDW) yields a still-functional gene. In addition, fusions of the specificity determining regions of the genes in Table 1 with catalytic domains of other acyl-ACP thioesterases can yield functional genes. Thus, in certain embodiments, the invention encompasses functional fragments (e.g., specificity determining regions) of the disclosed nucleic acid or amino acids fused to heterologous acyl-ACP thioesterase nucleic acid or amino acid sequences, respectively.
Prototheca
moriformis
Cinnamomum
camphora
Cinnamomum
camphora
Cinnamomum
camphora
Cuphea
hyssopifolia
Cuphea
hyssopifolia
Cuphea
hyssopifolia
Cuphea
hyssopifolia
Cuphea
hyssopifolia
Cuphea
Cuphea
wrightii
Cuphea
wrightii
Cuphea
wrightii
Cuphea
wrightii
Cuphea
heterophylla
Cuphea
heterophylla
Cuphea
heterophylla
Cuphea
heterophylla
Cuphea
heterophylla
Cuphea
heterophylla
Cuphea
heterophylla
Cuphea
heterophylla
Cuphea
heterophylla
Cuphea
heterophylla
Cuphea
heterophylla
Cuphea
viscosissima
Cuphea
viscosissima
Cuphea
viscosissima
Cuphea
calcarata
Cuphea
painteri
Cuphea
hookeriana
Cuphea
avigera var.
pulcherrima
Cuphea
paucipetala
Cuphea
procumbens
Cuphea
procumbens
Cuphea
procumbens
Cuphea ignea
In certain embodiments, a host cell (e.g. plant or microalgal cell) is transformed to produce a recombinant FATB protein falling into one of clades 1-12 of Table 1a. These clades were determined by sequence alignment and observation of changes in fatty acid profile when expressed in Prototheca. See Example 5. The FATB amino acid sequence can fall within x % amino acid sequence identity of each sequence in that clade listed in Table 1a, where x is a first second or third cutoff value, also listed in Table 1a.
Host Cells
The host cell can be a single cell (e.g., microalga, bacteria, yeast) or part of a multicellular organism such as a plant or fungus. Methods for expressing Fatb genes in a plant are given in U.S. Pat. Nos. 5,850,022; 5,723,761; 5,639,790; 5,807,893; 5,455,167; 5,654,495; 5,512,482; 5,298,421; 5,667,997; and 5,344,771; 5,304,481, or can be accomplished using other techniques generally known in plant biotechnology. Engineering of oleaginous microbes including those of Chlorophyta is disclosed in WO2010/063032, WO2011,150411, and WO2012/106560 and in the examples below.
Examples of oleaginous host cells include plant cells and microbial cells having a type II fatty acid biosynthetic pathway, including plastidic oleaginous cells such as those of oleaginous algae. Specific examples of microalgal cells include heterotrophic or obligate heterotrophic microalgae of the phylum Chlorophtya, the class Trebouxiophytae, the order Chlorellales, or the family Chlorellacae. Examples of oleaginous microalgae are provided in Published PCT Patent Applications WO2008/151149, WO2010/06032, WO2011/150410, and WO2011/150411, including species of Chlorella and Prototheca, a genus comprising obligate heterotrophs. The oleaginous cells can be, for example, capable of producing 25, 30, 40, 50, 60, 70, 80, 85, or about 90% oil by cell weight, ±5%. Optionally, the oils produced can be low in DHA or EPA fatty acids. For example, the oils can comprise less than 5%, 2%, or 1% DHA and/or EPA. The above-mentioned publications also disclose methods for cultivating such cells and extracting oil, especially from microalgal cells; such methods are applicable to the cells disclosed herein and incorporated by reference for these teachings. When microalgal cells are used they can be cultivated autotrophically (unless an obligate heterotroph) or in the dark using a sugar (e.g., glucose, fructose and/or sucrose). In any of the embodiments described herein, the cells can be heterotrophic cells comprising an exogenous invertase gene so as to allow the cells to produce oil from a sucrose feedstock. Alternately, or in addition, the cells can metabolize xylose from cellulosic feedstocks. For example, the cells can be genetically engineered to express one or more xylosc metabolism genes such as those encoding an active xylose transporter, a xylulose-5-phosphate transporter, a xylose isomerase, a xylulokinase, a xylitol dehydrogenase and a xylose reductase. See WO2012/154626, “GENETICALLY ENGINEERED MICROORGANISMS THAT METABOLIZE XYLOSE”, published Nov. 15, 2012.
Oils and Related Products
The oleaginous cells express one or more exogenous genes encoding fatty acid biosynthesis enzymes. As a result, some embodiments feature natural oils that were not obtainable from a non-plant or non-seed oil, or not obtainable at all.
The oleaginous cells produce a storage oil, which is primarily triacylglyceride and may be stored in storage bodies of the cell. A raw oil may be obtained from the cells by disrupting the cells and isolating the oil. WO2008/151149, WO2010/06032, WO2011/150410, and WO2011/1504 disclose heterotrophic cultivation and oil isolation techniques. For example, oil may be obtained by cultivating, drying and pressing the cells. The oils produced may be refined, bleached and deodorized (RBD) as known in the art or as described in WO2010/120939. The raw or RBD oils may be used in a variety of food, chemical, and industrial products or processes. After recovery of the oil, a valuable residual biomass remains. Uses for the residual biomass include the production of paper, plastics, absorbents, adsorbents, as animal feed, for human nutrition, or for fertilizer.
Where a fatty acid profile of a triglyceride (also referred to as a “triacylglyceride” or “TAG”) cell oil is given here, it will be understood that this refers to a nonfractionated sample of the storage oil extracted from the cell analyzed under conditions in which phospholipids have been removed or with an analysis method that is substantially insensitive to the fatty acids of the phospholipids (e.g. using chromatography and mass spectrometry). The oil may be subjected to an RBD process to remove phospholipids, free fatty acids and odors yet have only minor or negligible changes to the fatty acid profile of the triglycerides in the oil. Because the cells are oleaginous, in some cases the storage oil will constitute the bulk of all the TAGs in the cell.
The stable carbon isotope value δ13C is an expression of the ratio of 13C/12C relative to a standard (e.g. PDB, carbonite of fossil skeleton of Belemnite americana from Peedee formation of South Carolina). The stable carbon isotope value δ13C (0/00) of the oils can be related to the δ13C value of the feedstock used. In some embodiments, the oils are derived from oleaginous organisms heterotrophically grown on sugar derived from a C4 plant such as corn or sugarcane. In some embodiments the δ13C (0/00) of the oil is from −10 to −17 0/00 or from −13 to −16 0/00.
The oils produced according to the above methods in some cases are made using a microalgal host cell. As described above, the microalga can be, without limitation, fall in the classification of Chlorophyta, Trebouxiophyceae, Chlorellales, Chlorellaceae, or Chlorophyceae. It has been found that microalgae of Trebouxiophyceae can be distinguished from vegetable oils based on their sterol profiles. Oil produced by Chlorella protothecoides was found to produce sterols that appeared to be brassicasterol, ergosterol, campesterol, stigmasterol, and β-sitosterol, when detected by GC-MS. However, it is believed that all sterols produced by Chlorella have C2413 stereoohemistry. Thus, it is believed that the molecules detected as campesterol, stigmasterol, and β-sitosterol, are actually 22,23-dihydrobrassicasterol, proferasterol and clionasterol, respectively. Thus, the oils produced by the microalgae described above can be distinguished from plant oils by the presence of sterols with C24β stereochemistry and the absence of C24α stereochemistry in the sterols present. For example, the oils produced may contain 22, 23-dihydrobrassicasterol while lacking campesterol; contain clionasterol, while lacking in β-sitosterol, and/or contain poriferasterol while lacking stigmasterol. Alternately, or in addition, the oils may contain significant amounts of Δ7-poriferasterol.
In one embodiment, the oils provided herein are not vegetable oils. Vegetable oils are oils extracted from plants and plant seeds. Vegetable oils can be distinguished from the non-plant oils provided herein on the basis of their oil content. A variety of methods for analyzing the oil content can be employed to determine the source of the oil or whether adulteration of an oil provided herein with an oil of a different (e.g. plant) origin has occurred. The determination can be made on the basis of one or a combination of the analytical methods. These tests include but are not limited to analysis of one or more of free fatty acids, fatty acid profile, total triacylglycerol content, diacylglycerol content, peroxide values, spectroscopic properties (e.g. UV absorption), sterol profile, sterol degradation products, antioxidants (e.g. tocopherols), pigments (e.g. chlorophyll), d13C values and sensory analysis (e.g. taste, odor, and mouth feel). Many such tests have been standardized for commercial oils such as the Codex Alimentarius standards for edible fats and oils.
Sterol profile analysis is a particularly well-known method for determining the biological source of organic matter. Campesterol, b-sitosterol, and stigamsterol are common plant sterols, with b-sitosterol being a principle plant sterol. For example, b-sitosterol was found to be in greatest abundance in an analysis of certain seed oils, approximately 64% in corn, 29% in rapeseed, 64% in sunflower, 74% in cottonseed, 26% in soybean, and 79% in olive oil (Gul et al. J. Cell and Molecular Biology 5:71-79, 2006).
Oil isolated from Prototheca moriformis strain UTEX1435 were separately clarified (CL), refined and bleached (RB), or refined, bleached and deodorized (RBD) and were tested for sterol content according to the procedure described in JAOCS vol. 60, no. 8, August 1983. Results of the analysis are shown below (units in mg/100 g):
These results show three striking features. First, ergosterol was found to be the most abundant of all the sterols, accounting for about 50% or more of the total sterols. The amount of ergosterol is greater than that of campesterol, β-sitosterol, and stigmasterol combined. Ergosterol is steroid commonly found in fungus and not commonly found in plants, and its presence particularly in significant amounts serves as a useful marker for non-plant oils. Secondly, the oil was found to contain brassicasterol. With the exception of rapeseed oil, brassicasterol is not commonly found in plant based oils. Thirdly, less than 2% β-sitosterol was found to be present. β-sitosterol is a prominent plant sterol not commonly found in microalgae, and its presence particularly in significant amounts serves as a useful marker for oils of plant origin. In summary, Prototheca moriformis strain UTEX1435 has been found to contain both significant amounts of ergosterol and only trace amounts of β-sitosterol as a percentage of total sterol content. Accordingly, the ratio of ergosterol: β-sitosterol or in combination with the presence of brassicasterol can be used to distinguish this oil from plant oils.
In some embodiments, the oil content of an oil provided herein contains, as a percentage of total sterols, less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% β-sitosterol. In other embodiments the oil is free from β-sitosterol.
In some embodiments, the oil is free from one or more of β-sitosterol, campesterol, or stigmasterol. In some embodiments the oil is free from β-sitosterol, campesterol, and stigmasterol. In some embodiments the oil is free from campesterol. In some embodiments the oil is free from stigmasterol.
In some embodiments, the oil content of an oil provided herein comprises, as a percentage of total sterols, less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% 24-ethylcholest-5-en-3-ol. In some embodiments, the 24-ethylcholest-5-en-3-ol is clionasterol. In some embodiments, the oil content of an oil provided herein comprises, as a percentage of total sterols, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% clionasterol.
In some embodiments, the oil content of an oil provided herein contains, as a percentage of total sterols, less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% 24-methylcholest-5-en-3-ol. In some embodiments, the 24-methylcholest-5-en-3-ol is 22, 23-dihydrobrassicasterol. In some embodiments, the oil content of an oil provided herein comprises, as a percentage of total sterols, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% 22,23-dihydrobrassicasterol.
In some embodiments, the oil content of an oil provided herein contains, as a percentage of total sterols, less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% 5,22-cholestadien-24-ethyl-3-ol. In some embodiments, the 5, 22-cholestadien-24-ethyl-3-ol is poriferasterol. In some embodiments, the oil content of an oil provided herein comprises, as a percentage of total sterols, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% poriferasterol.
In some embodiments, the oil content of an oil provided herein contains ergosterol or brassicasterol or a combination of the two. In some embodiments, the oil content contains, as a percentage of total sterols, at least 5%, 10%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% ergosterol. In some embodiments, the oil content contains, as a percentage of total sterols, at least 25% ergosterol. In some embodiments, the oil content contains, as a percentage of total sterols, at least 40% ergosterol. In some embodiments, the oil content contains, as a percentage of total sterols, at least 5%, 10%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% of a combination of ergosterol and brassicasterol.
In some embodiments, the oil content contains, as a percentage of total sterols, at least 1%, 2%, 3%, 4% or 5% brassicasterol. In some embodiments, the oil content contains, as a percentage of total sterols less than 10%, 9%, 8%, 7%, 6%, or 5% brassicasterol.
In some embodiments the ratio of ergosterol to brassicasterol is at least 5:1, 10:1, 15:1, or 20:1.
In some embodiments, the oil content contains, as a percentage of total sterols, at least 5%, 10%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% ergosterol and less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% β-sitosterol. In some embodiments, the oil content contains, as a percentage of total sterols, at least 25% ergosterol and less than 5% β-sitosterol. In some embodiments, the oil content further comprises brassicasterol.
Sterols contain from 27 to 29 carbon atoms (C27 to C29) and are found in all eukaryotes. Animals exclusively make C27 sterols as they lack the ability to further modify the C27 sterols to produce C28 and C29 sterols. Plants however are able to synthesize C28 and C29 sterols, and C28/C29 plant sterols are often referred to as phytosterols. The sterol profile of a given plant is high in C29 sterols, and the primary sterols in plants are typically the C29 sterols b-sitosterol and stigmasterol. In contrast, the sterol profile of non-plant organisms contain greater percentages of C27 and C28 sterols. For example the sterols in fungi and in many microalgae are principally C28 sterols. The sterol profile and particularly the striking predominance of C29 sterols over C28 sterols in plants has been exploited for determining the proportion of plant and marine matter in soil samples (Huang, Wen-Yen, Meinschein W. G., “Sterols as ecological indicators”; Geochimica et Cosmochimia Acta. Vol 43. pp 739-745).
In some embodiments the primary sterols in the microalgal oils provided herein are sterols other than b-sitosterol and stigmasterol. In some embodiments of the microalgal oils, C29 sterols make up less than 50%, 40%, 30%, 20%, 10%, or 5% by weight of the total sterol content.
In some embodiments the microalgal oils provided herein contain C28 sterols in excess of C29 sterols. In some embodiments of the microalgal oils, C28 sterols make up greater than 50%, 60%, 70%, 80%, 90%, or 95% by weight of the total sterol content. In some embodiments the C28 sterol is ergosterol. In some embodiments the C28 sterol is brassicasterol.
In embodiments of the present invention, oleaginous cells expressing one or more of the genes of Table 1 can produce an oil with at least 20, 40, 60 or 70% of C8, C10, C12, C14 or C16 fatty acids. In a specific embodiment, the level of myristate (C14:0) in the oil is greater than 30%.
Thus, in embodiments of the invention, there is a process for producing an oil, triglyceride, fatty acid, or derivative of any of these, comprising transforming a cell with any of the nucleic acids discussed herein. In another embodiment, the transformed cell is cultivated to produce an oil and, optionally, the oil is extracted. Oil extracted in this way can be used to produce food, oleochemicals or other products.
The oils discussed above alone or in combination are useful in the production of foods, fuels and chemicals (including plastics, foams, films, etc). The oils, triglycerides, fatty acids from the oils may be subjected to C—H activation, hydroamino methylation, methoxy-carbonation, ozonolysis, enzymatic transformations, epoxidation, methylation, dimerization, thiolation, metathesis, hydro-alkylation, lactonization, or other chemical processes.
After extracting the oil, a residual biomass may be left, which may have use as a fuel, as an animal feed, or as an ingredient in paper, plastic, or other product. For example, residual biomass from heterotrophic algae can be used in such products.
Sequences of novel plant acyl-ACP thioesterases involved in seed-specific mid-chain (C8-C16) fatty acid biosynthesis in higher plants were isolated. Seed-specific lipid production genes were isolated through direct interrogation of RNA pools accumulating in oilseeds. Based on phylogenetic analysis, novel enzymes can be classified as members of FatB family of acyl-ACP thioesterases.
Seeds of oleaginous plants were obtained from local grocery stores or requested through USDA ARS National Plant Germplasm System (NPGS) from North Central Regional Plant Introduction Station (NCRIS) or USDA ARS North Central Soil Conservation Research Laboratory (Morris, Mich.). Dry seeds were homogenized in liquid nitrogen to powder, resuspended in cold extraction buffer containing 6-8M Urea and 3M LiCl and left on ice for a few hours to overnight at 4° C. The seed homogenate was passed through NucleoSpin Filters (Macherey-Nagel) by centrifugation at 20,000 g for 20 minutes in the refrigerated microcentrifuge (4° C.). The resulting RNA pellets were resuspended in the buffer containing 20 mM Tris HCl, pH7.5, 0.5% SDS, 100 mM NaCl, 25 mM EDTA, 2% PVPP) and RNA was subsequently extracted once with Phenol-Chloroform-Isoamyl Alcohol (25:24:1, v/v) and once with chloroform. RNA was finally precipitated with isopropyl alcohol (0.7 Vol.) in the presence of 150 mM of Na Acetate, pH51.2, washed with 80% ethanol by centrifugation, and dried. RNA samples were treated with Turbo DNAse (Lifetech) and purified further using RNeasy kits (Qiagen) following manufacturers' protocols. The resulting purified RNA samples were converted to pair-end cDNA libraries and subjected to next-generation sequencing (2×100 bp) using Illumina Hiseq 2000 platform. RNA sequence reads were assembled into corresponding seed transcriptomes using Trinity or Oases packages. Putative thioesterase-containg cDNA contigs were identified by mining transcriptomes for sequences with homology to known thioesterases. These in silico identified putative thioesterase cDNAs have been further verified by direct reverse transcription PCR analysis using seed RNA and primer pairs targeting full-length thioesterase cDNAs. The resulting amplified products were cloned and sequenced de novo to confirm authenticity of identified thioesterase genes.
To interrogate evolutionary and functional relationship between novel acyl-ACP thioesterases and the members of two existing thioesterase classes (FatA and FatB), we performed a phylogenetic analysis using published full-length (Mayer and Shanklin, 2007) and truncated (THYME database) amino acid thioesterase sequences. Novel proteins appear to group with known acyl-ACP FatB thioesterases involved in biosynthesis of C8-C16 fatty acids. Moreover, novel thioesterases appear to cluster into 3 predominant out-groups suggesting distinct functional similarity and evolutionary relatedness among members of each cluster.
The amino acid sequences of the FatB genes follow are shown in Table 4.
ChsFATB3j:
In the example below, we detail the effect of expressing plant oilseed transcriptome-derived, heterologous thioesterases in the UTEX1435 (web.biosci.utexas.edu/utex/) strain, Strain A.
As in Example 1, RNA was extracted from dried plant seeds and submitted for paired-end sequencing using the Illumina Hiseq 2000 platform. RNA sequence reads were assembled into corresponding seed transcriptomes using Trinity or Oases packages and putative thioesterase-containing cDNA contigs were identified by mining transcriptomes for sequences with homology to known thioesterases. These in silico identified putative thioesterase cDNAs were verified by direct reverse transcription PCR analysis using seed RNA and primer pairs targeting full-length thioesterase cDNAs. The resulting amplified products were cloned and sequenced de novo to confirm authenticity of identified thioesterase genes and to identify sequence variants arising from expression of different gene alleles or diversity of sequences within a population of seeds. The resulting amino acid sequences were subjected to phylogenetic analysis using published full-length (Mayer and Shanklin, 2007) and truncated (THYME database) FatB sequences. The thioesterases that clustered with acyl-ACP FatB thioesterases, which are involved in biosynthesis of C8-C16 fatty acids, were pursued.
Construction of Transforming Vectors Expressing Acyl-ACP FatB Thioesterases
27 putative acyl-ACP FatB thioesterases from the species Cinnamomum camphora, Cuphea hyssopifolia, Cuphea PSR23, Cuphea wrightii, Cuphea heterophylla, and Cuphea viscosissima were synthesized in a codon-optimized form to reflect Prototheca moriformis (UTEX 1435) codon usage. Of the 27 genes synthesized, 24 were identified by our transcriptome sequencing efforts and the 3 genes from Cuphea viscosissima, were from published sequences in GenBank.
Transgenic strains were generated via transformation of the base strain Strain A (Prototheca moriformis, derived from UTEX 1435 by classical mutation and screening for high oil production) with a construct encoding 1 of the 27 FatB thioesterases. The construct pSZ2760 encoding Cinnamomum camphora (Cc) FATB1b is shown as an example, but identical methods were used to generate each of the remaining 26 constructs encoding the different respective thioesterases. Construct pSZ2760 can be written as 6S::CrTUB2:ScSUC2:CvNR::PmAMT3:CcFATB1b:CvNR::6S. The sequence of the transforming DNA is provided in Table 5 (pSZ2760). The relevant restriction sites in the construct from 5′-3′, BspQ1, KpnI, AscI, MfeI, EcoRI, SpeI, XhoI, SacI, BspQ1, respectively, are indicated in lowercase, bold, and underlined. BspQ1 sites delimit the 5′ and 3′ ends of the transforming DNA. Bold, lowercase sequences at the 5′ and 3′ end of the construct represent genomic DNA from UTEX 1435 that target integration to the 6S locus via homologous recombination. Proceeding in the 5′ to 3′ direction, the selection cassette has the C. reinhardtii β-tubulin promoter driving expression of the S. cerevisiae gene SUC2 (conferring the ability to grow on sucrose) and the Chlorella vulgaris Nitrate Reductase (NR) gene 3′ UTR. The promoter is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for ScSUC2 are indicated by bold, uppercase italics, while the coding region is indicated with lowercase italics. The 3′ UTR is indicated by lowercase underlined text. The spacer region between the two cassettes is indicated by upper case text. The second cassette containing the codon optimized CcFATB1b gene (Table 5; pSZ2760) from Cinnamomum camphora is driven by the Prototheca moriformis endogenous AMT3 promoter, and has the Chlorella vulgaris Nitrate Reductase (NR) gene 3′ UTR. In this cassette, the AMT3 promoter is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for the CcFATB1b gene are indicated in bold, uppercase italics, while the coding region is indicated by lowercase italics and the spacer region is indicated by upper case text. The 3′ UTR is indicated by lowercase underlined text. The final construct was sequenced to ensure correct reading frame and targeting sequences.
gctcttcgccgccgccactcctgctcgagcgcgcccgcgcgtgcgccgccagcgccttggccttttcgccgcgctcgtgcgcgtcgctgat
cgccaagatcagcgcctccatgacgaacgagacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgacc
ccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtctgggggacgccc
ttgttctggggccacgccacgtccgacgacctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactccgg
cgccttctccggctccatggtggtggactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggcca
tctggacctacaacaccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccgagtaccagaaga
accccgtgctggccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaagtggatcatgaccgcggc
caagtcccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccgcgttcgccaacgagggcttc
ctcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtcctactgggtgatgttcatctccat
caaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccacttcgaggccttcgacaaccag
tcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacaccgacccgacctacgggagcgccctgggcatcg
cgtgggcctccaactgggagtactccgccttcgtgcccaccaacccctggcgctcctccatgtccctcgtgcgcaagttctccctcaaca
ccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagccgatcctgaacatcagcaacgccggcccctgga
gccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacctgtccaacagcaccggcaccctggagttcgag
ctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgttcgcggacctctccctctggttcaagggcctggaggaccccga
ggagtacctccgcatgggcttcgaggtgtccgcgtcctccttcttcctggaccgcgggaacagcaaggtgaagttcgtgaaggagaac
ccctacttcaccaaccgcatgagcgtgaacaaccagcccttcaagagcgagaacgacctgtcctactacaaggtgtacggcttgctgg
accagaacatcctggagctgtacttcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccgggaacgccctgggctc
cgtgaacatgacgacgggggtggacaacctgttctacatcgacaagttccaggtgcgcgaggtcaagTGA
caattggcagcagcag
ctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgctttt
atcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatcccctt
ccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgca
cagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatggg
ggcctgaagccccgctcctccgacctgcagctgcgcgccggcaacgcccagacctccctgaagatgatcaacggcaccaagttctcc
tacaccgagtccctgaagaagctgcccgactggtccatgctgttcgccgtgatcaccaccatcttctccgccgccgagaagcagtggac
caacctggagtggaagcccaagcccaaccccccccagctgctggacgaccacttcggcccccacggcctggtgttccgccgcacctt
cgccatccgctcctacgaggtgggccccgaccgctccacctccatcgtggccgtgatgaaccacctgcaggaggccgccctgaacca
cgccaagtccgtgggcatcctgggcgacggcttcggcaccaccctggagatgtccaagcgcgacctgatctgggtggtgaagcgcac
ccacgtggccgtggagcgctaccccgcctggggcgacaccgtggaggtggagtgctgggtgggcgcctccggcaacaacggccgc
cgccacgacttcctggtgcgcgactgcaagaccggcgagatcctgacccgctgcacctccctgtccgtgatgatgaacacccgcaccc
aagaagccccagaagctgaacgactccaccgccgactacatccagggcggcctgaccccccgctggaacgacctggacatcaacc
agcacgtgaacaacatcaagtacgtggactggatcctggagaccgtgcccgactccatcttcgagtcccaccacatctcctccttcacc
atcgagtaccgccgcgagtgcacccgcgactccgtgctgcagtccctgaccaccgtgtccggcggctcctccgaggccggcctggtgt
gcgagcacctgctgcagctggagggcggctccgaggtgctgcgcgccaagaccgagtggcgccccaagctgtccttccgcggcatct
ccgtgatccccgccgagtcctccgtgatggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacga
cgacgacaagTGActcgaggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacac
ttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgctt
gtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctc
agcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcact
gcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaAAGCTGTATAGGGATAACAGGGTAATga
gctcttgttttccagaaggagttgctccttgagcctttcattctcagcctcgataacctccaaagccgctctaattgtggagggggttcgaattta
Constructs encoding the identified heterologous FatB genes, such as CcFATB1b from pSZ2760 in Table 6, were transformed into Strain A, and selected for the ability to grow on sucrose. Transformations, cell culture, lipid production and fatty acid analysis were all carried out as previously described. After cultivating on sucrose under low nitrogen conditions to accumulate oil, fatty acid profiles were determined by FAME-GC. The top performer from each transformation, as judged by the ability to produce the highest level of midchain fatty acids, is shown in Table 4
Cinnamomum camphora
Cinnamomum camphora
Cinnomomum camphora
Cuphea hyssopifolia
Cuphea hyssopifolia
Cuphea hyssopifolia
Cuphea hyssopifolia
Cuphea hyssopifolia
Cuphea PSR23
Cuphea wrightii
Cuphea wrightii
Cuphea wrightii
Cuphea wrightii
Cuphea heterophylla
Cuphea heterophylla
Cuphea heterophylla
Cuphea heterophylla
Cuphea heterophylla
Cuphea heterophylla
Cuphea heterophylla
Cuphea heterophylla
Cuphea heterophylla
Cuphea heterophylla
Cuphea heterophylla
Cuphea viscosissima
Cuphea viscosissima
Cuphea viscosissima
Cinnamomum camphora
Cinnamomum camphora
Cinnomomum camphora
Cuphea hyssopifolia
Cuphea hyssopifolia
Cuphea hyssopifolia
Cuphea hyssopifolia
Cuphea hyssopifolia
Cuphea PSR23
Cuphea wrightii
Cuphea wrightii
Cuphea wrightii
Cuphea wrightii
Cuphea heterophylla
Cuphea heterophylla
Cuphea heterophylla
Cuphea heterophylla
Cuphea heterophylla
Cuphea heterophylla
Cuphea heterophylla
Cuphea heterophylla
Cuphea heterophylla
Cuphea heterophylla
Cuphea heterophylla
Cuphea viscosissima
Cuphea viscosissima
Cuphea viscosissima
Many of the acyl-ACP FatB thioesterases were found to exhibit midchain activity when expressed in Prototheca moriformis. For example, expression of CcFATB1b causes an increase in myristate levels from 2% of total fatty acids in the parent, Strain A, to ˜15% in the DI670-13 primary transformant. Other examples include CcFATB4, which exhibits an increase in laurate levels from 0% in Strain A to ˜33%, and ChsFATB3, which exhibits an increase in myristate levels to ˜34%. Although some of the acyl-ACP thioesterases did not exhibit dramatic effects on midchain levels in the current incarnation, efforts will likely develop to optimize some of these constructs.
Sequences of the Heterologous Acyl-ACP Thioesterases Identified and Transformed into P. moriformis (UTEX 1435)
A complete listing of relevant sequences for the transforming constructs, such as the deduced amino acid sequence of the encoded acyl-ACP thioesterase, the native CDS coding sequence, the Prototheca moriformis codon-optimized coding sequence, and the nature of the sequence variants examined, is provided as SEQ ID NOS: 1-78.
Additional FATB genes were obtained from seeds as described above. The species and number of FatB genes identified were:
Cuphea calcarata
Cuphea painteri
Cuphea hookeriana
Cuphea avigera var.
pulcherrima
Cuphea paucipetala
Cuphea procumbens
Cuphea ignea
The thioesterases that clustered with acyl-ACP FatB thioesterases, which are involved in biosynthesis of C8-C16 fatty acids, were pursued. The native, putative plastid-targeting transit peptide sequence is indicated by underlining.
Construction of Transforming Vectors Expressing Acyl-ACP FatB Thioesterases. The nine putative Acyl-ACP FatB Thioesterases from the species Cuphea calcarata, Cuphea painter, Cuphea hookeriana, Cuphea avigera var. pulcherrima, Cuphea paucipetala, Cuphea procumbens, and Cuphea ignea were synthesized in a codon-optimized form to reflect UTEX 1435 codon usage. In contrast to the previous example, the new Acyl-ACP FatB thioesterases were synthesized with a modified transit peptide from Chlorella protothecoides (Cp) in place of the native transit peptide. The modified transit peptide derived from the CpSAD1 gene, “CpSAD1tp_trimmed”, was synthesized as an in-frame, N-terminal fusion to the FatB acyl-ACP thioesterases in place of the native transit peptide; the resulting sequences are listed below. The novel FatB genes were cloned into Prototheca moriformis as described above. Constructs encoding heterologous FatB genes were transformed into strain S6165 (a descendant of S3150/Strain A) and selected for the ability to grow on sucrose. Transformations, cell culture, lipid production and fatty acid analysis were all carried out as previously described. The results for the nine novel FatB acyl-ACP thioesterases are displayed in the table immediately below.
Cuphea colcarata
Cuphea painteri
Cuphea
hookeriana
Cuphea ovigera
Cuphea
paucipetala
Cuphea
procumbens
Cuphea
procumbens
Cuphea
procumbens
Cuphea ignea
Of particular note are: CpaiFATB1, which exhibits 17% C10:0 and 8% C8:0 fatty acid levels; CpauFATB1, which exhibits 9% C10:0 and 1% C12:0 fatty acid levels; CigneaFATB1, which exhibits 8% C10:0 and 1% C12:0 fatty acid levels; CcalcFATB1, which exhibits 18% C14:0 and 12% C12:0 levels; and CaFATB1, which exhibits 22% C8:0 and 9% C10:0 fatty acid levels.
CaFATB1, which exhibits high C8:0 and C10:0 levels, is of particular interest. CaFATB1 arose from two separate contigs that were assembled from the Cupha avigera var. pulcherrima transcriptome, S17_Cavig_trinity_7406 and S17_Cavig_trinity_7407. Although the two partial contigs exhibit only 17 nucleotides of overlap, we were able to assemble a putative full length transcript encoding CaFATB1 from the two contigs and then subsequently confirm the existence of the full-length transcript by direct reverse transcription PCR analysis using seed RNA and primer pairs targeting the full-length CaFATB1 thioesterase cDNA. Tjellstrom et al. (2013) discloses the expression of a newly identified fatty acyl-ACP thioesterase from Cuphea pulcherrima that they named “CpuFATB3” (Genbank accession number KC675178). The coding sequence of CpuFATB3 is 100% identical to the CaFATB1 gene we identified and contains one nucleotide difference in the RNA sequence outside the predicted coding region. Tjellstrom et al. (2013) showed that CpuFATB3 produces an average of 4.8% C8:0 when expressed in Arabidopsis, and further requires deletion of two acyl-ACP synthetases, AAE15/16, to produce an average of 9.2% C8:0 with a maximum level of ˜12% C8.0. The CaFATB1 gene we identified was codon-optimized for expression in UTEX1435 and generated as a CpSAD1tp-trimmed transit peptide fusion before introduction into 56165. The CpSAD1tp_trimmed:CaFATB1 gene produces an average C8:0 level of 14% and a maximum level of 22% C8:0 without requiring the deletion of endogenous acyl-ACP synthetases.
SEQ ID NO: 172
SEQ ID NO: 173
SEQ ID NO: 174
SEQ ID NO: 175
SEQ ID NO: 176
SEQ ID NO: 177
SEQ ID NO: 178
SEQ ID NO: 179
SEQ ID NO: 180
SEQ ID NO: 181
SEQ ID NO: 182
SEQ ID NO: 183
In the course of testing several new putative midchain FatB thioesterases in UTEX1435, S3150 (Strain A above), we identified several thioesterases with increased C10:0 and C16:0 activity above the background midchain levels found in the strain. We reasoned that a consensus sequence could be obtained for an idealized C10:0 thioesterase and C16:0 thioesterase from aligning the best-performing C10:0 and C16:0 thioesterases. A consensus C10:0 specific thioesterase sequence was generated using the C. palustris FatB1 (CpFATB1), C. PSR23 FatB3 (CuPSR23FATB3), C. viscosissima FatB1 (CvisFATB1), C. glossostoma FatB1 (CgFATB1), and C. carthagenensis FatB2 (CcrFATB2) sequences as inputs resulting in a C10:0 specific consensus sequence termed JcFATB1/SzFATB1. A consensus C16:0 specific thioesterase sequence was generated using the C. heterophylla FatB3a (ChtFATB3a), C. carthagenensis FatB1 (CcrFATB1), C. viscosissima FatB2 (CvisFATB2), C. hookeriana FatB1 (ChFATB1; AAC48990), C. hyssopifolia FatB2 (ChsFATB2), C. calophylla FatB2 (CcalFATB2; ABB71581), C. hookeriana FatB1-1 (ChFATB1-1; AAC72882), C. lanceolata FatB1 (CIFATB1; CAC19933), and C. wrightii FatB4a (CwFATB4a) sequences as inputs resulting in a C16:0 specific consensus sequence termed JcFATB2/SzFATB2. The resulting consensus sequences were synthesized, cloned into a vector identical to that used to test other FatB thioesterases, and introduced into 53150 as described above. The consensus amino acid sequences are given as SEQ ID NOs. 106 and 107; the nucleic acid sequences were based on these amino acid sequences using codon optimization for Prototheca moriformis. The transformants were selected, cultivated and the oil was extracted and analyzed by FAME-GC-FID. The fatty acid profiles obtained are given in the table below.
Various novel FATB thioesterases were clustered according to a neighbor joining algorithm. These were found to form twelve clades as listed in Table 1a. Putative function was assigned based on expression in Prototheca as described above.
The described embodiments of the invention are intended to be merely exemplary and numerous variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to be within the scope of the present invention.
Sequence Listing
Cinnamomum camphora (Cc) FATB1b variant M25L, M322R, ΔT367-D368 amino
Cinnamomum camphora (Cc) FATB1b variant M25L, M322R, ΔT367-D368 coding
Cinnamomum camphora (Cc) FATB1b variant M25L, M322R, ΔT367-D368 coding
Cinnamomum camphora (Cc) FATB4 amino acid sequence
Cinnamomum camphora (Cc) FATB4 coding DNA sequence
Cinnamomum camphora (Cc) FATB4 coding DNA sequence codon optimized for
Prototheca moriformis
Cinnamomum camphora (Cc) FATB3 amino acid sequence
Cinnamomum camphora (Cc) FATB3 coding DNA sequence
Cinnamomum camphora (Cc) FATB4 coding DNA sequence codon optimized for
Prototheca moriformis
Cuphea hyssopifolia (Chs) FATB I amino acid sequence
Cuphea hyssopifolia (Chs) FATB1 coding DNA sequence
Cuphea hyssopifolia (Chs) FATB1 coding DNA sequence codon optimized for
Prototheca moriformis
Cuphea hyssopifolia (Chs) FATB2 amino acid sequence
Cuphea hyssopifolia (Chs) FATB2 coding DNA sequence
Cuphea hyssopifolia (Chs) FATB2 coding DNA sequence codon optimized for
Prototheca moriformis
Cuphea hyssopifolia (Chs) FATB2b + a.a. 248-259 variant amino acid
Cuphea hyssopifolia (Chs) FATB2b + a.a. 248-259 variant coding DNA
Cuphea hyssopifolia (Chs) FATB2b + a.a. 248-259 variant coding DNA
Cuphea hyssopifolia (Chs) FATB3 amino acid sequence
Cuphea hyssopifolia (Chs) FATB3 coding DNA sequence
Cuphea hyssopifolia (Chs) FATB3 coding DNA sequence codon optimized for
Prototheca moriformis
Cuphea hyssopifolia (Chs) FATB3b (V204I, C239F, E243D, M251V variant)
Cuphea hyssopifolia (Chs) FATB3b (V204I, C239F, E243D, M251V variant)
Cuphea hyssopifolia (Chs) FATB3b (V204I, C239F, E243D, M251V variant)
Cuphea PSR23 (Cu) FATB3 amino acid sequence
Cuphea PSR23 (Cu) FATB3 coding DNA sequence
Cuphea PSR23 (Cu) FATB3 coding DNA sequence codon optimized for
Prototheca moriformis
Cuphea wrightii (Cw) FATB3 amino acid sequence
Cuphca wrightii (Cw) FATB3 coding DNA sequence
Cuphea wrightii (Cw) FATB3 coding DNA sequence codon optimized for
Prototheca moriformis
Cuphea wrightii (Cw) FATB4a amino acid sequence
Cuphea wrightii (Cw)FATB4a coding DNA sequence
Cuphea wrightii (Cw) FATB4a coding DNA sequence codon optimized for
Prototheca moriformis
Cuphea wrightii (Cw) FATB4b amino acid sequence
Cuphea wrightii (Cw) FATB4b coding DNA sequence
Cuphea wrightii (Cw) FATB4b coding DNA sequence codon optimized for
Prototheca moriformis
Cuphea wrightii (Cw) FATB5 amino acid sequence
Cuphea wrightii (Cw) FATB5 coding DNA sequence
Cuphea wrightii (Cw) FATB5 coding DNA sequence codon optimized for
Prototheca moriformis
Cuphea heterophylla (Cht) FATBla amino acid sequence
Cuphea heterophylla (Cht) FATBla coding DNA sequence
Prototheca moriformis
Cuphea heterophylla (Cht) FATB1b (P16S, T20P, G94S, G105W, S293F, L305F
Cuphea heterophylla (Cht) FATB1b(P16S, T20P, G94S, G105W, S293F, L305F
Cuphea heterophylla (Cht) FATBlb (P16S, T20P, G94S, G105W, S293F, L305F
Cuphea heterophylla (Cht) FATB2b amino acid sequence
Cuphea heterophylla (Cht) FATB2b coding DNA sequence
Cuphea heterophylla (Cht) FATB2b coding DNA sequence codon optimized for
Prototheca moriformis
Cuphea heterophylla (Cht) FATB2a (S17P, P21S, T28N, L30P, S33L, G76D,
Cuphea heterophylla (Cht) FATB2a (S17P, P21S, T28N, L30P, S33L, G76D,
Cuphea heterophylla (Cht) FATB2a (S17P, P21S, T28N, L30P, S33L, G76D,
Prototheca moriformis
Cuphea heterophylla (Cht) FATB2c (G76D, S78P variant) amino acid sequence
Cuphea heterophylla (Cht) FATB2c (G76D, S78P variant) coding DNA sequence
Cuphea heterophylla (Cht) FATB2c (G76D, S78P variant) coding DNA sequence
Cuphea heterophylla (Cht) FATB2d (S21P, T28N, L30P, S33L, G76D, R97L,
Cuphea heterophylla (Cht) FATB2d (S21P, T28N, L30P, S33L, G76D, R97L,
Cuphea heterophylla (Cht) FATB2d (S21P, T28N, L30P, S33L, G76D, R97L,
Cuphea heterophylla (Cht) FATB2e (G76D, R97L, H124L, I132S, G152S, H165L,
Cuphea heterophylla (Cht) FATB2e (G76D, R97L, H124L, I132S, G152S, H165L,
Cuphea heterophylla (Cht) FATB2e (G76D, R97L, H124L, I132S, G152S, H165L,
Cuphea heterophylla (Cht) FATB2f (R97L, H124L, I132S, G152S, H165L, T211N
Cuphea heterophylla (Cht) FATB2f (R97L, H124L, I132S, G152S, H165L, T211N
Cuphea heterophylla (Cht) FATB2f (R97L, H124L, I132S, G152S, H165L, T211N
Cuphea heterophylla (Cht) FATB2g (A6T, A16V, S17P, G76D, R97L, H124L,
Cuphea heterophylla (Cht) FATB2g (A6T, A16V, S17P, G76D, R97L, H124L,
Cuphea heterophylla (Cht) FATB3aamino acid sequence
Cuphea heterophylla (Cht) FATB3a coding DNA sequence
Cuphea heterophylla (Cht) FATB3a coding DNA sequence codon optimized for
Prototheca moriformis
Cuphea heterophylla (Cht) FATB3b (C67G, H72Q, L128F, N179I variant) amino
Cuphea heterophylla (Cht) FATB3b (C67G, H72Q, L128F, N179I variant)
Cuphea viscosissima (Cvis) FATB1 amino acid sequence
Cuphea viscosissima (Cvis) FATB1 coding DNA sequence codon optimized for
Prototheca moriformis
Cuphea viscosissima (Cvis) FATB2 amino acid sequence
Cuphea viscosissima (Cvis) FATB2 coding DNA sequence codon optimized for
Prototheca moriformis
Cuphea viscosissima (Cvis) FATB3 amino acid sequence
Cuphea viscosissima (Cvis) FATB3 coding DNA sequence codon optimized for
Prototheca moriformis
Cuphea calcarata (Ccalc) FATB1 amino acid sequence
Cuphea calcarata (Ccalc) FATB1 coding DNA sequence
Cuphea calcarata (Ccalc) FATB1 coding DNA sequence codon optimized for
Prototheca moriformis
Cuphea painteri (Cpai) FATB1 amino acid sequence
Cuphea painteri (Cpai) FATB1 coding DNA sequence
Cuphea painteri (Cpai) FATB1 coding DNA sequence codon optimized for
Prototheca moriformis
Cuphea hookeriana (Chook) FATB4 amino acid sequence
Cuphea hookeriana (Chook) FATB4 coding DNA sequence
Cuphea hookeriana (Chook) FATB4 coding DNA sequence codon optimized for
Prototheca moriformis
Cuphea avigera var. pulcherrima (Ca) FATB1 coding DNA sequence
Cuphea avigera var. pulcherrima (Ca) FATB1 coding DNA sequence codon
Cuphea paucipetala (Cpau) FATB1 amino acid sequence
Cuphea paucipetala (Cpau) FATB1 coding DNA sequence
Cuphea paucipetala (Cpau) FATB1 coding DNA sequence codon optimized for
Prototheca moriformis
Cuphea procumbens (Cproc) FATB1 amino acid sequence
Cuphea procumbens (Cproc) FATB1 coding DNA sequence
Cuphea procumbens (Cproc) FATB1 coding DNA sequence codon optimized for
Prototheca moriformis
Cuphea procumbens (Cproc) FATB2 amino acid sequence
Cuphea procumbens (Cproc) FATB2 coding DNA sequence
Cuphea procumbens (Cproc) FATB2 coding DNA sequence codon optimized for
Prototheca moriformis
Cuphea procumbens (Cproc) FATB3 amino acid sequence
Cuphea procumbens (Cproc) FATB3 coding DNA sequence
Cuphea procumbens (Cproc) FATB3 coding DNA sequence codon optimized for
Prototheca moriformis
Cuphea ignea (Cignea) FATB1 amino acid sequence
Cuphea ignea (Cignea) FATB1 coding DNA sequence
Cuphea ignea (Cignea) FATB1 coding DNA sequence codon optimized for
Prototheca moriformis
MVAAAATSAFFPVPAPGTSPNPRKFGSWPSSLSPSLPKSIPNGGFQVKANASAFIPKANGSAVSLKSGSLNTQ
MVAAAATSAFFPVPAPGTSPNPRKFGSWPSSLSPSLPNSIPNGGFQVKANASAHPKANGSAVSLKSGSLNTQ
MVAAAASSAFFSVPVPGTSPKPGKFRIWPSSLSPSFKPKPIPNGGLQVKANSRAHPKANGSAVSLKSGSLNT
MVAAAASSAFFPVPAPGTSPKPGKSGNWPSSLSPSIKPMSIPNGGFQVKANASAHPKANGSAVNLKSGSLN
MVAAAASSAFFPAPAPGSSPKPGKSGNWPSSLSPSFKSKSIPYGRFQVKANASAHPKANGSAVNLKSGSLN
MVAAAASSAFFPAPAPGSSPKPGKSGNWPSSLSPSFKSKSIPYGRFQVKANASAHPKANGSAVNLKSGSLN
MVAAAASSAFFPAPAPGSSPKPGKSGNVVPSSLSPSFKSKSIPYGRFQVKANASAHPKANGSAVNLKSGSLN
PGTSRKTGKFGNWPSSLSPSFKPKSIPNGGFQVKANARAHPKANGSAVSLKSVSLNTQEDTSLSPPPRAFLN
Umbellularia californica UcFATB3 amino acid sequence
Cuphea carthagenensis CCrFATB2c (V138L variant of FATB2)
Cuphea carthagenensis CCrFATB2
This application is a continuation of U.S. application Ser. No. 14/209,931, filed on Mar. 13, 2014 and issued as U.S. Pat. No. 9,783,836 on Oct. 10, 2017, which is a continuation-in-part of U.S. patent application Ser. No. 13/837,996, filed Mar. 15, 2013 and issued as U.S. Pat. No. 9,290,749 on Mar. 22, 2016, and claims the benefit of U.S. Provisional Patent Application Ser. No. 61/791,861, filed Mar. 15, 2013, and U.S. Provisional Patent Application Ser. No. 61/917,217, filed Dec. 17, 2013, all of which are hereby incorporated by reference herein in their entireties for all purposes.
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| WO 2014151904 | Sep 2014 | WO |
| WO 2015051319 | Apr 2015 | WO |
| WO 2016014968 | Jan 2016 | WO |
| WO 2016044779 | Mar 2016 | WO |
| Entry |
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| Number | Date | Country | |
|---|---|---|---|
| 20180208953 A1 | Jul 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61917217 | Dec 2013 | US | |
| 61791861 | Mar 2013 | US |
| Number | Date | Country | |
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| Parent | 14209931 | Mar 2014 | US |
| Child | 15727624 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13837996 | Mar 2013 | US |
| Child | 14209931 | US |
