# Patent

## Title
DGAT genes comprising pleckstrin homology domains and methods of use for triglyceride production in recombinant microorganisms

## Abstract

The present invention provides novel diacylglycerol acyltransferase (DGAT) genes comprising Pleckstrin Homology (PH) domains. The present invention also provides for recombinant cells, such as algae, transformed with acyltransferase genes, such as DGAT, comprising PH domains, and methods of using such recombinant cells to produce increased triglyceride levels.

## Background
REFERENCE TO A SEQUENCE LISTING
This application contains references to amino acid sequences and/or nucleic acid sequences which have been submitted concurrently herewith as the sequence listing text file “61038985—1. txt”, file size 150 KiloBytes (KB), created on 6 Dec. 2012. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R.§1.52(e)(iii)(5).
FIELD
The present invention relates to diacylglycerol acyltransferase (DGAT) genes and the production of triglyceride (TAG) in a recombinant microorganism or host cell engineered to increase and/or maximize TAG synthesis; and to methods of producing TAG using such recombinant microorganisms or host cells. The present invention also relates to Pleckstrin homology (PH) domains and to methods for increasing TAG synthesis by expressing a DGAT comprising a PH domain.
BACKGROUND
Producing renewable sources for a variety of fuels and chemicals is of great importance to a world with increasing demand for such products. While petroleum is a product of decayed plant and other matter that has been incubated beneath the earth's surface for millions of years, some efforts today focus on the direct use of plants and other organisms to generate, e.g., lipids, which can include fatty acids and derivatives thereof, for use in the fuel and chemical industries. Specifically, recent effort has been directed to designing algae to produce lipids for biofuel production because algae can proliferate over a wide range of environmental conditions and because algae do not compete with food crops for arable growth space. See, Hu et al. (2008) Plant J. 54:621-39.
Algal cells are a promising source of biofuels. Wijffels & Barbosa (2010) Science 329:796-99. Their ability to harness solar energy to convert carbon dioxide into carbon-rich lipids already exceeds the abilities of oil-producing agricultural crops, with the added advantage that algae grown for biofuel do not compete with crops for agricultural land (Wijffels & Barbosa, 2010). In order to maximize algal fuel production, new algal strains will need to be engineered for growth and carbon fixation at an industrial scale (Wijffels & Barbosa, 2010).
Triacylglycerol or triglyceride (TAG), a heterogeneous group of molecules with a glycerol backbone and three fatty acids attached by ester bonds, is an excellent molecule for high-concentration metabolic-energy storage. TAG is the major form of energy storage in many eukaryotic algae under stress conditions, such as under nutrient limitation or depletion. Nitrogen depletion (where there is essentially no available nitrogen in the culture medium) is particularly effective in increasing TAG production in many eukaryotic algal species. However, culturing algae under nitrogen deficiency simultaneously limits overall lipid productivity of the culture by limiting overall biomass accumulation (Brennan & Owende (2010) Renewable and Sustainable Energy Reviews 14:557-77). Improving the scalability, controllability, and cost-effectiveness of TAG production would be beneficial to the development of renewable energy and chemical sources.
One means of boosting TAG production is to grow algae in a two-step process alternating between nutrient-rich and nutrient-limited conditions. The nutrient-rich growth phase allows the algae to proliferate, while nutrient limitation (e.g., nitrogen depletion) results in the production of storage lipids. See, Rodolfi et al. (2009) Biotechnol. Bioeng. 102:100-12. This process makes TAG production more expensive, because it requires long periods of growth during which the algae are producing little to no TAG.
Another means of boosting TAG production is to grow the algae heterotrophically by supplying extra organic carbon. For example, in various scenarios, organic carbon may be supplied as glycerol, one or more sugars, one or more organic acids, or other reduced carbon sources added to the growth medium. See, Allnutt et al. (WO 2011/026008). This heterotrophic growth technique not only increases the expense of TAG production, it also risks the contamination of the algal cultures with exogenous bacteria or fungi whose growth can be stimulated by the added carbohydrates. See, Scott et al. (2010) Curr. Opin. Biotechnol. 21:277-86.
The biosynthesis pathways leading to the production of TAG have been studied. In the final reaction of the Kennedy pathway, diacylglycerol (DAG), a precursor to both membrane and storage lipids, is covalently linked to a fatty acyl to produce TAG. This reaction is catalyzed by the diacylglycerol acyltransferase (DGAT) enzyme (Kennedy (1961) Fed. Pro. Fed. Am. Soc. Exp. Biol. 20:934-40). There are two distinct gene families in eukaryotic organisms which encode enzymes which catalyze this reaction, DGAT1 and DGAT2, which have little sequence similarity. Evidence from higher plants as well as mammals suggests that the two gene families have different functions, although the exact role of each type of DGAT has not been elucidated, and may differ in different species (Yen et al. (2008) J. Lipid Res. 49:2283-301).
Although the use of recombinant DGAT enzymes to enhance TAG production in oleaginous organisms is known to the art (Xu et al. (2008) Plant Biotechnol. J. 6:799-818), comparatively little attention has been given to the subcellular localization of these recombinant DGAT enzymes.
It has recently been reported that a DGAT1 gene in the diatom species Phaeodactylum tricornutum contains a PH domain-encoding sequence. However, PH domains are not found in known plant DGATs, despite close evolutionary relationships to orthologous algal DGATs. See, FIGS. 1 & 2.
Guiheneuf et al. (WO 2012/059925) reports a PH domain at the amino-terminal end of a DGAT1 in Phaeodactylum tricornutum. 
Further, Liu et al. (CN 102492672) report a DGAT1 sequence from the diatom Thalassiosira pseudonana with a PH domain at the amino-terminal end.
SUMMARY
The present invention provides novel acyltransferase genes that include Pleckstrin Homology (PH) domains. The presence of a PH domain in a DGAT gene introduced into a cell is demonstrated to improve lipid production in the recombinant cell as compared with a cell that includes a DGAT gene that lacks the PH domain. DGAT1 genes from a variety of algal species, each of which includes a sequence encoding a PH domain, are disclosed herein. As demonstrated herein, in addition to the diatoms Thalassiosira, Phaeodactylum, Navicula, Fragilariopsis, and Cyclotella, the PH domain is found in DGAT1 genes of the green algae Botryococcus, Chlorella, and Tetraselmis. This is a surprising since green algae (division Chlorophyta) are more closely related to higher plants than to the diatoms (division Heterokontophyta) (FIG. 1), and yet documented higher plant DGATs do not have PH domains. The present invention also provides novel PH domains, which may be operably linked to acyltransferases, such as DGATs, including acyltransferases that do not naturally include PH domains, to provide acyltransferases having heterologous PH domains, whose expression may enhance TAG production. The present invention also provides recombinant cells, such as fungi, heterokonts, and algae, transformed with acyltransferase genes, such as DGAT genes, that include sequences encoding homologous or heterologous PH domains, and their use in producing TAG. Novel methods of TAG production are provided herein that result in increased amounts of TAG being produced by a culture of a recombinant microorganism with respect to the amount produced by a control microorganism.
In one aspect, an isolated or recombinant DNA molecule is provided which comprises a nucleotide sequence encoding a DGAT that comprises an amino acid sequence selected from the group consisting of: an amino acid sequence having at least 80% identity to SEQ ID NO:2 or a functional fragment thereof; an amino acid sequence having at least 80% identity to SEQ ID NO:8 or a functional fragment thereof; an amino acid sequence having at least 80% identity to SEQ ID NO:14 or a functional fragment thereof; an amino acid sequence having at least 80% identity to SEQ ID NO:20 or a functional fragment thereof; an amino acid sequence having at least 80% identity to SEQ ID NO:26 or a functional fragment thereof; and an amino acid sequence having at least 80% identity to SEQ ID NO:32 or a functional fragment thereof.
Also provided is an isolated or recombinant DNA molecule which comprises a nucleotide sequence encoding a PH domain selected from the group consisting of: an amino acid sequence having at least 80% identity to SEQ ID NO:6 or a functional fragment thereof; an amino acid sequence having at least 80% identity to SEQ ID NO:12 or a functional fragment thereof; an amino acid sequence having at least 80% identity to SEQ ID NO:18 or a functional fragment thereof; an amino acid sequence having at least 80% identity to SEQ ID NO:24 or a functional fragment thereof; an amino acid sequence having at least 80% identity to SEQ ID NO:30 or a functional fragment thereof; and an amino acid sequence having at least 80% identity to SEQ ID NO:36 or a functional fragment thereof.
An isolated or recombinant DNA molecule as provided herein can comprise a nucleotide sequence encoding an amino acid sequence having at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to any one or more of SEQ ID NOs:2, 4, 8, 10, 14, 16, 20, 22, 26, 28, 32, 34, 38, 40, 44, and 46.
Also provided is an isolated or recombinant DNA molecule which comprises a nucleotide sequence encoding a PH domain, operably linked to a nucleotide sequence encoding a polypeptide of interest, where the PH domain is heterologous with respect to the polypeptide of interest. For example, an isolated DNA molecule encoding a PH domain described herein can be operably linked to a nucleotide sequence encoding a polypeptide of interest, such as but not limited to an acyltransferase, such as, for example, a monoacylglycerol acyltransferase (MGAT), a diacylglycerol acyltransferase type 1 (DGAT1), a diacylglycerol acyltransferase type 2 (DGAT2), a diacylglycerol acyltransferase type 3 (DGAT3), a wax synthase (WS) that has DGAT activity (WS/DGAT), a diacylglycerol acetyltransferase (DGAcT), a diacylglycerol transacylase (DGTA), a phospholipid:diacylglycerol acyltransferase (PDAT), a lysophospholipid acyltransferase (LPLAT), a lysophosphatidic acid acyltransferase (LPAAT), a glycerolphosphate acyltransferase (GPAT), or a combination thereof. In particular examples, the PH domain has at least 80% identity to SEQ ID NO:6 or a functional fragment thereof, at least 80% identity to SEQ ID NO:12 or a functional fragment thereof, at least 80% identity to SEQ ID NO:18 or a functional fragment thereof, at least 80% identity to SEQ ID NO:24 or a functional fragment thereof, at least 80% identity to SEQ ID NO:30 or a functional fragment thereof, at least 80% identity to SEQ ID NO:36 or a functional fragment thereof, at least 80% identity to SEQ ID NO:42 or a functional fragment thereof, and/or at least 80% identity to SEQ ID NO:48 or a functional fragment thereof.
Also provided herein is an expression cassette. The expression cassette comprises a gene as provided herein operably linked to a heterologous promoter. The heterologous gene can encode an acyltransferase that includes a PH domain, for example, a DGAT having of an amino acid sequence with at least 80% identity to SEQ ID NO:2 or a functional fragment thereof; an amino acid sequence with at least 80% identity to SEQ ID NO:8 or a functional fragment thereof; an amino acid sequence with at least 80% identity to SEQ ID NO:14 or a functional fragment thereof; an amino acid sequence with at least 80% identity to SEQ ID NO:20 or a functional fragment thereof; an amino acid sequence with at least 80% identity to SEQ ID NO:26 or a functional fragment thereof; an amino acid sequence with at least 80% identity to SEQ ID NO:32 or a functional fragment thereof; an amino acid sequence with at least 80% identity to SEQ ID NO:38 or a functional fragment thereof; and/or an amino acid sequence with at least 80% identity to SEQ ID NO:44 or a functional fragment thereof. Alternatively or additionally, the expression cassette can comprise an isolated DNA molecule encoding a PH domain, such as, for example, a PH domain as described herein having at least 80% identity to SEQ ID NOs:6, 12, 18, 24, 30, 36, 42, or 48, any of which can be operably linked to a nucleotide sequence encoding a protein of interest, for example an acyltransferase enzyme such as a MGAT, DGAT1, DGAT2, DGAT3, WS/DGAT, DGAcT, DGTA, PDAT, LPLAT, LPAAT, or GPAT. For example, the expression cassette can include a nucleotide sequence encoding a PH domain upstream of, and in frame with, a sequence encoding a heterologous DGAT. The expression cassette can be provided in a vector, e.g., an expression vector, which can optionally include one or more of an origin of replication, sequences mediating recombination into a host genome, and a selectable marker.
Further provided herein is a method of targeting an ectopically-expressed protein to a membrane surface using a PH domain as described herein. For example, the method can include targeting an ectopically-expressed protein to endoplasmic reticulum membrane, the chloroplast envelope, or the plasma membrane of a cell, using a PH domain, such as, for example, any as described herein. The method comprises transfecting a cell with an expression vector comprising a nucleic acid sequence encoding a PH domain as described herein, for example, a PH domain having at least 80% identity to SEQ ID NO:6 or a functional fragment thereof, at least 80% identity to SEQ ID NO:12 or a functional fragment thereof, at least 80% identity to SEQ ID NO:18 or a functional fragment thereof, at least 80% identity to SEQ ID NO:24 or a functional fragment thereof, at least 80% identity to SEQ ID NO:30 or a functional fragment thereof, at least 80% identity to SEQ ID NO:36 or a functional fragment thereof, at least 80% identity to SEQ ID NO:42 or a functional fragment thereof, and/or at least 80% identity to SEQ ID NO:48 or a functional fragment thereof, operably linked to a nucleic acid sequence encoding a protein for ectopic expression. In particular examples, the ectopically-expressed protein can be selected from the group consisting of a MGAT, DGAT1, DGAT2, DGAT3, WS/DGAT, DGAcT, DGTA, PDAT, LPLAT, LPAAT, or GPAT, and a combination thereof; and in a particular embodiment a DGAT1 comprises or is the ectopically-expressed protein.
Further provided herein is a recombinant eukaryotic microorganism that includes a non-native gene encoding an acyltransferase that includes a PH domain, in which the eukaryotic microorganism produces a greater amount of triglyceride than is produced by a eukaryotic microorganism substantially identical to the recombinant eukaryotic microorganism, but lacking a non-native gene encoding the acyltransferase that includes a PH domain. The acyltransferase can be, for example, a monoacylglycerol acyltransferase (MGAT), a diacylglycerol acyltransferase type 1 (DGAT1), a diacylglycerol acyltransferase type 2 (DGAT2), a diacylglycerol acyltransferase type 3 (DGAT3), a wax synthase (WS) that has DGAT activity (WS/DGAT), a diacylglycerol acetyltransferase (DGAcT), a diacylglycerol transacylase (DGTA), a phospholipid:diacylglycerol acyltransferase (PDAT), a lysophospholipid acyltransferase (LPLAT), a lysophosphatidic acid acyltransferase (LPAAT), or a glycerolphosphate acyltransferase (GPAT). In some examples, the acyltransferase is a DGAT, for example, a DGAT1, a DGAT2, WS/DGAT, or a DGAT3. The PH domain-encoding sequence of a non-native acyltransferase gene as provided herein can be naturally-occurring in the acyltransferase, e.g., DGAT1, gene (i.e., homologous with respect to the acyltransferase gene) or can be heterologous with respect to the acyltransferase gene, i.e., not naturally occurring in the acyltransferase gene. In some examples, the non-native gene can encode a DGAT that includes a PH domain, where the DGAT comprises the sequence of a naturally occurring DGAT that includes a PH domain or is a variant of a naturally-occurring DGAT that includes a PH domain, such as, for example, a DGAT having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to a naturally-occurring DGAT or an active fragment thereof. Additionally or alternatively, the acyltransferase can be a PH domain-containing DGAT1 of a green alga or derived from the sequence of a PH domain-containing DGAT1 of a green alga, e.g., an alga of the Chlorophyte division.
Additionally or alternatively, a recombinant eukaryotic microorganism can include a non-native gene encoding an acyltransferase that has at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to a naturally-occurring acyltransferase or an active fragment thereof, where the acyltransferase-encoding sequence is operably linked to a heterologous sequence encoding a PH domain, such as, for example, a PH domain having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to any of SEQ ID NOs:6, 12, 18, 24, 30, 36, 42, or 48. For example, a recombinant eukaryotic microorganism or alga can include a non-native gene encoding a DGAT that has at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to a naturally-occurring DGAT or an active fragment thereof where the DGAT encoding sequence is operably linked to a heterologous sequence encoding a PH domain, such as, for example, a PH domain having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to any of SEQ ID NOs:6, 12, 18, 24, 30, 36, 42, or 48.
The non-native gene can additionally or alternatively encode a PH-domain including DGAT that is a truncated variant of a naturally-occurring DGAT, such as an N-terminally or C-terminally truncated variant of a naturally-occurring DGAT or a DGAT having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to a naturally-occurring DGAT. In further examples, the acyltransferase can be a DGAT1 having at least 80% identity to SEQ ID NO:2 or a functional fragment thereof; an amino acid sequence having at least 80% identity to SEQ ID NO:8 or a functional fragment thereof; an amino acid sequence having at least 80% identity to SEQ ID NO:14 or a functional fragment thereof; an amino acid sequence having at least 80% identity to SEQ ID NO:20 or a functional fragment thereof; an amino acid sequence having at least 80% identity to SEQ ID NO:26 or a functional fragment thereof; or an amino acid sequence having at least 80% identity to SEQ ID NO:32 or a functional fragment thereof.
For example, a recombinant eukaryotic alga can include a non-native gene encoding a DGAT that has at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to a naturally-occurring DGAT or an active fragment thereof where the DGAT encoding sequence is operably linked to a heterologous sequence encoding a PH domain, such as, for example, a PH domain having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to any of SEQ ID NOs:6, 12, 18, 24, 30, 36, 42, or 48.
For example, a recombinant microorganism as provided herein can comprise a non-native gene encoding a PH domain, wherein the PH domain comprises an amino acid sequence having at least 80% identity to SEQ ID NOs:6, 12, 18, 24, 30, or 36, and/or to an active fragment of any thereof. For example, the non-native gene can encode a PH domain having an amino acid sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one or more of SEQ ID NOs:6, 12, 18, 24, 30, or 36. Within the recombinant microorganism, the PH domain can be linked to any protein of interest, for example, a DGAT.
A recombinant microorganism that includes a gene encoding a non-native DGAT comprising a PH domain can produce a greater amount of at least one lipid than a substantially identical microorganism lacking the recombinant DGAT gene that includes a PH domain-encoding sequence. For example, the recombinant microorganism, that includes a gene encoding a non-native DGAT comprising a native or heterologous PH domain, can produce a greater amount of TAG in a twenty-four hour, three day, and/or seven day time period than a substantially identical microorganism lacking the recombinant DGAT gene.
Further, the recombinant microorganism that includes a gene encoding a non-native DGAT comprising a PH domain, which may optionally be a heterologous PH domain with respect to the DGAT, can be a eukaryotic microalga, and preferably can, under photoautotrophic culture conditions, produce a greater amount of lipid than a substantially identical microorganism lacking the recombinant DGAT gene. Additionally or alternatively, the recombinant eukaryotic microalga can, under nutrient replete culture conditions, or when cultured under nitrogen-replete conditions, produce a greater amount of lipid than a substantially identical microorganism lacking the recombinant DGAT gene. Preferably, the recombinant eukaryotic microalga can, under photoautotrophic nutrient replete culture conditions and/or under photoautotrophic nitrogen replete culture conditions, produce a greater amount of lipid than a substantially identical microorganism lacking the recombinant DGAT gene.
Further additionally or alternatively, the recombinant eukaryotic microorganism that includes a non-native gene encoding a DGAT that includes a PH domain can produce a greater amount of triglyceride than is produced by a microorganism substantially identical to the recombinant microorganism, when the microorganisms are cultured under nitrogen replete conditions.
The recombinant eukaryotic microorganism that includes a non-native gene encoding an acyltransferase having a PH domain can be an alga, for example, a microalga such as for example, a species of a genus selected from the group consisting of Achnanthes, Amphiprora, Amphora, Ankistrodesmus, Asteromonas, Boekelovia, Bolidomonas, Borodinella, Botrydium, Botryococcus, Bracteococcus, Chaetoceros, Carteria, Chlamydomonas, Chlorococcum, Chlorogonium, Chlorella, Chroomonas, Chrysosphaera, Cricosphaera, Crypthecodinium, Cryptomonas, Cyclotella, Dunaliella, Ellipsoidon, Emiliania, Eremosphaera, Ernodesmius, Euglena, Eustigmatos, Franceia, Fragilaria, Fragilaropsis, Gloeothamnion, Haematococcus, Halocafeteria, Heterosigma, Hymenomonas, Isochrysis, Lepocinclis, Micractinium, Monoraphidium, Nannochloris, Nannochloropsis, Navicula, Neochloris, Nephrochloris, Nephroselmis, Nitzschia, Ochromonas, Oedogonium, Oocystis, Ostreococcus, Pavlova, Parachlorella, Pascheria, Pelagomonas, Phaeodactylum, Phagus, Picochlorum, Platymonas, Pleurochrysis, Pleurococcus, Prototheca, Pseudochlorella, Pseudoneochloris, Pseudostaurastrum, Pyramimonas, Pyrobotrys, Scenedesmus, Schizochlamydella, Skeletonema, Spyrogyra, Stichococcus, Tetrachlorella, Tetraselmis, Thalassiosira, Tribonema, Vaucheria, Viridiella, Vischeria, and Volvox. As nonlimiting examples, the microalga can be a Nannochloropsis species, a Tetraselmis species, a Chlorella species, or a diatom species (for example, a species of Amphora, Chaetoceros, Cyclotella, Fragilaria, Fragilaropsis, Navicula, Phaeodactylum, or Thalassiosira).
For example, a recombinant eukaryotic alga as provided herein can include a non-native gene encoding a type-1, type-2, or type-3 DGAT or WS/DGAT, such as, for example, a fungus, yeast, bacterium, protozoan, animal, alga, or plant, or a variant thereof having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the naturally-occurring DGAT1, DGAT2, DGAT3, WS/DGAT, or an active fragment thereof, and which can include a deletion, for example, a truncation, with respect to the naturally-occurring DGAT amino acid sequence, and/or can include one or more additional sequences, in addition to a PH domain. For example, the DGAT encoding sequence can be operably linked to a heterologous sequence encoding a PH domain, such as, for example, a PH domain having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to any of SEQ ID NOs:6, 12, 18, 24, 30, 36, 42, or 48.
In some examples, the DGAT1, DGAT2, WS/DGAT, or DGAT3 encoded by the non-native gene comprises a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:44, and/or an active portion thereof. For example, the non-native gene can encode a DGAT having an amino acid sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one or more of SEQ ID NOs:2, 8, 14, 20, 26, 32, 38, or 44. In some examples, the DGAT comprises or is a DGAT1.
Also provided is a method for producing TAG in a recombinant eukaryotic microorganism. The method comprises culturing a recombinant microorganism as described herein that comprises a gene encoding a non-native DGAT that includes a native or heterologous PH domain, such as any disclosed herein, under culture conditions such that a DGAT encoded by gene is expressed to produce TAG. The DGAT expressed can be encoded by any DGAT gene, such as any disclosed herein, and can be configured in an expression cassette optionally to comprise a PH as described herein (e.g. SEQ ID NOs:6, 12, 18, 24, 30, 36, 42, 48, and/or sequences having at least 80% identity thereto). In some examples, the culture conditions can be nitrogen replete. Additionally or alternatively, the culture conditions can be nutrient replete. Further additionally or alternatively, the culture conditions can be substantially or entirely photoautotrophic. Still further additionally or alternatively, the recombinant microorganisms described herein can produce a greater amount of TAG under nitrogen replete conditions than is produced by a control microorganism substantially identical to the recombinant microorganism in all respects and cultured under the substantially same conditions, except that the control microorganism does not include a recombinant DGAT gene. In some examples, the recombinant microorganism can be proliferating and generating TAG at levels higher than the TAG levels generated by a non-transfected control microorganism cultured under substantially the same conditions. For example, the recombinant microorganism can generate at least 50% more lipid in comparison to a non-transfected control cell or at least two fold, at least five fold, and/or at least ten fold the amount of TAG produced by a non-transfected control cell in a twenty-four hour period, a three day period, and/or a seven day period. In certain examples, the DGAT expressed can have an amino acid sequence having at least 80% identity to SEQ ID NOs:2, 8, 14, 20, 26, 32, 38, and/or 44. For example, an amino acid sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one or more of SEQ ID NOs:2, 8, 14, 20, 26, 32, 38, and 44. In some examples, the DGAT can comprise or be a DGAT1.
Also provided is a further method for producing TAG in a recombinant eukaryotic algal cell. The method comprises culturing a recombinant microorganism as provided herein, comprising a recombinant nucleic acid molecule encoding a polypeptide having acyltransferase activity, wherein the polypeptide comprises a heterologous PH domain, under conditions in which the recombinant nucleic acid molecule is expressed, to produce TAG. For example, the heterologous PH domain can be selected from SEQ ID NOs:6, 12, 18, 24, 30, 36, 42, 48, and/or sequences having at least 80% identity thereto and the polypeptide having acyltransferase activity can be a MGAT, DGAT1, DGAT2, DGAT3, WS/DGAT, DGAcT, DGTA, PDAT, LPLAT, LPAAT, or GPAT. Additionally or alternatively, the culture conditions can be nutrient replete. Further additionally or alternatively, the culture conditions can be substantially or entirely photoautotrophic. Still further additionally or alternatively, the recombinant microorganism described herein can produce a greater amount of TAG under nitrogen replete conditions than is produced by a control microorganism substantially identical to the recombinant microorganism in all respects and cultured under the substantially same conditions, except that the control microorganism does not comprise an acyltransferase comprising a heterologous PH domain that can direct the acyltransferase to a location within the cell. In some examples, the recombinant microorganism can be proliferating and generating TAG at levels higher than the TAG levels generated by a non-transfected control microorganism cultured under substantially the same conditions. For example, the recombinant microorganism can generate at least 50% more lipid in comparison to a non-transfected control cell or at least two fold, at least five fold, and/or at least ten fold the amount of TAG produced by a non-transfected control cell in a twenty-four hour period, a three day period, and/or a seven day period. In certain examples, the PH domain expressed can have an amino acid sequence having at least 80% identity to SEQ ID NOs:6, 12, 18, 24, 30, 36, 42, and/or 48. For example, an amino acid sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one or more of SEQ ID NOs:6, 12, 18, 24, 30, 36, 42, and 48. In some examples, the polypeptide having acyltransferase activity can comprise or be a DGAT1.
Further areas of applicability will become apparent from the description provided herein. The description and specific examples in this summary are intended for purposes of illustration only and are not intended to limit the scope of the present disclosure.



BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The drawings described herein are for illustrative purposes only of selected embodiments and not all possible implementations, and are not intended to limit the scope of the present disclosure.
FIG. 1 is a phylogenetic tree of DGAT1 genes identified from proprietary and public algal genomes. The plant genes shown in the lower third of the figure do not include the PH domain and are included for reference purposes.
FIG. 2A-F is an amino acid sequence alignment (constructed in Vector NTI, Life Technologies Inc.). It contains all of the novel fusion DGAT1 enzymes found by manually curating genes from public algal genomes. The PH domain is located from approximately position 53 to 205 of the Cyclotella sequence.
FIG. 3 is a schematic of the DGAT1 genes from algae that include the PH domain along with the MBOAT protein domain characteristic of DGATs.
FIG. 4 summarizes total TAG production from yeast transfected to express a full length Cyclotella DGAT1 (DGAT1-452 c13-p416) and a truncated Cyclotella DGAT1 lacking the PH domain (DGAT1-452T p416). TAG production from a yeast transfected with the Marinobacter WS1 wax-synthase (WS1 p416) is included as a positive control. TAG from wild-type yeast (WT-BY4741) and yeast transfected with empty vector (EV p416) are included as negative controls. Production of TAG is clearly improved when the full-length DGAT1, containing the PH domain, is expressed in the host strain.
FIG. 5 summarizes TAG production, normalized for cell count, from a full length Cyclotella DGAT1 (black bar) and a truncated Cyclotella DGAT1 lacking the PH domain (striped bar) in yeast. TAG from wild-type yeast (WT-BY4741) and yeast transfected with empty vector (EV p416) are included as negative controls. Production of TAG is clearly improved when the full-length DGAT1, containing the PH domain, is expressed in the host strain.
FIG. 6 summarizes TAG production from full-length Phaeodactylum DGAT1 clones (PhaeDGAT1-1 and -2) and truncated Phaeodactylum DGAT1 clones lacking a PH domain (PhaeDGAT1 T2 and T3), full length Thalassiosira DGAT1 clones (Tha1DGAT1-2) and truncated Thalassiosira DGAT1 clones lacking a PH domain (Tha1DGAT1-T2 and -T3). The negative control is the quadruple knockout yeast strains carrying the empty vector (EV), which produces no detectable TAG. The positive control (FFWS-1) is the quadruple knockout yeast strain expressing the Marinobacter WS1 wax-synthase.
FIG. 7 summarizes total fatty acid methyl-ester (FAME)s production from full-length Phaeodactylum DGAT1 clones (PhaeDGAT1-1 and -2) and truncated Phaeodactylum DGAT1 clones lacking a PH domain (PhaeDGAT1 T2 and T3), full length Thalassiosira DGAT1 clones (Tha1DGAT1-2) and truncated Thalassiosira DGAT1 clones lacking a PH domain (Tha1DGAT1-T2 and -T3). The negative control is the quadruple knockout yeast strains carrying the empty vector (EV), which produces no detectable TAG. The positive control (FFWS-1) is the quadruple knockout yeast strain expressing the Marinobacter WS1 wax-synthase.
FIG. 8 summarizes TAG per total organic carbon (TOC) obtained from Nannochloropsis transformed with full-length algal DGAT1 genes from: Cyclotella (−DGAT); Phaeodactylum (Phae); Thalassiosira (Thal); Marinobacter WS1 (FFWS1 3′FT) (positive control); Empty vector (EV) (negative control). For comparison sake, TAG/TOC from two untransformed, clonal wild-type cultures (E-3730) are shown, one on each side of the graph. Nannochloropsis cultures were grown for two days under nitrogen replete conditions prior to analyzing the cultures for TAG by HPLC. Expression of algal DGAT1 genes that included the PH domain resulted in increased levels of TAG with respect to wild type.
FIG. 9 summarizes FAME per TOC obtained from Nannochloropsis transformed with full-length algal DGAT1 genes from: Cyclotella (−DGAT); Phaeodactylum (Phae); Thalassiosira (Thal); Marinobacter WS1 (FFWS1 3′FT) (positive control); Empty vector (EV) (negative control). For comparison sake, TAG/TOC from two untransformed, clonal wild-type cultures (E-3730) are show, one on each side of the graph. Nannochloropsis cultures were grown for two days under nitrogen replete conditions prior to analyzing the cultures for TAG by HPLC. Expression of algal DGAT1 genes that included the PH domain resulted in increased levels of TAG with respect to wild type.



DETAILED DESCRIPTION
Definitions
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application including the definitions will control. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications, patents and other references mentioned herein are incorporated by reference in their entireties for all purposes as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.
Although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention, suitable methods and materials are described below. The materials, methods and examples are illustrative only and are not intended to be limiting. Other features and advantages of the invention will be apparent from the detailed description and from the claims.
To facilitate an understanding of the present invention, a number of terms and phrases are defined below.
As used in the present disclosure and claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise.
Wherever embodiments are described herein with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of” and/or “consisting essentially of” are also provided.
The term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B”, “A or B”, “A”, and “B”.
The terms “cells”, “cell cultures”, “cell line”, “recombinant host cells”, “recipient cells” and “host cells” as used herein include the primary subject cells and any progeny thereof, without regard to the number of transfers. It should be understood that not all progeny are exactly identical to the parental cell (due to deliberate or inadvertent mutations or differences in environment); however, such altered progeny are included in these terms, so long as the progeny retain substantially the same functionality as that of the originally transformed cell.
The term “gene” is used broadly to refer to any segment of a nucleic acid molecule (typically DNA, but optionally RNA) that encodes a protein or that can be transcribed into a functional RNA. Genes may include sequences that are transcribed but are not part of a final, mature, and/or functional RNA transcript, and genes that encode proteins may further comprise sequences that are transcribed but not translated, for example, 5′ untranslated regions, 3′ untranslated regions, introns, etc. Further, genes may optionally further comprise regulatory sequences required for their expression, and such sequences may be e.g., sequences that are not transcribed or translated. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.
The terms “nucleic acid”, “nucleic acid molecule”, or “polynucleotide” are used interchangeably herein and refer to, e.g., DNA or RNA (e.g., mRNA). The nucleic acid molecules can be double-stranded or single-stranded; single stranded RNA or DNA can be the coding (sense) strand or the non-coding (antisense) strand.
The terms “coding sequence” or “coding region” as used herein, refer to a region of a DNA sequence that can be transcribed to produce an mRNA transcript that can be translated into an amino acid sequence, e.g., of a peptide or polypeptide or an RNA transcript that can be translated into an amino acid sequence, e.g., of a peptide or a polypeptide. The term “non-coding sequence” or “non-coding region” refers to (1) a region of a DNA sequence that, if transcribed, is not translated into an amino acid sequence (e.g., introns, untranslated regions, etc.); or (2) a region of an RNA sequence that is not translated into amino acids.
A “functional RNA molecule” is an RNA molecule that can interact with one or more proteins or nucleic acid molecules to perform or participate in a structural, catalytic, or regulatory function that affects the expression or activity of a gene or gene product other than the gene that produced the functional RNA. A functional RNA can be, for example, a transfer RNA (tRNA), ribosomal RNA (rRNA), anti-sense RNA (asRNA), microRNA (miRNA), short-hairpin RNA (shRNA), small interfering RNA (siRNA), small nucleolar RNAs (snoRNAs), piwi-interacting RNA (piRNA), or a ribozyme.
A biomolecule may be “derived from” an indicated source, which includes the isolation (in whole or in part) of a nucleic acid segment from an indicated source or the purification of a polypeptide from an indicated source. A nucleic acid molecule may also be derived from an indicated source by, for example, direct cloning, PCR amplification, or artificial synthesis from the indicated polynucleotide source or based on a sequence associated with the indicated polynucleotide source. Genes or nucleic acid molecules derived from a particular source or species also include genes or nucleic acid molecules having sequence modifications with respect to the source nucleic acid molecules. For example, a gene or nucleic acid molecule derived from a source (e.g., a particular referenced gene) can incur one or more mutations with respect to the source gene or nucleic acid molecule that are unintended or that are deliberately introduced. If one or more mutations, including substitutions, deletions, or insertions, are deliberately introduced the sequence alterations can be introduced by random or targeted mutation of cells or nucleic acids, by amplification or other molecular biology techniques, or by chemical synthesis. A gene or nucleic acid molecule that is derived from a referenced gene or nucleic acid molecule that encodes a functional RNA or polypeptide can encode a functional RNA or polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the referenced or source functional RNA or polypeptide, or to a functional fragment thereof.
The term “isolated”, such as an isolated protein or nucleic acid as used herein, refers to a biomolecule removed from the context in which the biomolecule exists in nature. An isolated biomolecule can be, in some instances, partially or substantially purified. For example, an isolated nucleic acid molecule can be a nucleic acid sequence that has been excised from the chromosome, genome, or episome into which it is integrated in nature.
A “purified” nucleic acid molecule or nucleotide sequence, or protein or polypeptide sequence, is substantially free of cellular material and cellular components. The purified nucleic acid molecule or protein may be free of chemicals beyond buffer or solvent, for example. “Substantially free” is not intended to mean that other components beyond the novel nucleic acid molecules are undetectable.
The terms “naturally-occurring” and “wild-type” (WT) refer to a form found in nature. For example, a naturally occurring or wild-type nucleic acid molecule, nucleotide sequence, or protein may be present in, and isolated from, a natural source, and is not intentionally modified by human manipulation.
As used herein “attenuated” means reduced in amount, degree, intensity, or strength. Attenuated gene expression may refer to a significantly reduced amount and/or rate of transcription of the gene in question, or of translation, folding, or assembly of the encoded protein. As nonlimiting examples, an attenuated gene may be a mutated or disrupted gene (e.g., a gene disrupted by partial or total deletion, insertional mutation, or meganuclease disruption), or a gene having decreased expression resulting from alteration of gene regulatory sequences. An attenuated gene may also be a gene that is targeted by a “gene knockdown” construct, such as, for example, a construct encoding an antisense RNA, a microRNA, a short hairpin RNA, or a ribozyme. In the case of both expression of transgenes and suppression of endogenous genes (e.g., by antisense or sense suppression) one of ordinary skill will recognize that the inserted polynucleotide sequence need not be identical, but may be only substantially identical to a sequence of the gene from which it was derived. These substantially identical variants are specifically covered by reference to a specific nucleic acid sequence.
“Exogenous nucleic acid molecule” or “exogenous gene” refers to a nucleic acid molecule or gene that has been introduced (“transformed”) into a cell. A transformed cell may be referred to as a “recombinant” cell, into which additional exogenous gene(s) may be introduced. A descendent of a cell transformed with a nucleic acid molecule is also referred to as “transformed” if it has inherited the exogenous nucleic acid molecule. The exogenous gene may be from a different species (and may in this context be described as “heterologous” with respect to the host organism), or from the same species (and so may in this context be described as “homologous” with respect to the host organism), relative to the cell being transformed. An “endogenous” nucleic acid molecule, gene, or protein is a native nucleic acid molecule, gene, or protein as it occurs in, or is naturally produced by, the host.
Further, the term “exogenous” as used herein in the context of a gene or protein, refers to a gene or protein that is not derived from the host organism species.
The term “transgene” as used herein refers to an exogenous gene, that is, a gene introduced into a microorganism or its progenitor by human intervention.
The term “ortholog” of a gene or protein as used herein refers to its functional equivalent in another species.
The term “native” is used herein to refer to nucleic acid sequences or amino acid sequences as they naturally occur in the host. The term “non-native” is used herein to refer to nucleic acid sequences or amino acid sequences that do not occur naturally in the host. A nucleic acid sequence or amino acid sequence that has been removed from a host cell, subjected to laboratory manipulation, and reintroduced into a host cell is considered “non-native.” Non-native genes include genes endogenous to the host microorganism operably linked to one or more heterologous regulatory sequences that have been inserted into the host genome.
The term “heterologous gene” or “heterologous nucleic acid sequence” as used herein, refers to a gene or nucleic acid sequence from a different species than the species of the host organism into which it is introduced. When used in reference to a polynucleotide, a gene, a nucleic acid, a polypeptide, or an enzyme, the term “heterologous” refers to a polynucleotide, gene, a nucleic acid, polypeptide, or an enzyme not derived from the host species. Further, when referring to a gene regulatory sequence or to an auxiliary nucleic acid sequence used for maintaining or manipulating a gene sequence (e.g. a 5′ untranslated region, 3′ untranslated region, Kozak sequence, poly A addition sequence, intron sequence, splice site, ribosome binding site, internal ribosome entry sequence, genome homology region, recombination site, etc.), “heterologous” means that the regulatory sequence or auxiliary sequence is from a different source than the gene with which the regulatory or auxiliary nucleic acid sequence is juxtaposed in a construct, genome, chromosome, or episome. When referring to a protein functional domain, such as a localization sequence or a receptor binding site, “heterologous” can also mean that the protein functional domain is from a different source than the rest of the protein region with which it is juxtaposed in an engineered protein. Thus, a Pleckstrin Homology domain operably linked to a polypeptide of which it is not part in its natural state (i.e. in the proteome of the non-genetically engineered organism) is referred to herein as a “heterologous Pleckstrin Homology domain,” even though the Pleckstrin Homology domain may be derived from the same species (or, in some cases, the same organism) as the polypeptide to which it is linked. Similarly, when referring to a promoter sequence of an engineered gene, “heterologous” means that the promoter is derived from a different gene than that to which it is linked by genetic engineering.
The terms “recombinant” or “engineered” as used herein in reference to a nucleic acid molecule, refer to a nucleic acid molecule that has been altered through human intervention. As non-limiting examples, a cDNA is a recombinant DNA molecule, as is any nucleic acid molecule that has been generated by in vitro polymerase reaction(s), or to which linkers have been attached, or that has been integrated into a vector, such as a cloning vector or expression vector. As non-limiting examples, a recombinant nucleic acid molecule: 1) has been synthesized or modified in vitro, for example, using chemical or enzymatic techniques (for example, by use of chemical nucleic acid synthesis, or by use of enzymes for the replication, polymerization, exonucleolytic digestion, endonucleolytic digestion, ligation, reverse transcription, transcription, base modification (including, e.g., methylation), or recombination (including homologous and site-specific recombination)) of nucleic acid molecules; 2) includes conjoined nucleotide sequences that are not conjoined in nature; 3) has been engineered using molecular cloning techniques such that it lacks one or more nucleotides with respect to the naturally occurring nucleic acid molecule sequence; and/or 4) has been manipulated using molecular cloning techniques such that it has one or more sequence changes or rearrangements with respect to the naturally occurring nucleic acid sequence.
The term “recombinant protein” as used herein refers to a protein produced by genetic engineering.
When applied to organisms, the terms “transgenic” or “recombinant” or “engineered” or “genetically engineered,” used interchangeably herein, refer to organisms that have been manipulated by introduction into the organism of an exogenous or recombinant nucleic acid sequence. For example, a transgenic microorganism can include an introduced exogenous regulatory sequence operably linked to an endogenous gene of the transgenic microorganism. Non-limiting examples of such manipulations include gene knockouts, targeted mutations and gene replacement, promoter replacement, deletion, or insertion, as well as introduction of transgenes into the organism. Recombinant or genetically engineered organisms can also be organisms into which constructs for gene “knock down” have been introduced. Such constructs include, but are not limited to, RNAi, microRNA, shRNA, antisense, and ribozyme constructs. Also included are organisms whose genomes have been altered by the activity of meganucleases or zinc finger nucleases. A heterologous or recombinant nucleic acid molecule can be integrated into a recombinant/genetically engineered organism's genome or, in other instances, not integrated into a recombinant/genetically engineered organism's genome. As used herein, “recombinant microorganism” or “recombinant host cell” includes progeny or derivatives of the recombinant microorganisms of the invention. Because certain modifications may occur in succeeding generations from either mutation or environmental influences, such progeny or derivatives may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
The terms “regulatory sequence”, “regulatory element”, or “regulatory element sequence” are used interchangeable herein and refer to a nucleotide sequence located upstream (5′), within, or downstream (3′) of a coding sequence. Transcription of the coding sequence and/or translation of an RNA molecule resulting from transcription of the coding sequence are typically affected by the presence or absence of the regulatory sequence. These regulatory element sequences may comprise promoters, cis-elements, enhancers, Kozak sequences, terminators, or introns. Regulatory elements may be isolated or identified from UnTranslated Regions (UTRs) from a particular polynucleotide sequence. Any of the regulatory elements described herein may be present in a chimeric or hybrid regulatory expression element. Any of the regulatory elements described herein may be present in a recombinant construct of the present invention.
The terms “promoter”, “promoter region”, or “promoter sequence” are used interchangeably herein and refer to a nucleic acid sequence capable of binding RNA polymerase to initiate transcription of a gene in a 5′ to 3′ (“downstream”) direction. A gene is “under the control of” or “regulated by” a promoter when the binding of RNA polymerase to the promoter is the proximate cause of said gene's transcription. The promoter region typically provides a recognition site for RNA polymerase and other factors necessary for proper initiation of transcription. A promoter may be isolated from the 5′ untranslated region (5′ UTR) of a genomic copy of a gene. Alternatively, a promoter may be synthetically produced or designed by altering known DNA elements. Also considered are chimeric promoters that combine sequences of one promoter with sequences of another promoter. Promoters may be defined by their expression pattern based on, for example, metabolic, environmental, or developmental conditions. A promoter can be used as a regulatory element for modulating expression of an operably linked transcribable polynucleotide molecule, e.g., a coding sequence. Promoters may contain, in addition to sequences recognized by RNA polymerase and (preferably) other transcription factors, regulatory sequence elements such as cis-elements or enhancer domains that affect the transcription of operably linked genes. An “algal promoter” is a native or non-native promoter that is functional in algal cells.
A promoter can include a transcription initiation site as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters often, but not always, contain “TATA” boxes and “CAT” boxes. Prokaryotic promoters may contain −10 and −35 prokaryotic promoter consensus sequences. A large number of promoters, including constitutive, inducible and repressible promoters, from a variety of different sources are well known in the art. Representative sources include for example, viral, mammalian, insect, plant, yeast, and bacterial cell types, and suitable promoters from these sources are readily available, or can be made synthetically, based on sequences publicly available on line or, for example, from depositories such as the ATCC as well as other commercial or individual sources. Promoters can be unidirectional (i.e., initiate transcription in one direction) or bi-directional (i.e., initiate transcription in both directions off of opposite strands). A promoter may be a constitutive promoter, a repressible promoter, or an inducible promoter. Non-limiting examples of promoters include, for example, the T7 promoter, the cytomegalovirus (CMV) promoter, the SV40 promoter, and the RSV promoter. Examples of inducible promoters include the lac promoter, the pBAD (araA) promoter, the Tet promoter (U.S. Pat. Nos. 5,464,758 and 5,814,618), and the Ecdysone promoter (No et al. (1996) Proc. Natl. Acad. Sci. 93:3346-51). Promoters specific to Nannochloropsis are disclosed in co-pending U.S. patent application Ser. No. 13/486,930, entitled “Promoters and Terminators for Use in Eukaryotic Cells” filed on 1 Jun. 2012.
The term “constitutive” as used herein, refers to a promoter that is active under most environmental and developmental conditions. A constitutive promoter is active regardless of external environment, such as light and medium. In some examples, a constitutive promoter is active in the presence and in the absence of a nutrient. For example, a constitutive promoter may be a promoter that is active (mediates transcription of a gene to which it is operably-linked) under conditions of nitrogen depletion as well as under conditions in which nitrogen is not limiting (nitrogen replete conditions). In contrast, an “inducible” promoter is a promoter that is active in response to particular environmental conditions, such as the presence or absence of a nutrient or regulator, the presence of light, etc.
The term “terminator” or “terminator sequence” or “transcription terminator” as used herein refers to a regulatory section of genetic sequence that causes RNA polymerase to cease transcription.
The term “operably linked” as used herein denotes a configuration in which a regulatory sequence is placed at an appropriate position relative to a polynucleotide or polypeptide sequence such that the regulatory sequence affects or directs expression of the polynucleotide sequence, for example, to produce a polypeptide and/or functional RNA. Thus, a promoter is in operable linkage with a nucleic acid sequence if it can mediate transcription of the nucleic acid sequence. In the context of a polypeptide, an amino acid sequence that comprises a functional domain, such as a targeting sequence or other functional domain, is “operably linked” to the rest of the polypeptide when it is linked in such a way as to effectuate targeting to a subcellular location or otherwise becomes integrated into the polypeptide to affect protein function.
When introduced into a host cell, an expression cassette can result in transcription and/or translation of an encoded RNA or polypeptide under appropriate conditions. Antisense or sense constructs that are not or cannot be translated are not excluded by this definition.
The term “expression cassette” as used herein refers to a nucleic acid construct that contains a nucleic acid sequence, encoding for a protein or functional RNA (e.g., a tRNA, a short hairpin RNA, one or more microRNAs, a ribosomal RNA, etc.) operably linked to expression control elements, such as a promoter, and optionally, any or a combination of other nucleic acid sequences that affect transcription or translation, such as, but not limited to, a transcriptional terminator, a ribosome binding site, a splice site or splicing recognition sequence, an intron, an enhancer, a polyadenylation signal, an internal ribosome entry site, etc.
The term “vector” as used herein refers to a nucleic acid construct that is structured so as to facilitate movement of nucleic acids from one environment, intracellular or extracellular, to another environment, intracellular or extracellular. A vector optimized for use in modifying portions of the nucleic acid is a “cloning vector.” A vector optimized for use in transforming a cell or expressing a gene of interest in a cell is a “transformation vector” or an “expression vector,” these two terms being used interchangeably herein. A vector may optionally include one or more of: an origin of replication; a sequence mediating recombination into a host genome; or a selectable marker.
The term “microorganism” as used herein refers to any organism that is microscopic, i.e. too small to be seen by the naked eye. As used herein, the term “microorganism” may also refer to macroscopic organisms taxonomically related to microscopic organisms within the categories of yeast, heterokonts, algae, plants, bacteria, and fungi (including lichens). A microorganism may be unicellular or multicellular. A microorganism may be a bacterium, rickettsia, protozoon, or fungus. The term “microorganism” also includes microscopic plants and animals such as plankton, planaria, and amoebae, as well as arthropods such as dust mites, spider mites, etc.
The term “photosynthetic organism” as used herein is any prokaryotic or eukaryotic organism that can perform photosynthesis. Photosynthetic organisms include but are not limited to, higher plants (i.e., vascular plants), bryophytes, algae, and photosynthetic bacteria.
The terms “eukaryotic” and “eukaryote” are used in their broadest sense to include any organisms containing membrane bound nuclei and membrane bound organelles. Examples of eukaryotes include but are not limited to plants, yeast, animals, algae, diatoms, and fungi.
The terms “prokaryote” and “prokaryotic” are used in their broadest sense to include any organisms without a distinct nucleus. Examples of prokaryotes include but are not limited to bacteria, blue-green algae, archaebacteria, actinomycetes, and mycoplasma.
The term “algae” includes, but is not limited to, a species of Bacillariophyceae (diatoms), Bolidomonas, Chlorophyceae (green algae), Chrysophyceae (golden algae), Cyanophyceae (cyanobacteria), Eustigmatophyceae (pico-plankton), Glaucocystophytes, Pelagophytes, Phaeophyceae (brown algae), Prasinophyceae (pico-plankton), Raphidophytes, Rhodophyceae (red algae), Synurophyceae, and Xanthophyceae (yellow-green algae). The term “microalgae” as used herein refers to microscopic, single-celled algae species including, but not limited to, Bacillariophyceae (diatoms), Chlorophyceae, Prasinophyceae, Trebouxiophyceae, and Eustigmatophyceae. The term “algae” includes microalgae. The term “photosynthetic bacteria” includes, but is not limited to, cyanobacteria, green sulfur bacteria, purple sulfur bacteria, purple non-sulfur bacteria, and green non-sulfur bacteria.
The term “selectable marker” or “selectable marker gene” as used herein includes any gene that confers a phenotype on a cell in which it is expressed to facilitate the selection of cells that are transfected or transformed with a nucleic acid construct of the invention. The term may also be used to refer to gene products that effectuate said phenotypes. Examples of selectable markers include:

    
    
        genes conferring resistance to antibiotics such as amikacin (aphA6), ampicillin (Amp®), blasticidin (bls, bsr, bsd), bleomicin or phleomycin (ZEOCINTM) (ble), chloramphenicol (cat), emetine (RBS14p or cry1-1), erythromycin (ermE), G418 or neomycin (GENETICINTM) (neo), gentamycin (aac3 or aacC4), hygromycin B (aphIV, hph, hpt), kanamycin (val), methotrexate (DHFR Mtx®), penicillin and other β-lactams (β-lactamases), streptomycin or spectinomycin (aadA, spec/strep), and tetracycline (tetA, tetM, tetQ);
        genes conferring tolerance to herbicides such as aminotriazole, amitrole, andrimid, aryloxyphenoxy propionates, atrazines, bipyridyliums, bromoxynil, cyclohexandione oximes, dicamba, diclfop, dichlorophenyl dimethyl urea (DCMU), difunone, diketonitriles, diuron, fluridone, glufosinate, glyphosate, halogenated hydrobenzonitriles, haloxyfop, 4-hydroxypyridines, imidazolinones, isoxasflutole, isoxazoles, isoxazolidinones, miroamide B, p-nitrodiphenylethers, norflurazon, oxadiazoles, m-phenoxybenzamides, N-phenyl imides, pinoxadin, protoporphyrionogen oxidase inhibitors, pyridazinones, pyrazolinates, sulfonylureas, 1,2,4-triazol pyrimidine, triketones, urea;
        genes that may be used in auxotrophic strains or to confer other metabolic effects, such as arg7, his3, hisD, hisG, lysA, manA, metE, nit1, trpB, ura3, xylA, a dihydrofolate reductase gene, a mannose-6-phosphate isomerase gene, a nitrate reductase gene, or an ornithine decarboxylase gene; a negative selection factor such as thymidine kinase; or toxin resistance factors such as a 2-deoxyglucose resistance gene; and
        acetyl CoA carboxylase (ACCase), acetohydroxy acid synthase (ahas), acetolactate synthase (als, csr1-1, csr1-2, imr1, imr2), aminoglycoside phosphotransferase (apt), anthranilate synthase, bromoxynil nitrilase (b×n), cytochrome P450-NADH-cytochrome P450 oxidoreductase, dalapon dehalogenase (dehal), dihydropteroate synthase (sul), class I 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), class II EPSPS (aroA), non-class I/II EPSPS, glutathione reductase, glyphosate acetyltransferase (gat), glyphosate oxidoreductase (gox), hydroxyphenylpyruvate dehydrogenase, hydroxy-phenylpyruvate dioxygenase (hppd), isoprenyl pyrophosphate isomerase, lycopene cyclase, phosphinothricin acteyl transferase (pat, bar), phytoene desaturase (crtI), prenyl transferase, protoporphyrin oxidase, the psbA photosystem II polypeptide (psbA), and SMM esterase (SulE), and superoxide dismutase (sod).
    
    


A “reporter gene” is a gene encoding a protein that is detectable or has an activity that produces a detectable product. A reporter gene can encode a visual marker or enzyme that produces a detectable signal, such as cat, lacZ, uidA, xylE, an alkaline phosphatase gene, an α-amylase gene, an α-galactosidase gene, a β-glucuronidase gene, a β-lactamase gene, a horseradish peroxidase gene, a luciferin/luciferase gene, an R-locus gene, a tyrosinase gene, or a gene encoding a fluorescent protein, including but not limited to a blue, cyan, green, red, or yellow fluorescent protein, a photoconvertible, photoswitchable, or optical highlighter fluorescent protein, or any of variant thereof, including codon-optimized, rapidly folding, monomeric, increased stability, and enhanced fluorescence variants.
The term “transformation” as used herein refers to the introduction of one or more exogenous nucleic acid sequences or polynucleotides into a host cell or organism by using one or more physical, chemical, or biological methods. Physical and chemical methods of transformation (i.e., “transfection”) include, by way of non-limiting example, electroporation and liposome delivery. Biological methods of transformation (i.e., “transduction”) include transfer of DNA using engineered viruses or microbes (e.g., Agrobacterium).
As used herein, “up-regulated” or “up-regulation” includes an increase in expression of a gene or nucleic acid molecule of interest or the activity of an enzyme, e.g., an increase in gene expression or enzymatic activity as compared to the expression or activity in an otherwise identical gene or enzyme that has not been up-regulated.
As used herein, “down-regulated” or “down-regulation” includes a decrease in expression of a gene or nucleic acid molecule of interest or the activity of an enzyme, e.g., a decrease in gene expression or enzymatic activity as compared to the expression or activity in an otherwise identical gene or enzyme that has not been down-regulated.
As used herein, the generic category of enzymes known as “diacylglycerol acyltransferase” (abbreviated as “DGAT”) includes the specific category of enzymes known as “diacylglycerol acetyltransferase” (abbreviated as “DGAcT”).
The terms, “identical” or percent “identity”, in the context of two or more nucleic acids or polypeptide sequences, refer 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 over a comparison window. The degree of amino acid or nucleic acid sequence identity can be determined by various computer programs for aligning the sequences to be compared based on designated program parameters. For example, sequences can be aligned and compared using the local homology algorithm of Smith & Waterman (1981) Adv. Appl. Math. 2:482-89, the homology alignment algorithm of Needleman & Wunsch (1970) J. Mol. Biol. 48:443-53, or the search for similarity method of Pearson & Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444-48, and can be aligned and compared based on visual inspection or can use computer programs for the analysis (for example, GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.).
The BLAST algorithm, described in Altschul et al. (1990) J. Mol. Biol. 215:403-10, is publicly available through software provided by the National Center for Biotechnology Information (at the web address www.ncbi.nlm.nih.gov). This algorithm identifies high scoring sequence pairs (HSPS) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra.). Initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated for nucleotides sequences using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. For determining the percent identity of an amino acid sequence or nucleic acid sequence, the default parameters of the BLAST programs can be used. For analysis of amino acid sequences, the BLASTP defaults are: word length (W), 3; expectation (E), 10; and the BLOSUM62 scoring matrix. For analysis of nucleic acid sequences, the BLASTN program defaults are word length (W), 11; expectation (E), 10; M=5; N=−4; and a comparison of both strands. The TBLASTN program (using a protein sequence to query nucleotide sequence databases) uses as defaults a word length (W) of 3, an expectation (E) of 10, and a BLOSUM 62 scoring matrix. See, Henikoff & Henikoff (1992) Proc. Nat'l. Acad. Sci. USA 89:10915-19
In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (1993) Proc. Nat'l. Acad. Sci. USA 90:5873-87). The smallest sum probability (P(N)), provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, preferably less than about 0.01, and more preferably less than about 0.001.
Nucleic Acid Molecules
The nucleic acid molecules and encoded polypeptides described herein can be used in any of the methods of the invention, and may be included in any of the expression cassettes, vectors, or recombinant microorganisms of the invention. Nucleic acid molecules comprising sequences that encode DGATs and PH domains are provided for use in host microorganisms and methods for producing TAG. A nucleic acid molecule as disclosed herein can be isolated, recombinant and/or purified.
Diacylglycerol acyltransferases (DGATs) are members of the O-acyltransferase superfamily, which esterify either sterols or diacylglycerols in an oleoyl-CoA-dependent manner. The DGAT enzyme performs the final step in TAG biosynthesis by transferring an acyl group from acyl-coenzyme-A to the sn-3 position of 1,2-diacylglycerol (DAG) to form TAG. Eukaryotes have two types of DGAT, abbreviated as DGAT1 and DGAT2, that belong to different gene families and share little homology. Eukaryotic DGAT1 polypeptides typically contain a FY×DWWN (SEQ ID NO:63) amino acid sequence motif, as well as a histidine (or tyrosine)-serine-phenylalanine (H/YSF) tripeptide motif, as described in Guo et al. (2001) J. Lipid Res. 42:1282-91. DGAT2 polypeptides typically include a HPHG, EPHSV, or PPHGV. A third type of DGAT, known as DGAT3, has been identified in peanut (Saha et al. (2006) Plant Physiol. 141:1533-43) and Arabidopsis (Hernandez et al. (2012) Plant Physiol. 160:215-25). Further considered as a DGAT useful for expression in microoranisms as disclosed herein is a diacylglycerol acetyltransferase (DGAcT) as has been described in Euonymous alatus (Durrett et al. (2010) Proc. Nat'l. Acad. Sci. USA 107:9464-69). In contrast, some prokaryotes that accumulate neutral lipids have genes encoding acyltransferase enzymes that form wax esters or TAG known as “WS/DGATs” (e.g., Acinetobacter baylii, A. baumanii, and Mycobacterium avium, and M. tuberculosis CDC1551, (see, e.g., Daniel et al. (2004) J. Bacteriol. 186:5017-30; see also Kalscheuer et al. (2003) J. Biol. Chem. 287:8075-82).
For example, an isolated or recombinant nucleic acid molecule as provided herein can encode a polypeptide having DGAT activity, in which the polypeptide comprises an amino acid sequence having:

    
    
        at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:2;
        at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:4;
        at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:8;
        at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:10;
        at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:14;
        at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:16;
        at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:20;
        at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:22;
        at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:26;
        at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:28;
        at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:32;
        at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:34;
        at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:38;
        at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:40;
        at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:44;
        at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:46;
        and/or to a functional fragment of any of the provided amino acid sequences.
    
    


Assays for determining the activity of DGATs are known in the art and include, in addition to in vivo expression to evaluate increased production of TAG (see, e.g., the Examples provided in co-pending U.S. patent application Ser. No. 13/652,934, entitled “DGAT Genes and Methods of Use for Triglyceride Production in Recombinant Microorganisms” filed on 16 Oct. 2012), in vivo or in vitro assays that measure TAG production using radiolabeled substrates or detection of TAG by staining of thin layer chromatograms (see, e.g., Cases et al. (1998) Proc. Nat'l. Acad. Sci. USA 95:13018-23; Cases et al. (2001) J. Biol. Chem. 276:38870-76; Durrett et al. (2010) Proc. Nat'l. Acad. Sci. USA 107:9464-69; Beopoulos et al. (2012) Appl. Microbiol. Biotechnol. 93:1523-37).
An isolated or recombinant nucleic acid molecule encoding a DGAT can comprise a nucleic acid sequence that encodes a polypeptide having DGAT activity that has at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of a eukaryotic DGAT (e.g., a DGAT1), such as but not limited to: DGATs from Cyclotella species (SEQ ID NO:2) and orthologs in other algal species; DGATs from Navicula species (SEQ ID NO:8) and orthologs in other algal species; DGATs from Fragilariopsis cylindrus (SEQ ID NO:14) and orthologs in other algal species; DGATs from Botryococcus species (SEQ ID NO:20) and orthologs in other algal species; DGATs from Tetraselmis species (SEQ ID NO:26) and orthologs in other algal species; DGATs from Chlorella species (SEQ ID NO:32) and orthologs in other algal species; DGATs from Thalassiosira pseudonana (Genbank accession ADV58933, GI:340772255, SEQ ID NO:38) and orthologs in other algal species; and DGATs from Phaeodactylum tricornutum (Genebank accession XP—002177753, GI:219112003, SEQ ID NO:44) and orthologs in other algal species. For example, in some instances an isolated or recombinant nucleic acid molecule encoding a DGAT can comprise a nucleic acid sequence that encodes a polypeptide having DGAT activity that has at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence of a prokaryotic DGAT, such as but not limited to: DGATs from Cyclotella species (SEQ ID NO:2) and orthologs in other algal species; DGATs from Navicula species (SEQ ID NO:8) and orthologs in other algal species; DGATs from Fragilariopsis cylindrus (SEQ ID NO:14) and orthologs in other algal species; DGATs from Botryococcus species (SEQ ID NO:20) and orthologs in other algal species; DGATs from Tetraselmis species (SEQ ID NO:26) and orthologs in other algal species; DGATs from Chlorella species (SEQ ID NO:32) and orthologs in other algal species; DGATs from Thalassiosira pseudonana (Genbank accession ADV58933, GI:340772255, SEQ ID NO:38) and orthologs in other algal species; and DGATs from Phaeodactylum tricornutum (Genebank accession XP—002177753, GI:219112003, SEQ ID NO:44) and orthologs in other algal species.
Additional DGAT genes that may find use in the microorganisms and methods of the invention (e.g., by engineering of such DGAT genes to include PH domain-encoding sequences) can include those encoding DGAT enzymes of animals, higher plants, algae, bacteria, or fungii, such as, for example, those encoding DGAT enzymes of: Mycobacterium smegmatis (Genbank accession ABK74273, GI:118173377); Alcanivorax borkumensis (Genbank accession YP—694462, GI:110835603); Marinobacter hydrocarbonoclasticus (Genbank accession ABM17275, GI:120322960); Rhodococcus opacus (Genbank accession GQ923886, GI:261411835); Homo sapiens (NP—036211.2, GI:145864459); Danio rerio (NP—956024.1, GI:41054343); Arabidopsis thaliana (Genebank accession Q9SLD2, GI:75206653; Genebank accession Q9ASU1.1, GI:75167729); Brassica juncea (AAY40784.1, GI:63376226); Brassica napus (AAD40881.1, GI:5225382); Ostreococcus tauri (Wagner et al. (2010) Plant Physiol. & Biochem. 48:407-16); Chlamydomonas reinhardtii (La Russa et al. (2012) J. Biotechnol. 162:13-20); Zea mays (ABV91586.1, GI:157885767); Yarrowia lipolytica (Genebank accession XP504700); Mus musculus (Genbank accession NP—080660, GI:16975490); Nannochloropsis gaditana (SEQ ID NOs:18, 24, and 36 in co-pending U.S. patent application Ser. No. 13/652,934); Arachis hypogaea (Genbank accession AY875644, GI:62084564); Euonymus alatus (Genbank accession GU594061, GI:294992377); Rhodotorula glutinis (ABC41546.1, GI:83702260); Ricinus communis (XP—002519339.1, GI:255556610); and Paracoccidiodioides brasiliensis (EEH17170.1, GI:225678886). Also included are DGAT genes encoding DGAT enzymes having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to these enzymes, orthologs in other species, and/or functional fragments thereof.
Additionally or alternatively, the invention encompasses nucleic acid molecules encoding truncations of a DGAT where one or more amino acids have been deleted from the protein. For example, the encoded polypeptide can lack at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, or at least 80 amino acids from the N- and/or C-terminus and can have an amino acid sequence at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% identical to the corresponding amino acid sequence of SEQ ID NOs:4, 10, 16, 22, 28, 34, 40, and/or 46. In some examples, the deleted sequences may include at least a portion of a functional domain, such as but not limited to a targeting or localization sequence, for example, at least a portion of a chloroplast transit peptide, at least a portion of a mitochondrial targeting sequence, at least a portion of an endoplasmic reticulum targeting sequence, at least a portion of a Pleckstrin Homology domain, etc. By way of nonlimiting example, in some instances an isolated or recombinant nucleic acid molecule encoding a DGAT can comprise a nucleic acid sequence that encodes a polypeptide having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of a eukaryotic DGAT (e.g., a DGAT1), such as but not limited to: DGATs from Cyclotella species (SEQ ID NO:4) and orthologs in other species; DGATs from Navicula species (SEQ ID NO:10) and orthologs in other species; DGATs from Fragilariopsis cylindrus (SEQ ID NO:16) and orthologs in other species; DGATs from Botryococcus species (SEQ ID NO:22) and orthologs in other species; DGATs from Tetraselmis species (SEQ ID NO:28) and orthologs in other species; DGATs from Chlorella species (SEQ ID NO:34) and orthologs in other species; DGATs from Thalassiosira pseudonana (SEQ ID NO:40) and orthologs in other species; and DGATs from Phaeodactylum tricornutum (SEQ ID NO:46) and orthologs in other 1 species. For example, in some instances an isolated or recombinant nucleic acid molecule encoding a DGAT can comprise a nucleic acid sequence that encodes a polypeptide having DGAT activity that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence of a DGAT1, such as but not limited to: a DGAT1 from Cyclotella species (SEQ ID NO:4) and orthologs in other algal species; a DGAT1 from Navicula species (SEQ ID NO:10) and orthologs in other algal species; a DGAT1 from Fragilariopsis cylindrus (SEQ ID NO:16) and orthologs in other algal species; a DGAT1 from Botryococcus species (SEQ ID NO:22) and orthologs in other algal species; a DGAT1 from Tetraselmis species (SEQ ID NO:28) and orthologs in other algal species; a DGAT1 from Chlorella species (SEQ ID NO:34) and orthologs in other algal species; a DGAT1 from Thalassiosira pseudonana (SEQ ID NO:40) and orthologs in other algal species; and a DGAT1 from Phaeodactylum tricornutum (SEQ ID NO:46) and orthologs in other algal species. The DGAT that includes a PH domain can be, for example, from a species of the Chlorophyta or Heterokontophyta division, and can be, for example, a species of the class Bacillariophyceae (diatoms), Prasinophyceae, or Trebouxiophyceae.
Pleckstrin Homology (PH) domains are small (100-120 amino acid) structural domains common to a wide variety and large number of proteins. Although they were first identified in the amino- and carboxy-termini of pleckstrin, they have since been identified in more than 670 proteins. Lemmon (2008) Nat. Rev. Mol. Cell Biol. 9:99-111. There is little primary sequence conservation within this domain, although there is significant secondary and tertiary structure conserved among the various members of this domain family (see, FIG. 2A-F). Philip et al. (2002) FEBS Lett. 531:28-32. Among the many roles that these domains play, one is to target and bind proteins to specific phosphatidylinositol lipids on membrane surfaces. Scheffzek & Welti (2012) FEBS Lett. 586:2662-73.
The present invention also provides nucleic acid molecules encoding PH domains. Without being bound by theory, PH domains may target acyltransferases of the present invention to membrane surfaces, for example to membrane surfaces, such as, for example, membrane surfaces of the endoplasmic reticulum or the chloroplast envelope. Such isolated or recombinant nucleic acid molecules as provided herein can encode a PH domain, in which the polypeptide comprises an amino acid sequence having:

    
    
        at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:6;
        at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:12;
        at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:18;
        at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:24;
        at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:30;
        at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:36;
        at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:42;
        at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:48;
        and/or to a functional fragment of any of the provided amino acid sequences.
    
    


For example, the nucleic acid molecules encoding PH domains can comprise a nucleic acid sequence that encodes a polypeptide that has at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the PH domains of the present invention (e.g., SEQ ID NOs:6, 12, 18, 24, 30, 36, 42, and 48). The Pleckstrin Homology domain can optionally be derived from a gene, such as an algal DGAT1 gene, or from a eukaryotic algal species. Additionally, the nucleic acid molecules encoding PH domains can be fused in frame to a nucleic acid molecule encoding an acyltransferase, such as, for example a MGAT, DGAT1, DGAT2, DGAT3, WS/DGAT, DGAcT, DGTA, PDAT, LPLAT, LPAAT, or GPAT, where the acyltransferase may be derived from any species. For example, a nucleic acid molecule encoding a DGAT of Saccharomyces cerevisiae can be fused in frame to a nucleic acid molecule encoding a PH domain of the present invention. Additionally or alternatively, any nucleic acid molecule encoding a PH domain can be operably linked a nucleotide sequence encoding a MGAT, DGAT1, DGAT2, DGAT3, WS/DGAT, DGAcT, DGTA, PDAT, LPLAT, LPAAT, and/or GPAT.
The invention also provides DGAT genes that include PH domains that include sequences encoding fragments and variants of naturally-occurring DGATs. A substitution, insertion or deletion can adversely affect the protein when the altered sequence substantially inhibits a biological function associated with the protein. In certain embodiments, a variant of a DGAT may have activity that is reduced by not more than about 1%, not more than about 2%, not more than about 3%, not more than about 4%, not more than about 5%, not more than about 6%, not more than about 7%, not more than about 8%, not more than about 9%, not more than about 10%, not more than about 15%, not more than about 20%, not more than about 30%, not more than about 40%, not more than about 50%, or not more than about 90% in comparison to the activity of the DGAT from which the variant is derived (e.g., any of SEQ ID NOs:2, 8, 14, 20, 26, 32, 38, and/or 44). In some embodiments, the amount of a TAG produced by a host cell expressing the DGAT variant is not less than about 99%, not less than about 98%, not less than about 97%, not less than about 96%, not less than about 95%, not less than about 94%, not less than about 93%, not less than about 92%, not less than about 91%, not less than about 90%, not less than about 85%, not less than about 80%, not less than about 75%, or not less than about 50% of the amount or the fatty acid product produced by a host cell expressing the DGAT from which the variant is derived (e.g., any of SEQ ID NOs:2, 8, 14, 20, 26, 32, 38, and/or 44).
The invention also provides fragments and variants of a DGAT that have increased activity in comparison to the reference polypeptides. In certain embodiments, the DGAT fragment or variant may have activity that is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% in comparison to the activity of the DGAT from which the variant is derived. In certain embodiments, the amount of TAG produced by a host cell expressing the fragment or variant is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% of the amount of TAG made by a host cell expressing the DGAT from which the fragment or variant is derived.
The invention described herein also relates to fragments of the isolated nucleic acid molecules described herein encompassing a portion of a nucleotide sequence described herein which is from at least 20 contiguous nucleotides to at least 50 contiguous nucleotides or longer in length. Such fragments may be useful as probes and primers. In particular, primers and probes may selectively hybridize to the nucleic acid molecule encoding the polypeptides described herein. For example, fragments which encode polypeptides that retain activity, as described below, are particularly useful.
The invention also provides nucleic acid molecules that hybridize under high stringency hybridization conditions, such as for selective hybridization, to the nucleotide sequences described herein (e.g. nucleic acid molecules which specifically hybridize to a nucleotide sequence encoding polypeptides described herein and encode a DGAT and/or a PH domain). Hybridization probes include synthetic oligonucleotides which bind in a base-specific manner to a complementary strand of nucleic acid. Suitable probes include polypeptide nucleic acids (PNAs), as described in Nielsen (1991) Science, 254:1497-500.
Such nucleic acid molecules can be detected and/or isolated by specific hybridization e.g. under high stringency conditions. “Stringency conditions” for hybridization is a term of art that refers to the incubation and wash conditions, e.g. conditions of temperature and buffer concentration, which permit hybridization of a particular nucleic acid to a second nucleic acid; the first nucleic acid may be perfectly complementary, i.e. 100%, to the second, or the first and second may share some degree of complementarity, which is less than perfect, e.g. 60%, 75%, 85%, 95% or more. For example, certain high stringency conditions can be used which distinguish perfectly complementary nucleic acids from those of less complementarity.
“High stringency conditions”, “moderate stringency conditions” and “low stringency conditions” for nucleic acid hybridizations are explained in Current Protocols in Molecular Biology (2011) John Wiley & Sons). The exact conditions which determine the stringency of hybridization depend not only on ionic strength, e.g. 0.2×SSC, 0.1×SSC of the wash buffers, temperature, e.g. 23° C., 42° C., 68° C., etc. and the concentration of destabilizing agents such as formamide or denaturing agents such as SDS, but also on factors such as the length of the nucleic acid sequence, base composition, percent mismatch between hybridizing sequences and the frequency of occurrence of subsets of that sequence within other non-identical sequences. Thus, high, moderate or low stringency conditions may be determined empirically.
By varying hybridization conditions from a level of stringency at which no hybridization occurs to a level at which hybridization is first observed, conditions which will allow a given sequence to hybridize with the most similar sequences in the sample can be determined.
Exemplary conditions are described in Krause (1991) Methods in Enzymology, 200:546-56. Washing is the step in which conditions are usually set so as to determine a minimum level of complementarity of the hybrids. Generally, starting from the lowest temperature at which only homologous hybridization occurs, each degree (° C.) by which the final wash temperature is reduced, while holding SSC concentration constant, allows an increase by 1% in the maximum extent of mismatching among the sequences that hybridize. Generally, doubling the concentration of SSC results in an increase in melting temperature (Tm). Using these guidelines, the washing temperature can be determined empirically for high, moderate or low stringency, depending on the level of mismatch sought. Exemplary high stringency conditions include, but are not limited to, hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60° C. Example of progressively higher stringency conditions include, after hybridization, washing with 0.2×SSC and 0.1% SDS at about room temperature (low stringency conditions); washing with 0.2×SSC, and 0.1% SDS at about 42° C. (moderate stringency conditions); and washing with 0.1×SSC at about 68° C. (high stringency conditions). Washing can be carried out using only one of these conditions, e.g. high stringency conditions, washing may encompass two or more of the stringency conditions in order of increasing stringency. Optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically.
Equivalent conditions can be determined by varying one or more of the parameters given as an example, as known in the art, while maintaining a similar degree of identity or similarity between the target nucleic acid molecule and the primer or probe used. Hybridizable nucleotide sequences are useful as probes and primers for identification of organisms comprising a nucleic acid of the invention and/or to isolate a nucleic acid of the invention, for example.
The nucleic acid molecules of the invention can optionally comprise additional non-coding sequences such as non-coding 3′ and 5′ sequences (including, e.g., regulatory sequences) that may be homologous or heterologous to a DGAT gene. Alternatively or in addition, any of the provided nucleic acid molecules can optionally further comprise an additional nucleic acid sequence of at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 700, at least 800, at least 900, at least 1000, or at least 1500 nucleotides from a photosynthetic organism. The nucleic acid molecules and polypeptides described herein can be used in any of the methods of the invention, and may be included in any of the vectors or recombinant microorganisms of the invention. Nucleic acid molecules comprising sequences that encode DGAT are provided for use in host microorganisms and methods for producing TAG.
Other Modifications
The invention also provides further variants of the nucleotide sequences of the invention. In some embodiments, the nucleotide sequence variants encode fragments or variants of the polypeptides as described herein. In some embodiments, the nucleotide sequence variants are naturally-occurring. In other embodiments, the nucleotide sequence variants are non-naturally-occurring, such as those induced by various mutagens and mutagenic processes. In certain embodiments, the nucleotide sequence variants are a combination of naturally- and non-naturally-occurring. A given nucleic acid sequence may be modified, for example, according to standard mutagenesis or artificial evolution or domain swapping methods to produce modified sequences. Accelerated evolution methods are described, e.g. by Stemmer (1994) Nature 370:389-91, and Stemmer (1994) Proc. Nat'l. Acad. Sci. USA 91:10747-51. Chemical or enzymatic alteration of expressed nucleic acids and polypeptides can be performed by standard methods. For example, a sequence can be modified by addition of phosphate groups, methyl groups, lipids, sugars, peptides or organic or inorganic compounds, by the inclusion of modified nucleotides or amino acids, or the like.
For optimal expression of a recombinant protein, in certain instances it may be beneficial to employ coding sequences that produce mRNA with codons preferentially used by the host cell to be transformed (“codon optimization”). Thus, for enhanced expression of transgenes, the codon usage of the transgene can be matched with the specific codon bias of the organism in which the transgene is desired to be expressed. Methods of recoding genes for expression in microalgae are described in, e.g., U.S. Pat. No. 7,135,290. The precise mechanisms underlying this effect are believed to be many, but can include the proper balancing of available aminoacylated tRNA pools with proteins being synthesized in the cell, coupled with more efficient translation of the transgenic messenger RNA (mRNA) when this need is met. In some examples, only a portion of the codons is changed to reflect a preferred codon usage of a host microorganism. In certain examples, one or more codons are changed to codons that are not necessarily the most preferred codon of the host microorganism encoding a particular amino acid. Additional information for codon optimization is available, e.g. at the codon usage database of GenBank. The coding sequences may be codon optimized for optimal production of a desired product in the host organism selected for expression. In certain examples, the non-native nucleic acid sequence encoding a DGAT is codon optimized for expression in a photosynthetic microorganism, e.g., a cyanobacterium or a eukaryotic microalga. In some aspects, the nucleic acid molecules of the invention encode fusion proteins that comprise a DGAT. For example, the nucleic acids of the invention may comprise polynucleotide sequences that encode a heterologous PH domain, the amino-terminus of a putative chloroplast inner envelope glucose-6-phosphate/phosphate antiporter or a portion thereof, glutathione-S-transferase (GST) or a portion thereof, thioredoxin or a portion thereof, maltose binding protein or a portion thereof, poly-histidine (e.g. His6), poly-HN, poly-lysine, a FLAG tag sequence, a hemagglutinin tag sequence, HSV-Tag and/or at least a portion of HIV-Tat fused to the DGAT-encoding sequence.
Nucleic Acid Constructs
The invention also provides constructs, such as an isolated or recombinant nucleic acid molecule comprising a nucleotide sequence as provided herein encoding a polypeptide having acyltransferase activity, wherein the polypeptide comprises a PH domain. Additionally, the construct can further include one or more sequences that regulate or mediate transcription, translation, or integration of nucleotide sequences into a host genome. For example, the invention also provides expression constructs that comprise one or more “expression control elements” or sequences that regulate transcription of an operably linked gene, or translation of the transcribed RNA. For example, an expression control element can be a promoter that may be operably linked to the gene of interest (e.g., a DGAT gene) in an expression construct or “expression cassette.” In some examples of the foregoing, the promoter is regulatable, e.g., inducible. In other examples of the foregoing, the promoter may be constitutive. The promoter in some examples can be an algal promoter or derived from an algal promoter.
In examples where the nucleic acid construct does not contain a promoter in operable linkage with the nucleic acid sequence encoding the polypeptide having acyltransferase activity (e.g., a DGAT gene) the nucleic acid sequence can be transformed into the cells such that it becomes operably linked to an endogenous promoter by, e.g., homologous recombination, site specific integration, and/or vector integration. In some examples, genomic host sequences included in a nucleic acid construct for mediating homologous recombination into the host genome can include gene regulatory sequences, for example, a promoter sequence, that can regulate expression of a DGAT gene of the nucleic acid construct. In such embodiments, the transgene(s) of the construct can become operably linked to a promoter that is endogenous to the host microorganism. In some embodiments, the endogenous promoter(s) are regulatable, e.g., inducible. Alternatively the DGAT gene can be operably linked to an endogenous promoter that is constitutive and/or active under nitrogen replete conditions.
In one aspect, the present invention provides isolated or recombinant nucleic acid molecules encoding a polypeptide having acyltransferase activity, wherein the polypeptide comprises a heterologous PH domain derived from a microorganism, for example, a microalga. In certain embodiments, the heterologous PH domain can be selected from the group consisting of: a PH domain having at least 80% identity to SEQ ID NO:6 or a functional fragment thereof; a PH domain having at least 80% identity to SEQ ID NO:12 or a functional fragment thereof; a PH domain having at least 80% identity to SEQ ID NO:18 or a functional fragment thereof; a PH domain having at least 80% identity to SEQ ID NO:24 or a functional fragment thereof; a PH domain having at least 80% identity to SEQ ID NO:30 or a functional fragment thereof; a PH domain having at least 80% identity to SEQ ID NO:36 or a functional fragment thereof; a PH domain having at least 80% identity to SEQ ID NO:42 or a functional fragment thereof; and a PH domain having at least 80% identity to SEQ ID NO:48 or a functional fragment thereof.
Alternatively or additionally, the recombinant nucleic acid molecule encoding a polypeptide having acyltransferase activity can comprise a heterologous PH domain selected from the group consisting of: a PH domain having at least 95% identity to SEQ ID NO:6 or a functional fragment thereof; a PH domain having at least 95% identity to SEQ ID NO:12 or a functional fragment thereof; a PH domain having at least 95% identity to SEQ ID NO:18 or a functional fragment thereof; a PH domain having at least 95% identity to SEQ ID NO:24 or a functional fragment thereof; a PH domain having at least 95% identity to SEQ ID NO:30 or a functional fragment thereof; a PH domain having at least 95% identity to SEQ ID NO:36 or a functional fragment thereof; a PH domain having at least 95% identity to SEQ ID NO:42 or a functional fragment thereof; and a PH domain having at least 95% identity to SEQ ID NO:48 or a functional fragment thereof.
In certain embodiments, the polypeptide having acyltransferase activity can be a MGAT, a DGAT1, a DGAT2, a WS/DGAT, a DGAT3, a DGTA, a PDAT, a LPLAT, a LPAAT, and/or a GPAT. For example, the polypeptide having acyltransferase activity can be a DGAT1 or a DGAT2. For example, the polypeptide having acyltransferase activity can be a DGAT1.
A promoter operably linked to a nucleic acid sequence encoding a acyltransferase may be a promoter that is heterologous with respect to the acyltransferase gene. Promoters considered for use in regulating acyltransferase genes in eukaryotes can include, without limitation, inducible promoters such as a GAL, MET, Lys, or Leu promoter, or a nmt1 thiamine-repressible promoter, a uracil regulatable promoter (e.g., Watt et al. (2008) PLoS One 3:e1428) for example from a yeast or fungus, or a Tet-On or Tet-Off promoter. Other eukaryotic promoters as they are known in the art can also be employed, such as the SV40 promoter and the Cauliflower Mosaic Virus (CaMV) promoter, active fragments thereof, or hybrid promoters that include fragments of known eukaryotic promoters such as the SV40, CaMV, and nopaline synthase promoters.
In some embodiments of the foregoing invention, the promoter may be an inducible promoter, i.e., a promoter that mediates transcription of an operably linked gene in response to a particular stimulus. Such promoters may be advantageous, e.g., to minimize any deleterious effects on the growth of the host cell and/or to maximize production of TAG. An inducible promoter can be responsive to, e.g., light or dark or high or low temperature, and/or can be responsive to specific compounds. The inducible promoter may be a hormone-responsive promoter (e.g., an ecdysone-responsive promoter, such as described in U.S. Pat. No. 6,379,945), a metallothionien promoter (e.g., U.S. Pat. No. 6,410,828), a pathogenesis-related (PR) promoter that can be responsive to a chemical such as, for example, salicylic acid, ethylene, thiamine, and/or BTH (U.S. Pat. No. 5,689,044), or the like, or some combination thereof. An inducible promoter can also be responsive to light or dark (U.S. Pat. Nos. 5,750,385, 5,639,952), metals (Quinn et al. (2003) Eukaryot. Cell 2:995-1002) or temperature (U.S. Pat. No. 5,447,858; Abe et al. (2008) Plant Cell Physiol. 49:625-32; Shroda et al. (2000) Plant J. 21:121-31). The foregoing list is exemplary and not limiting. The promoter sequence can be from any organism, provided that it is functional in the host organism. In certain embodiments, inducible promoters are formed by fusing one or more portions or domains from a known inducible promoter to at least a portion of a different promoter that can operate in the host cell, e.g. to confer inducibility on a promoter that operates in the host species.
Inducible promoters from eukaryotic algae include a NR promoter (ammonia regulated; Wang et al. (2004) J. Appl. Phycol. 16:11-16), nia promoter (U.S. Pat. No. 7,642,405), CYC6 (copper inducible, nickel and cobalt repressible) or CA1 promoter (CO2-regulated; Ferrante et al. (2008) PLos One 3:e3200), as well as an algal Pnr (nitrogen-regulated) promoter (Poulsen and Kroger (2005) FEBS J. 272:3413-23), an algal inorganic phosphate transporter promoter (Wurch et al. (2011) Environ. Microbiol. 13:468-81), or other phosphate-status regulated promoter from algae, e.g, a PNP or PSR promoter (Yehudai-Resheff et al. (2007) Plant Cell. 19:1023-38). Further examples of promoters that may be induced by nitrogen limitation or depletion include but are not limited to: ammonium or ammonia transporter gene promoters (see, for example, Wurch et al., 2011); glutamine synthetase transporters (e.g., Miller et al. (2010) Plant Physiol. 154:737-52) or other promoters of genes upregulated at the transcriptional level during nitrogen depletion, including those disclosed in co-pending U.S. patent application Ser. No. 13/536,345, entitled “Regulation of Toxin and Antitoxin Genes for Biological Containment” filed Jun. 28, 2012, or active fragments of any thereof. The promoter can alternatively or in addition be regulated by phosphate depletion, and can be, for example, a PNPase gene promoter (Yehudai-Reseheff et al., 2007); an inorganic phosphate transporter gene promoter (Wurch et al., 2011), a phosphate permease gene promoter, or any disclosed in co-pending U.S. patent application Ser. No. 13/536,345, entitled “Regulation of Toxin and Antitoxin Genes for Biological Containment” filed Jun. 28, 2012, or an active fragment thereof. Candidate copper depletion-regulated promoters include those of CTR-type copper ion transporter genes (Castruita et al. (2011) Plant Cell. 23:1273-92), as well as CYC6 and CPX1 algal promoters (Quinn et al. (2000) J. Biol. Chem. 275:6080-89). Promoters regulated by iron deficiency may include, for example, those from the FOX1 gene or the FTR1 gene (La Fontaine et al. (2002) Eukaryotic Cell 1:736-57).
Specifically considered are promoters of the genes known to to be transcribed under nitrogen replete conditions. For example, promoters used to regulate acyltransferase genes, such as DGAT genes, can be active under conditions where the algae are proliferating, e.g., promoters that are active in the absence of nutrient limitation, such as, for example the Nannochloropsis promoters disclosed in co-pending U.S. patent application Ser. No. 13/486,930, entitled “Promoters and Terminators for Use in Eukaryotic Cells” filed 1 Jun. 2012 and co-pending U.S. patent application Ser. No. 13/693,585, entitled “Tetraselmis Promoters and Terminators for Use in Eukaryotic Cells” filed 4 Dec. 2012, as well as in co-pending U.S. patent application Ser. No. 13/536,345, entitled “Regulation of Toxin and Antitoxin Genes for Biological Containment” filed 28 Jun. 2012. Additional examples of algal promoters include the Nannochloropsis oceanica vcp promoter sequence, which is regulated by light-exposure, (U.S. 2009/317,904); the a light-responsive fcpA promoter from Phaeodactylum tricornutum (U.S. Pat. No. 6,027,900); the Chlamydomonas psaD promoter (Fisher & Rochaix (2001) Mol. Genet. Genomics 265:888-94), as well as RuBisCo small subunit (ssu) promoters (Walker et al. (2004) Plant Cell Reports 23:727-35; Chen et al. (2008) J. Phycol. 44:768-76); the Cyclotella cryptica acc promoter, and Chlorella viral promoters (U.S. Pat. Nos. 6,252,140; 6,316,224). Also considered are promoters that are associated with orthologous genes in other species. For example, a gene of one algal species that is transcribed under nutrient replete or nitrogen replete conditions can be used to identify an orthologous gene in a second algal species, and the promoter of the gene in the second species can be isolated and tested for its activity under the desired culture conditions.
The examples of promoters provided are not limiting with regard to the promoters that may be used in constructs for expression of acyltransferases such as DGATs. Specifically considered are active fragments of promoters provided herein or disclosed in the art and promoters that comprise multimers of promoters or promoter fragments, as well as hybrid promoters such as but not limited to promoters that may include sequences of two or more different algal promoters (e.g., the HSP70-RBCS promoter (Schroda et al. (2000) Plant J. 21:121-31)), or hybrid promoters that may comprise algal and non-algal sequences, such as, for example, at least a portion of an SV40 promoter or CaMV promoter juxtaposed with at least a portion of an algal promoter. A promoter may comprise multimers of a sequence, including multimers of a hybrid promoter sequence.
A promoter used to regulate a DGAT or acyltransferase gene in a eukaryote can also be a synthetic promoter, for example, a promoter that includes a DNA binding domain that can be recognized and bound by an engineered transcription factor positioned upstream of a minimal promoter that is operable in the host microorganism. The microorganism can include an exogenous gene encoding a synthetic transcription factor that binds the synthetic promoter. The synthetic transcription factor can include, in addition to a DNA binding domain that recognizes the synthetic promoter, an activation domain (e.g., VP16, CREB, GAL10, GCN4) and a regulatory domain, where the regulatory domain may bind one or more compounds that can be added to the culture medium to induce or repress transcription (Weber & Fussenegger (2011) Curr. Opin. Chem. Biol. 15:414-20).
Likewise, a wide variety of transcriptional terminators can be used for expression vector construction. Nonlimiting examples of possible terminators can include, but are not limited to, those disclosed in co-pending U.S. patent application Ser. No. 13/486,930, entitled “Promoters and Terminators for Use in Eukaryotic Cells” filed Jun. 1, 2012.
Further, the present invention also provides for a nucleotide construct such as an isolated or recombinant nucleic acid molecule comprising a promoter operably linked to a nucleotide sequence encoding a polypeptide having DGAT activity, as already described herein. In a particular aspect, the nucleotide sequence encoding a polypeptide having DGAT activity is selected from the group consisting of nucleotide sequences encoding an amino acid having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of any one or more of SEQ ID NOs:2, 8, 14, 20, 26, and 32.
In addition to a gene having acytransferase activity, such as a DGAT gene, one or more additional genes can optionally be included in a recombinant microorganism as provided herein, where the one or more additional genes may include, for example, one or more genes encoding enzymes or proteins of the fatty acid synthesis pathway and/or one or more genes encoding enzymes or proteins that may enhance TAG synthesis, one or more genes that may enhance photosynthesis or carbon-fixation, and/or one or more reporter genes or selectable markers. For example, the construct or expression cassette may further comprise a heterologous protein coding sequence, for example, encoding a MGAT, a DGAT1, a DGAT2, a DGAT3, a DGTA, a PDAT, a LPLAT, a LPAAT, and/or a GPAT.
Further, the present invention also provides for a nucleotide construct such as an isolated or recombinant nucleic acid molecule comprising a nucleotide sequence encoding a PH domain as already described herein. The nucleotide sequence encoding a PH domain can be fused in frame with any heterologous gene for ectopic expression in a recombinant microorganism. As nonlimiting examples the PH domain can direct a polypeptide to a chloroplast or a region thereof. In one aspect, the nucleotide sequence encoding a PH domain is fused in frame with a heterologous DGAT-encoding sequence. In a particular aspect, the nucleotide sequence encoding a PH domain is fused in frame with a nucleotide sequence encoding an amino acid having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of any one or more of SEQ ID NOs:4, 10, 16, 22, 28, 34, 40, and 46.
An isolated nucleic acid construct of the present invention can include the sequences disclosed herein that encode a DGAT or other polypeptide in a vector, such as, but not limited to, an expression vector. A vector can be a nucleic acid that has been generated via human intervention, including by recombinant means and/or direct chemical synthesis, and can include, for example, one or more of: 1) an origin of replication for propagation of the nucleic acid sequences in one or more hosts (which may or may not include the production host); 2) one or more selectable markers; 3) one or more reporter genes; 4) one or more expression control sequences, such as, but not limited to, promoter sequences, enhancer sequences, terminator sequences, sequence for enhancing translation, etc.; and/or 5) one or more sequences for promoting integration of the nucleic acid sequences into a host genome, for example, one or more sequences having homology with one or more nucleotide sequences of the host microorganism. A vector can be an expression vector that includes one or more specified nucleic acid “expression control elements” that permit transcription and/or translation of a particular nucleic acid in a host cell. The vector can be a plasmid, a part of a plasmid, a viral construct, a nucleic acid fragment, or the like, or a combination thereof.
The vector can be a high copy number vector, a shuttle vector that can replicate in more than one species of cell, a cloning vector, an expression vector, an integration vector, or a combination thereof. Typically, the expression vector can include a nucleic acid comprising a gene of interest operably linked to a promoter in an “expression cassette,” which can also include, but is not limited to, a PH domain encoding sequence, a transcriptional terminator, a ribosome binding site, a splice site or splicing recognition sequence, an intron, an enhancer, a polyadenylation signal, an internal ribosome entry site, and similar elements.
In one example, an expression cassette as provided herein comprises a promoter operably linked to a nucleotide sequence encoding a PH domain as described herein. The nucleotide sequence encoding a PH domain can further be operably linked to any gene of interest. In a particular aspect, the nucleotide sequence encoding a PH domain is selected from the group consisting of: a PH domain having at least 80% identity to SEQ ID NO:6 or a functional fragment thereof; a PH domain having at least 80% identity to SEQ ID NO:12 or a functional fragment thereof; a PH domain having at least 80% identity to SEQ ID NO:18 or a functional fragment thereof; a PH domain having at least 80% identity to SEQ ID NO:24 or a functional fragment thereof; a PH domain having at least 80% identity to SEQ ID NO:30 or a functional fragment thereof; a PH domain having at least 80% identity to SEQ ID NO:36 or a functional fragment thereof; a PH domain having at least 80% identity to SEQ ID NO:42 or a functional fragment thereof; and a PH domain having at least 80% identity to SEQ ID NO:48 or a functional fragment thereof.
Additionally, the present invention can involve recombinant microorganisms transformed with a vector comprising a gene of interest under control of a heterologous promoter. Alternatively, if the vector does not contain a promoter operably linked with an isolated nucleic acid comprising a gene of interest, the isolated nucleic acid can be transformed into the microorganisms or host cells such that it becomes operably linked to an endogenous promoter by homologous recombination, site specific integration, and/or vector integration.
Additionally, the present invention provides recombinant microorganisms or host cells transformed with a vector comprising a gene of interest that is operably linked to one or more expression control elements. In some instances, it can be advantageous to express the protein at a certain point during the growth of the recombinant microorganism, e.g., to minimize any deleterious effects on the growth of the recombinant microorganism and/or to maximize production of TAG. In such instances, one or more exogenous genes introduced into the recombinant microorganism or host cell can be operably linked to an inducible promoter, which mediates transcription of an operably linked gene in response to a particular stimulus.
Vectors provided herein can additionally or alternatively include a selectable marker. Transformed cells can be selected based upon the ability to grow in the presence of the antibiotic and/or other selectable marker under conditions in which cells lacking the resistance cassette or auxotrophic marker could not grow. Further, a non-selectable marker may be present on a vector, such as a gene encoding a fluorescent protein or enzyme that generates a detectable reaction product.
A vector comprising an isolated nucleic acid comprising a gene of interest can also be an integration vector that includes one or more sequences that promote integration of the gene of interest or a gene expression cassette into the genome of the host microorganism or host cell. For example, an integration vector can include at least one sequence of at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, or at least 600 nucleotides with homology to a sequence in the genome of the host organism to allow integration of the gene of interest or gene expression cassette into the genome of the host microorganism or host cell to occur via homologous recombination. In some examples, the gene or gene expression cassette is flanked by sequences homologous to a region of the host chromosome to promote integration of the gene of interest or gene expression cassette into the host chromosome. Alternatively or in addition, an integration vector can include one or more sequences that promote site-specific recombination or random integration such as, but not limited to, sequences recognized by recombinases, integrases, or transposases. In some embodiments, the integration vector can further include a gene encoding a recombinase, integrase, or transposase.
Microorganisms and Host Cells and Cultures
The present invention also provides recombinant microorganisms and host cells that comprise a nucleic acid molecule encoding a polypeptide having acyltransferase activity, wherein the polypeptide comprises a PH domain.
In one aspect, provided herein is a recombinant microorganism that includes a non-native gene encoding a polypeptide having acyltransferase activity and having a native or heterologous PH domain, such as a DGAT that includes a native or heterologous PH domain. The recombinant microorganism can produce a greater amount of TAG than is produced by a microorganism substantially identical to the recombinant microorganism, but lacking a non-native gene encoding a polypeptide having acyltransferase activity that includes a PH domain. The recombinant microorganism that includes a non-native gene encoding a polypeptide having acyltransferase activity, such as a DGAT, can produce a greater amount of TAG than is produced by a substantially identical microorganism when the microorganisms are cultured under conditions in which inorganic carbon is substantially the sole source of carbon in the growth medium. Additionally, the recombinant microorganism that includes a non-native gene encoding a polypeptide having acyltransferase activity, such as a DGAT, that includes a PH domain, can produce a greater amout of TAG than is produced by a microorganism substantially identical to the recombinant microorganism but lacking a non-native gene that encodes an acyltranferase that has a PH domain, when the microorganisms are cultured under nitrogen replete conditions. For example, a culture of a recombinant microorganism of the present invention can be maintained under nutrient replete conditions during the production period, and the culture can produce TAG during a culture period in which the cells of the culture are dividing.
A non-native gene encoding a polypeptide having acyltransferase activity that includes a PH domain can encode any polypeptide having acyltransferase activity, for example, a MGAT, DGAT1, DGAT2, DGAT3, WS/DGAT, DGAcT, DGTA, PDAT, LPLAT, LPAAT, or GPAT. The PH domain can be native to the acyltransferase (e.g., can be a DGAT1 gene that naturally includes a PH domain, or a variant thereof) or can be heterologous with respect to the acyltransferase, e.g., the gene encoding the acyltransferase is engineered to include a sequence encoding a PH domain. In some examples, the non-native gene can encode a DGAT that is a variant of a naturally-occurring DGAT, such as a DGAT having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to a naturally-occurring DGAT or an active fragment thereof. The non-native gene can encode a DGAT that is a truncated variant of a naturally-occurring DGAT, such as an N-terminally or C-terminally truncated variant of a naturally-occurring DGAT or a DGAT having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to a naturally-occurring DGAT. Additionally or alternatively, a recombinant microorganism or host cell can include a non-native gene encoding a DGAT that has at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to a naturally-occurring DGAT or an active fragment thereof and can further include one or more additional amino acid sequences, such as, but not limited to, one or more amino acid sequences that can direct the DGAT to a location within the cell, such as, for example, a plastid or a region thereof and/or the endoplasmic reticulum or a region thereof.
Acyltransferase genes utilized according to the present invention may be isolated from any organism, including eukaryotic and prokaryotic organisms, and can be any disclosed herein. Eukaryotic organisms having an acyltransferase gene are well-known in the art, and include various animals (e.g., mammals, fruit flies, nematodes), plants, parasites, and fungi (e.g., yeast such as Saccharomyces cerevisiae and Schizosaccharomyces pombe). Examples of prokaryotic organisms include certain actinomycetes, a group of Gram-positive bacteria with high G+C ratio, such as those from the representative genera Actinomyces, Arthrobacter, Corynebacterium, Frankia, Micrococcus, Mocrimonospora, Mycobacterium, Nocardia, Propionibacterium, Rhodococcus and Streptomyces. Particular examples of actinomycetes that have one or more genes encoding polypeptides having acyltransferase activity include, for example, Mycobacterium tuberculosis, M. avium, M. smegmatis, Micromonospora echinospora, Rhodococcus opacus, R. ruber, and Streptomyces lividans. Examples of prokaryotic organisms that encode one or more enzymes having a DGAT activity include members of the genera Acinetobacter, such as A. calcoaceticus, A. baumanii, and A. baylii. In certain embodiments, a DGAT enzyme is isolated from Acinetobacter baylii sp. ADP1, a gram-negative triglyceride forming prokaryote, which contains a well-characterized DGAT (AtfA).
In further examples, a recombinant microorganism or host cell as provided herein can include a non-native gene encoding a type-1 or a type-2 DGAT from a eukaryote, such as, for example, a fungus, yeast, protozoan, animal, alga, or plant, or a variant thereof having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the naturally-occurring DGAT1, DGAT2, or DGAT3 or an active fragment thereof, and can include a deletion, for example, a truncation, with respect to the naturally-occurring DGAT amino acid sequence, and/or can optionally include one or more additional sequences, such as, but not limited to, a PH domain. In some examples, the DGAT1, DGAT2, WS/DGAT, or DGAT3 encoded by the non-native gene comprises a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:44, and/or a functional fragment thereof. In some examples, the DGAT1, DGAT2, WS/DGAT, or DGAT3 encoded by the non-native gene comprises a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:22, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:40, SEQ ID NO:46, and/or a functional fragment thereof. Alternatively or additionally, a recombinant eukaryotic alga as provided herein can include a non-native gene encoding a wax synthase/diacylglycerol transferase (WS/DGAT) from a prokaryote or a variant thereof having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the naturally-occurring prokaryotic WS/DGAT or a functional fragment thereof, and may optionally comprise a PH domain sequence.
The recombinant host cell may comprise, e.g., any of the nucleic acid sequences encoding a DGAT described herein and may comprise any of the nucleic acid sequences encoding a PH domain described herein (e.g., SEQ ID NOs:6, 12, 18, 24, 30, 36, 42, and/or 48) or variants thereof. Further, the recombinant host cells may comprise, e.g., any of the vectors described herein. In some examples, the recombinant host cell includes a microalgal or heterokont DGAT1 gene that includes a PH domain or a variant thereof, and may include a DGAT gene of a green alga (Chlorophyte division) that includes a PH domain.
The recombinant microorganisms and host cells of the present invention may comprise any acyltransferase comprising any heterologous PH domain. By way of non-limiting example, a recombinant microorganism according to the present invention may comprise a polypeptide having acyltransferase activity, for example a DGAT1, comprising a PH domain selected from the group consisting of: a PH domain having at least 80% identity to SEQ ID NO:6; a PH domain having at least 80% identity to SEQ ID NO:12; a PH domain having at least 80% identity to SEQ ID NO:18; a PH domain having at least 80% identity to SEQ ID NO:24; a PH domain having at least 80% identity to SEQ ID NO:30; a PH domain having at least 80% identity to SEQ ID NO:36; a PH domain having at least 80% identity to SEQ ID NO:42; and a PH domain having at least 80% identity to SEQ ID NO:48.
Alternatively or additionally, the recombinant microorganisms and host cells of the present invention may comprise a polypeptide having acyltransferase activity, for example a DGAT1, comprising a PH domain selected from the group consisting of: a PH domain having at least 95% identity to SEQ ID NO:6; a PH domain having at least 95% identity to SEQ ID NO:12; a PH domain having at least 95% identity to SEQ ID NO:18; a PH domain having at least 95% identity to SEQ ID NO:24; a PH domain having at least 95% identity to SEQ ID NO:30; a PH domain having at least 95% identity to SEQ ID NO:36; a PH domain having at least 95% identity to SEQ ID NO:42; and a PH domain having at least 95% identity to SEQ ID NO:48.
In certain embodiments, the recombinant microorganism or host cell comprises a polypeptide having acyltransferase activity comprising a heterologous PH domain because the cell, or a progenitor, is transformed with a nucleic acid sequence encoding a heterologous PH domain, which integrates into an endogenous acyltransferase gene of the recombinant microorganism or host cell.
In some aspects, the nucleic acid sequence encoding the acyltransferase gene, for example a DGAT gene, is heterologous with respect to the recombinant host cell, and can be derived from any species, including plant, animal, or microbial species.
Additionally or alternatively, the recombinant microorganisms or host cells of the present invention may comprise an acyltransferase gene, for example a DGAT gene, that is homologous with respect to the host organism. For example, the acyltransferase gene may be native to the host microorganism and is introduced into the recombinant microorganism in an expression cassette that allows non-native patterns of regulated expression or overexpression of the endogenous acyltransferase gene. Additionally, the acyltransferase gene, for example a DGAT gene, may be endogenous to the microorganism and a heterologous promoter may be introduced into the host microorganism such that it becomes juxtaposed with and operably linked to the endogenous acyltransferase gene.
The recombinant microorganism can comprise a non-native gene encoding:

    
    
        a DGAT with at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:2;
        a DGAT with at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:4;
        a DGAT with at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:8;
        a DGAT with at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:10;
        a DGAT with at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:14;
        a DGAT with at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:16;
        a DGAT with at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:20;
        a DGAT with at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:22;
        a DGAT with at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:26;
        a DGAT with at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:28;
        a DGAT with at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:32;
        a DGAT with at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:34;
        a DGAT with at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:38;
        a DGAT with at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:40;
        a DGAT with at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:44;
        a DGAT with at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:46; and/or
        a functional fragment of any one of the DGAT listed sequences.
    
    


Additionally, the recombinant microorganism can comprise a non-native gene encoding the DGAT of SEQ ID NOs:2, 4, 8, 10, 14, 16, 20, 22, 26, 28, 32, 34, 38, 40, 44, and/or 46.
Alternatively, the recombinant microorganism can comprise a non-native gene encoding a DGAT with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs:2, 4, 8, 10, 14, 16, 20, 22, 26, 28, 32, 34, 38, 40, 44, and/or 46; and/or can encode a DGAT with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs:2, 4, 8, 10, 14, 16, 20, 22, 26, 28, 32, 34, 38, 40, 44, and/or 46.
Alternatively or additionally, the recombinant microorganisms or host cells of the present invention comprise a nucleic acid molecule encoding a polypeptide having DGAT1 activity comprising a sequence selected from the group consisting of: an amino acid sequence having at least 80% identity to SEQ ID NO:2 or a functional fragment thereof; an amino acid sequence having at least 80% identity to SEQ ID NO:8 or a functional fragment thereof; an amino acid sequence having at least 80% identity to SEQ ID NO:14 or a functional fragment thereof; an amino acid sequence having at least 80% identity to SEQ ID NO:20 or a functional fragment thereof; an amino acid sequence having at least 80% identity to SEQ ID NO:26 or a functional fragment thereof; and an amino acid sequence having at least 80% identity to SEQ ID NO:32 or a functional fragment thereof.
Alternatively or additionally, the recombinant microorganism or host cell of the present invention comprises a nucleic acid molecule encoding a polypeptide having DGAT1 activity comprising a sequence selected from the group consisting of: an amino acid sequence having at least 95% identity to SEQ ID NO:2 or a functional fragment thereof; an amino acid sequence having at least 95% identity to SEQ ID NO:8 or a functional fragment thereof; an amino acid sequence having at least 95% identity to SEQ ID NO:14 or a functional fragment thereof; an amino acid sequence having at least 95% identity to SEQ ID NO:20 or a functional fragment thereof; an amino acid sequence having at least 95% identity to SEQ ID NO:26 or a functional fragment thereof; and an amino acid sequence having at least 95% identity to SEQ ID NO:32 or a functional fragment thereof.
Recombinant microorganisms or host cells may be of prokaryotic or eukaryotic origin, including, without limitation, fungi, heterokonts, algae, eubacteria, archaebacteria, green nonsulfur bacteria, purple nonsulfur bacteria, or cyanobacteria.
Non-photosynthetic microorganisms and host cells such as fungi and non-algal stramenophiles are considered as hosts that can include non-native acyltransferase genes. Oleaginous yeasts, including but not limited to Aspergillus niger, Yarrowia lipolytica, Cryptococcus curvatus, Cryptococcus terricolus, Candida species, Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis vernalis, Rhodotorula glutinis, and Rhodotorula gracilis or other fungi or yeasts, including but not limited to species of Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete, Chrysosporium, Saccharomyces, and Schizosaccharomyces, are also encompassed as microorganisms and host cells. Further considered are Labyrinthulomycete species (e.g., Thraustichytrium, Ulkenia, and Schizochytrium species).
In some embodiments, the microorganism or host cell can be a bacterium, such as, but not limited to, an Acetobacter, Acinetobacter, Arthrobacter, Bacillus, Brevibacterium, Chromatium, Chlorobium, Clostridium, Corynebacterium, Deinococcus, Delftia, Desulfovibrio, Enterococcus, Escherichia, Kineococcus, Klebsiella, Lactobacillus, Lactococcus, Micrococcus, Mycobacterium, Jeotgalicoccus, Paenibacillus, Propionibacter, Pseudomonas, Rhodopseudomonas, Rhodobacter, Rhodococcus, Rhodospirillium, Rhodomicrobium, Salmonella, Serratia, Shewanella, Stenotrophomonas, Streptomyces, Streptococcus, Vibrio, or Zymomonas species. Photosynthetic bacteria, including for example, green sulfur bacteria, purple sulfur bacteria, green nonsulfur bacteria, purple nonsulfur bacteria, or cyanobacteria can be used.
Recombinant host cells can be photosynthetic organisms. Photosynthetic organisms include, by way of example, higher plants (i.e., vascular plants), bryophytes, algae, and photosynthetic bacteria. The term “algae” includes, without limitation, cyanobacteria (Cyanophyceae), green algae (Chlorophyceae), yellow-green algae (Xanthophyceae), golden algae (Chrysophyceae), brown algae (Phceophyceae), red algae (Rhodophyceae), diatoms (Bacillariophyceae), and “pico-plankton” (Prasinophyceae and Eustigmatophyceae). Also included in the term algae are members of the taxonomic classes Dinophyceae, Cryptophyceae, Euglenophyceae, Glaucophyceae, and Prymnesiophyceae. Microalgae are unicellular or colonial algae that can be seen as single organisms only with the aid of a microscope. Microalgae include both eukaryotic and prokaryotic algae (e.g., cyanobacteria).
Cyanobacterial species that can be used for production of TAG include, without limitation, Agmenellum, Anabaena, Anabaenopsis, Anacystis, Aphanizomenon, Arthrospira, Asterocapsa, Borzia, Calothrix, Chamaesiphon, Chroococcus, Chlorogloeopsis, Chroococcidiopsis, Chroococcus, Crinalium, Cyanobacterium, Cyanobium, Cyanocystis, Cyanospira, Cyanothece, Cylindrospermopsis, Cylindrospermum, Dactylococcopsis, Dermocarpella, Fischerella, Fremyella, Geitleria, Geitlerinema, Gloeobacter, Gloeocapsa, Gloeothece, Halospirulina, Iyengariella, Leptolyngbya, Limnothrix, Lyngbya, Microcoleus, Microcystis, Myxosarcina, Nodularia, Nostoc, Nostochopsis, Oscillatoria, Phormidium, Planktothrix, Pleurocapsa, Prochlorococcus, Prochloron, Prochlorothrix, Pseudanabaena, Rivularia, Schizothrix, Scytonema, Spirulina, Stanieria, Starria, Stigonema, Symploca, Synechococcus, Synechocystis, Thermosynechococcus, Tolypothrix, Trichodesmium, Tychonema and Xenococcus. For example, the recombinant photosynthetic microorganism can be a Cyanobium, Cyanothece, or Cyanobacterium species, or further alternatively, the recombinant photosynthetic microorganism can be a Gloeobacter, Lyngbya or Leptolyngba species. Alternatively, the recombinant photosynthetic microorganism can be a Synechococcus, Synechocystis, or Thermosynechococcus species. A number of cyanobacterial species are known and have been manipulated using molecular biological techniques, including the unicellular cyanobacteria Synechocystis sp. PCC6803 and Synechococcus elongates PCC7942, whose genomes have been completely sequenced.
Eukaryotic microalgae for use in the invention, include without limitation, Achnanthes, Amphiprora, Amphora, Ankistrodesmus, Asteromonas, Boekelovia, Borodinella, Botryococcus, Bracteococcus, Chaetoceros, Carteria, Chlamydomonas, Chlorococcum, Chlorogonium, Chlorella, Chroomonas, Chrysosphaera, Cricosphaera, Crypthecodinium, Cryptomonas, Cyclotella, Dunaliella, Ellipsoidon, Emiliania, Eremosphaera, Ernodesmius, Euglena, Franceia, Fragilaria, Gloeothamnion, Haematococcus, Halocafeteria, Hymenomonas, Isochrysis, Lepocinclis, Micractinium, Monoraphidium, Nannochloris, Nannochloropsis, Navicula, Neochloris, Nephrochloris, Nephroselmis, Nitzschia, Ochromonas, Oedogonium, Oocystis, Ostreococcus, Pavlova, Parachlorella, Pascheria, Phaeodactylum, Phagus, Picochlorum, Platymonas, Pleurochrysis, Pleurococcus, Prototheca, Pseudochlorella, Pseudoneochloris, Pyramimonas, Pyrobotrys, Scenedesmus, Schizochytrium, Skeletonema, Spyrogyra, Stichococcus, Tetraselmis, Viridiella, or Volvox species. In a particular aspect, Nannochloropsis is used as the host cell, i.e. a recombinant algal cell. Illustrative examples of recombinant microorganisms that express a non-native acyltransferase gene include recombinant microorganisms such as but not limited to Nannochloropsis gaditana that express a non-native gene encoding a DGAT having at least 85% identity, for example at least 90% or at least 95% identity to SEQ ID NOs:2, 4, 8, 10, 14, 16, 20, 22, 26, 28, 32, 34, 38, 40, 44, and/or 46.
Additionally or alternatively, the recombinant microorganism can comprise a non-native gene, in addition to the non-native acyltransferase, which encodes a polypeptide for the production of a lipid, such as, for example, a non-native gene encoding an enzyme for the production of fatty acids, fatty acid derivatives, and/or glycerolipids in the production of a fatty acid product not normally produced by the microorganism. For example, a recombinant microorganism as disclosed herein can include a non-native gene encoding a DGAT1 and can further include a non-native gene encoding an enzyme that participates in the production of glycerolipids, including, but not limited to, a MGAT, DGAT2, DGAT3, WS/DGAT, DGAcT, DGTA, PDAT, LPLAT, LPAAT, GPAT, and/or another DGAT1.
Additionally, the recombinant microorganism can comprise a non-native gene encoding the PH domain of SEQ ID NOs:6, 12, 18, 24, 30, 36, 42, and/or 48.
Alternatively, the recombinant microorganism can comprise a non-native gene encoding a PH domain with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs:6, 12, 18, 24, 30, 36, 42, and/or 48; and/or can encode a PH domain with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs:6, 12, 18, 24, 30, 36, 42, and/or 48.
Illustrative examples of recombinant microorganisms that express a PH domain gene include recombinant microorganisms such as but not limited to Nannochloropsis gaditana that express a gene encoding a polypeptide having an acyltransferase activity and comprising a heterologous PH domain having at least 85% identity, for example at least 90% or at least 95% identity to SEQ ID NOs:6, 12, 18, 24, 30, 36, 42, and/or 48.
Additionally or alternatively, the recombinant microorganism can comprise a gene which encodes a polypeptide for the production of a lipid, such as, for example, an enzyme for the production of fatty acids, fatty acid derivatives, and/or glycerolipids in the production of a fatty acid product not normally produced by the microorganism, wherein said polypeptide comprises a heterologous PH domain. For example, a recombinant microorganism as disclosed herein can include a gene encoding a non-native DGAT1 and can further optionally include a gene encoding an enzyme that participates in the production of glycerolipids, including, but not limited to, a MGAT, DGAT2, DGAT3, WS/DGAT, DGAcT, DGTA, PDAT, LPLAT, LPAAT, GPAT, and/or another DGAT1, wherein said enzyme comprises a heterologous PH domain.
In further examples, the recombinant microorganism produces a greater amount of TAG than is produced by a control algal cell. In some embodiments, a recombinant microorganism or host cell as provided herein can comprise a non-native gene encoding an acyltransferase, such as DGAT, wherein the recombinant microorganism can produce a greater amount of triglyceride than is produced by a control recombinant microorganism or host cell substantially identical to the recombinant recombinant microorganism or host cell, but lacking a non-native gene encoding an acyltransferase. For example the recombinant algal cell comprising a DGAT as described herein results in a higher production level of TAG by the recombinant microorganism than the production level in a control microorganism, where the control microorganism is cultured under substantially the same conditions and is substantially identical to the microorganism expressing the non-native DGAT gene in all respects, with the exception that the control microorganism does not express a non-native DGAT gene. In particular examples, the recombinant microorganism can be a photosynthetic microorganism such as a recombinant algal cell.
Alternatively or additionally, a recombinant microorganism or host cell as provided herein can comprise a non-native gene encoding an acyltransferase, such as DGAT, comprising a heterologous PH domain and can produce a greater amount of triglyceride than is produced by a control recombinant microorganism or host cell substantially identical to the recombinant microorganism or host cell, but lacking the heterologous PH domain.
In some aspects, the amount of protein produced by a culture of the recombinant microorganism expressing a non-native acyltransferase gene comprising a PH domain is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 225%, at least 250%, at least 275%, at least 300%, at least 325%, at least 350%, at least 375%, at least 400%, at least 425%, at least 450%, at least 475%, at least 500%, at least 525%, at least 550%, at least 575%, at least 600%, at least 625%, at least 650%, at least 675%, at least 700%, at least 725%, at least 750%, at least 775%, at least 800%, at least 825%, at least 850%, at least 875%, at least 900%, at least 925%, at least 950%, at least 975%, or at least 1000% greater than the amount of protein produced by a control host cell that does not express the acyltransferase gene comprising a heterologous PH domain.
In some aspects, the amount of TAG produced by a culture of the recombinant microorganism expressing a DGAT comprising a heterologous PH domain is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 225%, at least 250%, at least 275%, at least 300%, at least 325%, at least 350%, at least 375%, at least 400%, at least 425%, at least 450%, at least 475%, at least 500%, at least 525%, at least 550%, at least 575%, at least 600%, at least 625%, at least 650%, at least 675%, at least 700%, at least 725%, at least 750%, at least 775%, at least 800%, at least 825%, at least 850%, at least 875%, at least 900%, at least 925%, at least 950%, at least 975%, or at least 1000% greater than the amount of TAG produced by a control host cell that does not express the DGAT comprising a heterologous PH domain.
In certain aspects, the recombinant microorganism can produce more TAG when compared to a control host cell under nitrogen replete conditions. Additionally, in certain examples, the recombinant microorganism can produce a greater amount of TAG after about one, or two or three days of culturing under nitrogen replete conditions.
Additionally, a culture of a recombinant photosynthetic microorganism as described herein is provided. The culture can produce a greater amount of TAG than is produced by a control culture substantially identical in all respects except that the recombinant photosynthetic microorganism of the control culture does not include or does not express the gene encoding a non-native DGAT or the gene encoding a DGAT comprising a heterologous PH domain. Preferably, a culture of the recombinant photosynthetic microorganism that includes a gene encoding a non-native DGAT or a DGAT comprising a heterologous PH domain (and optionally a non-native gene encoding a polypeptide that participates in the production of a lipid) produces a greater amount of fatty acid product, for example TAG, than is produced by a culture of an otherwise substantially identical recombinant photosynthetic microorganism that lacks the non-native gene encoding a DGAT. For example, a photoautotrophic culture of the recombinant photosynthetic microorganism can preferably produce a greater amount of a fatty acid product, for example TAG, than is produced by a photoautotrophic culture of an otherwise identical photosynthetic microorganism that lacks the gene encoding the non-native DGAT or the DGAT comprising a heterologous PH domain. Additionally or alternatively, a culture of the recombinant photosynthetic microorganism can achieve a higher cell density while producing TAG under photoautotrophic conditions, e.g., using inorganic (non-reduced) carbon as the carbon source for production of the fatty acid product such as TAG.
Transformation of Microorganisms and Host Cells
A vector comprising an isolated nucleic acid comprising a gene of interest can be introduced into a host cell via conventional transformation and/or transfection techniques. The terms “transformation,” “transfection,” “conjugation,” and “transduction,” as used in the present context, are intended to comprise a multiplicity of methods known to those skilled in the art for the introduction of foreign nucleic acid (for example, exogenous DNA) into a host cell, including calcium phosphate and/or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipofection, natural competence, chemically mediated transfer, electroporation, particle bombardment, or the like, or combinations thereof. Examples of suitable methods for the transformation and/or transfection of host cells, e.g., can be found in Molecular Cloning—A Laboratory Manual (2010), Cold Spring Harbor Laboratory Press.
Host cells such as plants for use in the invention can be transformed by any feasible means, including, without limitation, the use of Agrobacterium, particle gun-mediated transformation, laser-mediated transformation, or electroporation. Algae and photosynthetic bacteria can be transformed by any suitable methods, including, as nonlimiting examples, natural DNA uptake (Chung et al. (1998) FEMS Microbiol. Lett. 164:353-61; Frigaard et al. (2004) Methods Mol. Biol. 274:325-40; Zang et al. (2007) J. Microbiol. 45:241-45), conjugation, transduction, glass bead transformation (Kindle et al. (1989) J. Cell Biol. 109:2589-601; Feng et al. (2009) Mol. Biol. Rep. 36:1433-39; U.S. Pat. No. 5,661,017), silicon carbide whisker transformation (Dunahay et al. (1997) Methods Mol. Biol. 62:503-09), biolistics (Dawson et al. (1997) Curr. Microbiol. 3: 356-62; Hallmann et al. (1997) Proc. Nat'l. Acad. USA 94:7469-74; Jakobiak et al. (2004) Protist 155:381-93; Tan et al. (2005) J. Microbiol. 43:361-65; Steinbrenner et al. (2006) Appl. Environ. Microbiol. 72:7477-84; Kroth (2007) Methods Mol. Biol. 390:257-67; U.S. Pat. No. 5,661,017) electroporation (Kjaerulff et al. (1994) Photosynth. Res. 41:277-83; Iwai et al. (2004) Plant Cell. Physiol. 45:171-75; Ravindran et al. (2006) J. Microbiol. Methods 66:174-76; Sun et al. (2006) Gene 377:140-49; Wang et al. (2007) Appl. Microbiol. Biotechnol. 76:651-57; Chaurasia et al. (2008) J. Microbiol. Methods 73:133-41; Ludwig et al. (2008) Appl. Microbiol. Biotechnol. 78:729-35), laser-mediated transformation, or incubation with DNA in the presence of or after pre-treatment with any of poly(amidoamine) dendrimers (Pasupathy et al. (2008) Biotechnol. J. 3:1078-82), polyethylene glycol (Ohnuma et al. (2008) Plant Cell. Physiol. 49:117-20), cationic lipids (Muradawa et al. (2008) J. Biosci. Bioeng. 105:77-80), dextran, calcium phosphate, or calcium chloride (Mendez-Alvarez et al. (1994) J. Bacteriol. 176:7395-97), optionally after treatment of the cells with cell wall-degrading enzymes (Perrone et al. (1998) Mol. Biol. Cell 9:3351-65). Agrobacterium-mediated transformation can also be performed on algal cells, for example after removing or wounding the algal cell wall (e.g., WO 2000/62601; Kumar et al. (2004) Plant Sci. 166:731-38). Biolistic methods are particularly successful for transformation of the chloroplasts of plant and eukaryotic algal species (see, for example, Ramesh et al. (2004) Methods Mol. Biol. 274:301-07; Doestch et al. (2001) Curr. Genet. 39:49-60; U.S. Pat. No. 7,294,506; WO 2003/091413; WO 2005/005643; WO 2007/133558; and WO 2011/034863, all incorporated herein by reference in their entireties).
Methods of Peptide Targeting/Localization
The invention also encompasses methods of targeting or localizing a peptide to a membrane. This can be achieved by fusing a nucleic acid coding for a PH domain in frame with a nucleic acid coding for desired peptide to be expressed, and then expressing this fused nucleic acid in a cell of interest. For example, one can fuse a nucleic acid coding for a PH domain with at least 80% sequence identity to SEQ ID NOs:6, 12, 18, 24, 30, 36, 42, and/or 48 in frame with a nucleic acid coding for a desired peptide to be expressed, and then expressing this fused nucleic acid in a cell of interest. As used herein, a fused nucleic acid can be “expressed” by means described elsewhere herein and by other means well known to those skilled in the art.
Additionally, a method is provided of targeting or localizing an acyltransferase to a membrane surface comprising expressing an isolated or recombinant nucleic acid molecule encoding a polypeptide having acyltransferase activity, wherein the polypeptide comprises a heterologous PH domain. This can be achieved by fusing a nucleic acid coding for a PH domain with at least 80% sequence identity to SEQ ID NOs:6, 12, 18, 24, 30, 36, 42, and/or 48 in frame with a nucleic acid coding for a polypeptide having acyltransferase activity, such as a DGAT, to be expressed, and then expressing this fused nucleic acid in a cell of interest.
SEQ ID NOs:6, 12, 18, 24, 30, 36, 42, and 48 comprise PH domains isolated from a variety of algal species. While the invention is not bound by any one theory, it is hypothesized that a protein comprising one or more of these PH domains is directed, by virtue of the PH domain's affinity for particular lipid compositions, to a membrane or membrane region enriched in substrates for lipid biosynthesis.
In some examples, the protein to which the PH domain is fused can be a MGAT, DGAT1, DGAT2, DGAT3, WS/DGAT, DGAcT, DGTA, PDAT, LPLAT, LPAAT, and/or GPAT. While the invention is not bound by any one theory, it is believed that the chloroplastic environment is rich in substrate pools for acetyl coenzyme A, so TAG production can be enhanced by targeting a DGAT to the endoplasmic reticulum or a region thereof, the chloroplast envelope, or a region of the plasma membrane.
Methods of Producing Triglyceride (TAG)
The invention also encompasses methods of producing TAG by culturing the recombinant microorganisms and host cells described herein, under conditions in which TAG is produced. The present invention further encompasses methods of producing TAG by culturing recombinant microorganisms and host cells under conditions in which TAG is produced, wherein the recombinant microorganisms and host cells comprise DGAT enzymes comprising heterologous PH domains.
For example, by culturing a recombinant microorganism comprising a recombinant DGAT selected from the group consisting of SEQ ID NOs:2, 8, 14, 20, 26, and 32, TAG production can be enhanced relative to production achieved from a substantially identical culture of a control microorganism that lacks the recombinant DGAT. For example, the recombinant microorganism to be used in the methods of the present invention can be selected from the group consisting of a recombinant microorganism that comprises a recombinant nucleic acid molecule that encodes a polypeptide having DGAT activity comprising a sequence selected from the group consisting of: an amino acid sequence having at least 80% identity to SEQ ID NO:2 or a functional fragment thereof; an amino acid sequence having at least 80% identity to SEQ ID NO:8 or a functional fragment thereof; an amino acid sequence having at least 80% identity to SEQ ID NO:14 or a functional fragment thereof; an amino acid sequence having at least 80% identity to SEQ ID NO:20 or a functional fragment thereof; an amino acid sequence having at least 80% identity to SEQ ID NO:26 or a functional fragment thereof; and an amino acid sequence having at least 80% identity to SEQ ID NO:32 or a functional fragment thereof. This microorganism can be cultured under conditions in which the recombinant nucleic acid molecule is expressed, to produce TAG.
Alternatively or additionally, the recombinant microorganism to be used in the methods of the present invention can be selected from the group consisting of a recombinant microorganism that comprises a recombinant nucleic acid molecule that encodes a polypeptide having DGAT activity comprising a sequence selected from the group consisting of: an amino acid sequence having at least 95% identity to SEQ ID NO:2 or a functional fragment thereof; an amino acid sequence having at least 95% identity to SEQ ID NO:8 or a functional fragment thereof; an amino acid sequence having at least 95% identity to SEQ ID NO:14 or a functional fragment thereof; an amino acid sequence having at least 95% identity to SEQ ID NO:20 or a functional fragment thereof; an amino acid sequence having at least 95% identity to SEQ ID NO:26 or a functional fragment thereof; and an amino acid sequence having at least 95% identity to SEQ ID NO:32 or a functional fragment thereof. This microorganism can be cultured under conditions in which the recombinant nucleic acid molecule is expressed, to produce TAG.
Alternatively or additionally, the recombinant microorganism to be used in the methods of the present invention can comprise a nucleic acid molecule encoding a polypeptide having acyltransferase activity, where the polypeptide comprises a heterologous PH domain. For example, the recombinant microorganism can comprising a nucleic acid molecule encoding a polypeptide having acyltransferase activity, where the polypeptide comprises: a PH domain having at least 80% identity to SEQ ID NO:6; a PH domain having at least 80% identity to SEQ ID NO:12; a PH domain having at least 80% identity to SEQ ID NO:18; a PH domain having at least 80% identity to SEQ ID NO:24; a PH domain having at least 80% identity to SEQ ID NO:30; a PH domain having at least 80% identity to SEQ ID NO:36; a PH domain having at least 80% identity to SEQ ID NO:42; and a PH domain having at least 80% identity to SEQ ID NO:48.
Alternatively or additionally, the recombinant microorganism to be used in the methods of the present invention can comprise a nucleic acid molecule encoding a polypeptide having acyltransferase activity, where the polypeptide comprises: a PH domain having at least 95% identity to SEQ ID NO:6; a PH domain having at least 95% identity to SEQ ID NO:12; a PH domain having at least 95% identity to SEQ ID NO:18; a PH domain having at least 95% identity to SEQ ID NO:24; a PH domain having at least 95% identity to SEQ ID NO:30; a PH domain having at least 95% identity to SEQ ID NO:36; a PH domain having at least 95% identity to SEQ ID NO:42; and a PH domain having at least 95% identity to SEQ ID NO:48.
In certain embodiments, the polypeptide having acyltransferase activity is a DGAT. In certain embodiments the recombinant microorganism is a microalga. In certain embodiments, the microalga belongs to a species of the genus Achnanthes, Amphiprora, Amphora, Ankistrodesmus, Asteromonas, Boekelovia, Bolidomonas, Borodinella, Botrydium, Botryococcus, Bracteococcus, Chaetoceros, Carteria, Chlamydomonas, Chlorococcum, Chlorogonium, Chlorella, Chroomonas, Chrysosphaera, Cricosphaera, Crypthecodinium, Cryptomonas, Cyclotella, Dunaliella, Ellipsoidon, Emiliania, Eremosphaera, Ernodesmius, Euglena, Eustigmatos, Franceia, Fragilaria, Gloeothamnion, Haematococcus, Halocafeteria, Heterosigma, Hymenomonas, Isochrysis, Lepocinclis, Micractinium, Monoraphidium, Nannochloris, Nannochloropsis, Navicula, Neochloris, Nephrochloris, Nephroselmis, Nitzschia, Ochromonas, Oedogonium, Oocystis, Ostreococcus, Pavlova, Parachlorella, Pascheria, Pelagomonas, Phaeodactylum, Phagus, Picochlorum, Platymonas, Pleurochrysis, Pleurococcus, Prototheca, Pseudochlorella, Pseudoneochloris, Pseudostaurastrum, Pyramimonas, Pyrobotrys, Scenedesmus, Schizochlamydella, Skeletonema, Spyrogyra, Stichococcus, Tetrachlorella, Tetraselmis, Thalassiosira, Tribonema, Vaucheria, Viridiella, Vischeria, or Volvox. In some particular embodiments, the microalga belongs to a species of the genus Nannochloropsis, particularly the species N. gaditana. 
The methods can further comprise isolating TAG. Additionally, the expression of a polypeptide encoded by the nucleic acid molecules described herein can be induced in the recombinant microorganism to produce the TAG. In some examples, the TAG production occurs during nitrogen-replete culture conditions. Additionally or alternatively, the TAG production occurs during photoautotrophic culture conditions. Additionally or alternatively, the TAG production occurs while the host organism is actively growing and dividing.
As discussed previously, the present invention further provides for a culture comprising the recombinant microorganisms producing TAG. Culturing refers to the intentional fostering of growth (e.g., increases in cell size, cellular contents, and/or cellular activity) and/or propagation (e.g., increases in cell numbers via mitosis) of one or more cells by use of selected and/or controlled conditions. The combination of both growth and propagation may be termed proliferation. Non-limiting examples of selected and/or controlled conditions can include the use of a defined medium (with known characteristics such as pH, ionic strength, nitrogen concentration, and/or carbon source), specified temperature, oxygen tension, carbon dioxide levels, growth in a bioreactor, or the like, or combinations thereof. In some embodiments, the microorganism or host cell can be grown heterotrophically, using a reduced carbon source, or mixotrophically, using both light and a reduced carbon source. Additionally or alternately, the microorganism or host cell can be cultured photoautotrophically. When growing photoautotrophically, the microorganism can advantageously use light as an energy source. An inorganic carbon source, such as CO2 or bicarbonate, can be used for synthesis of biomolecules by the microorganism. “Inorganic carbon”, as used herein, includes carbon-containing compounds or molecules that cannot be used as a sustainable energy source by an organism. Typically “inorganic carbon” can be in the form of CO2 (carbon dioxide), carbonic acid, bicarbonate salts, carbonate salts, hydrogen carbonate salts, or the like, or combinations thereof, which cannot be further oxidized for sustainable energy nor used as a source of reducing power by organisms. Under conditions in which inorganic carbon is substantially the sole source of carbon, if an organic carbon molecule or compound is provided in the culture medium, it generally cannot be taken up and/or metabolized by the cell for energy and/or typically is not present in an amount sufficient to provide sustainable energy for the growth of the cell culture. However, microorganisms growing heterotrophically do utilize organic carbon provided in the culture medium. Thus, the present invention includes a process for converting a carbon source to TAG comprising contacting the carbon source with a recombinant microorganism or host cell of the invention. In some aspects the carbon source is an inorganic carbon source and in other aspects the carbon source is an organic carbon source.
Microorganisms and host cells that can be useful in accordance with the methods of the present invention can be found in various locations and environments throughout the world. Without wishing to be bound by theory, it is observed that, perhaps as a consequence of their isolation from other species and their evolutionary divergence, the particular growth medium for optimal growth and generation of lipid and/or other hydrocarbon constituents can vary. In some cases, certain strains of microorganisms may be unable to grow in a particular growth medium because of the presence of some inhibitory component or the absence of some essential nutritional requirement of the particular strain of microorganism or host cell.
Solid and liquid growth media are generally available from a wide variety of sources, as are instructions for the preparation of particular media suitable for a wide variety of strains of microorganisms. For example, various fresh water and salt water media can include those described in Barsanti (2005) Algae: Anatomy, Biochemistry & Biotechnology (CRC Press) for media and methods for culturing algae. Algal media recipes can also be found at the websites of various algal culture collections, including, as nonlimiting examples, the UTEX Culture Collection of Algae (www.sbs.utexas.edu/utex/media.aspx) (visited 15 Nov. 2012); Culture Collection of Algae and Protozoa (www.ccap.ac.uk) (visited 15 Nov. 2012); and CAUP Culture Collection (botany.natur.cuni.cz/algo/caup-media.html) (visited 15 Nov. 2012).
In some embodiments, the nitrogen content of the medium can be “replete”, that is, the level of nitrogen is not limiting to culture propagation. The amount of nitrogen required in a replete nitrogen culture medium can vary depending on the algal strain and other culture conditions, but preferably is at least about 250 μM, at least 500 μM, or at least 800 μM, such as at least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, or at least 8 mM nitrogen, which is preferably supplied as ammonia and/or nitrate, but can be supplied as any utilizable nitrogen source compound. In some embodiments, the culture medium can be nitrogen replete and can lack a supplementary organic carbon source. In some embodiments, the culture medium can be nutrient replete, where no nutrients (not including a carbon source) are limiting for culture propagation, and the culture medium can lack a supplementary organic carbon source.
In some examples, a culture medium used during at least a portion of the production period may be nitrogen limited but not necessarily nitrogen depleted. For example, the amount of nitrogen provided in the culture medium might permit culture proliferation but be less than the amount required for optimal growth (e.g., optimal rates of biomass accumulation or cell division). A “nitrogen deplete” medium does not include a nitrogen source that can be utilized by the microorganism for growth or culture propagation. A microorganism cultured in a nitrogen deplete medium experiences nitrogen starvation conditions.
The culture methods can include inducing expression of a particular gene described herein for the production of TAG, and/or regulating a metabolic pathway in the microorganism. Inducing expression can include adding a nutrient or compound to the culture, removing one or more components from the culture medium, increasing or decreasing light and/or temperature, and/or other manipulations that promote expression of the gene of interest. Such manipulations can largely depend on the nature of the (heterologous) promoter operably linked to the gene of interest.
In some embodiments of the present invention, the recombinant microorganisms or host cells can be cultured in a bioreactor. “Bioreactor” refers to an enclosure or partial enclosure in which cells are cultured, optionally in suspension and, when suspended, preferably in an aqueous liquid. The bioreactor can be used to culture microalgal cells through the various phases of their physiological cycle. Bioreactors can offer many advantages for use in heterotrophic growth and propagation methods. To produce biomass for use as food, microorganisms or host cells are preferably fermented in large quantities in liquid, such as in suspension cultures as an example. Bioreactors such as steel fermentors can accommodate very large culture volumes (40 kiloliter and greater capacity bioreactors can be used in various embodiments of the invention). Bioreactors can also typically allow for the control of one or more culture conditions such as temperature, pH, oxygen tension, carbon dioxide levels, and the like, as well as combinations thereof. Bioreactors can typically be configurable, for example, using ports attached to tubing, to allow gaseous components, such as CO2, CO2-enriched air, oxygen, and/or nitrogen, to be contacted with (e.g., bubbled through) a liquid culture. Other culture parameters, such as the pH of the culture media, the identity and/or concentration of trace elements and/or nutrients, the identity and/or concentration of other media constituents, or the like, or combinations thereof, can typically be more readily manipulated using a bioreactor.
Microorganisms and host cells can additionally or alternately be cultured in a bioreactor equipped with an artificial light source, a “photobioreactor,” and/or can have one or more walls that is transparent enough to light, including sunlight, to enable, facilitate, and/or maintain acceptable microorganism growth. For TAG production, photosynthetic microorganisms or host cells can additionally or alternately be cultured in shake flasks, test tubes, vials, microtiter dishes, petri dishes, or the like, or combinations thereof.
Additionally or alternatively, recombinant photosynthetic microorganisms or host cells may be grown in ponds, canals, sea-based growth containers, trenches, raceways, channels, or the like, or combinations thereof. As with standard bioreactors, a source of inorganic carbon (such as, but not limited to, CO2, bicarbonate, carbonate salts, and the like), including, but not limited to, air, CO2-enriched air, flue gas, or the like, or combinations thereof, can be supplied to the culture. When supplying flue gas and/or other sources of inorganic that may contain CO in addition to CO2, it may be necessary to pre-treat such sources such that the CO level introduced into the (photo)bioreactor do not constitute a dangerous and/or lethal dose with respect to the growth and/or survival of the microorganisms.
The methods include culturing a recombinant microorganism, such as a photosynthetic microorganism, such as, for example, an algae, that expresses a protein as described herein to produce TAG, in which the method results in production of at least 0.1%, at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% more than the amount of the TAG produced by an otherwise substantially identical microorganism not including the protein(s), cultured under identical conditions. Additionally or alternatively, the methods include producing at least 100 mg, at least 110 mg, at least 120 mg, at least 130 mg, at least 140 mg, at least 150 mg, at least 160 mg, at least 170 mg, at least 180 mg, at least 190 mg, at least 200 mg, at least 210 mg, at least 220 mg, at least 230 mg, at least 240 mg, at least 250 mg, at least 260 mg, at least 270 mg, at least 280 mg, at least 290 mg, at least 300 mg, at least 310 mg, at least 320 mg, at least 330 mg, at least 340 mg, at least 350 mg, at least 360 mg, at least 370 mg, at least 380 mg, at least 390 mg, at least 400 mg, at least 450 mg, at least 500 mg, at least 550 mg, at least 600 mg, at least 650 mg, at least 700 mg, at least 750 mg, at least 800 mg, at least 850 mg, at least 900 mg, or at least 950 mg, per liter of culture of TAG by culturing the recombinant microorganisms described herein.
TAG can be recovered from culture by recovery means known to those of ordinary skill in the art, such as by whole culture extraction, for example, using organic solvents. In some cases, recovery of TAG can be enhanced by homogenization of the cells. For example, lipids such as TAG can be isolated from algae by extraction of the algae with a solvent at elevated temperature and/or pressure, as described in the co-pending, commonly-assigned U.S. patent application Ser. No. 13/407,817 entitled “Solvent Extraction of Products from Algae,” filed on 29 Feb. 2012, which is incorporated herein by reference in its entirety. Further, when TAG is sufficiently released or secreted from the microorganisms into the culture medium, the recovery method can be adapted to recover efficiently only the released TAG, only the TAG produced and stored within the microorganisms, or both the produced and released TAG.
It is to be understood that the disclosure of the present invention extends to methods, products and systems according to the various aspects of the invention which comprise combinations of one or more features discussed herein by reference to certain embodiments of the invention with one or more further features discussed herein by reference to certain other embodiments of the invention.
Additionally or alternatively, the present invention can include one or more of the following embodiments.
FURTHER EMBODIMENTS
Embodiment 1. An isolated or recombinant nucleic acid molecule encoding a polypeptide having acyltransferase activity comprising a sequence encoding a Pleckstrin Homology (PH) domain, optionally wherein one or more of the following are satisfied:

    
    
        a) the PH domain sequence is derived from a DGAT1 protein;
        b) the PH domain sequence is derived from a polypeptide of a eukaryotic microalga, optionally a eukaryotic microalga of the Heterokontophyta division or the Chlorophyta division, for example, a microalga of the Bacillariophyceae, Chlorophyceae, Prasinophyceae, or Trebouxiophyceae class;
        c) the PH domain is has at least 80% identity to a SEQ ID NO:6; at least 80% identity to SEQ ID NO:12; at least 80% identity to SEQ ID NO:18; at least 80% identity to SEQ ID NO:24; at least 80% identity to SEQ ID NO:30; at least 80% identity to SEQ ID NO:36; at least 80% identity to SEQ ID NO:42; or at least 80% identity to SEQ ID NO:48; and/or d) the polypeptide having acyltransferase activity is a monoacylglycerol acyltransferase (MGAT), a diacylglycerol acyltransferase type 1 (DGAT1), a diacylglycerol acyltransferase type 2 (DGAT2), a diacylglycerol acyltransferase type 3 (DGAT3), a diacylglycerol acetyltransferase (DGAcT), a diacylglycerol transacylase (DGTA), a phospholipid:diacylglycerol acyltransferase (PDAT), a lysophospholipid acyltransferase (LPLAT), a lysophosphatidic acid acyltransferase (LPAAT), or a glycerolphosphate acyltransferase (GPAT).
    
    


Embodiment 2. An isolated or recombinant nucleic acid molecule according to Embodiment 1, wherein the PH domain has:

    at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to a SEQ ID NO:6;
    at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO:12;
    at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO:18;
    at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO:24;
    at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO:30;
    at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO:36;
    at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO:42;
    or at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO:48


Embodiment 3. An isolated or recombinant nucleic acid molecule according Embodiment 1 or 2, wherein the polypeptide having acyltransferase activity has DGAT activity, and preferably is a DGAT1, a DGAT2, or a DGAT3.
Embodiment 4. An isolated or recombinant nucleic acid molecule according to any of Embodiments 1-3, wherein the PH domain is heterologous with respect to the polypeptide having acyltransferase activity.
Embodiment 5. An isolated or recombinant nucleic acid molecule according to any of Embodiments 1-3, wherein the PH domain is homologous with respect to the polypeptide having acyltransferase activity, optionally wherein the polypeptide having acyltransferase activity is a DGAT1, further optionally wherein the DGAT1 comprises an amino acid sequence having at least 80% identity to SEQ ID NO:2 or a functional fragment thereof; an amino acid sequence having at least 80% identity to SEQ ID NO:8 or a functional fragment thereof; an amino acid sequence having at least 80% identity to SEQ ID NO:14 or a functional fragment thereof; an amino acid sequence having at least 80% identity to SEQ ID NO:20 or a functional fragment thereof; an amino acid sequence having at least 80% identity to SEQ ID NO:26 or a functional fragment thereof; or an amino acid sequence having at least 80% identity to SEQ ID NO:32 or a functional fragment thereof.
Embodiment 6. An isolated or recombinant nucleic acid molecule according to Embodiment 5, wherein the DGAT1 comprises an amino acid sequence having:

    
    
        at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to a SEQ ID NO:2 or a functional fragment thereof;
        at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO:8 or a functional fragment thereof;
        at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO:14 or a functional fragment thereof;
        at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO:20 or a functional fragment thereof;
        at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO:26 or a functional fragment thereof; or
        at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO:32 or a functional fragment thereof.
    
    


Embodiment 7. A recombinant eukaryotic microorganism comprising a recombinant nucleic acid molecule according to any of Embodiments 1-6.
Embodiment 8. A recombinant microorganism according to Embodiment 7, wherein the recombinant microorganism is a fungus, heterokont, or microalga.
Embodiment 9. A recombinant microorganism according to Embodiment 8, wherein the recombinant microorganism is a species belonging to any of the following:

    
    
        the Heterokontophyta or Chlorophyta division;
        the Bacillariophyceae, Chlorophyceae, Eustigmatophyceae, Prasinophyceae, or Trebouxiophyceae class; or
        a Achnanthes, Amphiprora, Amphora, Ankistrodesmus, Asteromonas, Boekelovia, Bolidomonas, Borodinella, Botrydium, Botryococcus, Bracteococcus, Chaetoceros, Carteria, Chlamydomonas, Chlorococcum, Chlorogonium, Chlorella, Chroomonas, Chrysosphaera, Cricosphaera, Crypthecodinium, Cryptomonas, Cyclotella, Dunaliella, Ellipsoidon, Emiliania, Eremosphaera, Ernodesmius, Euglena, Eustigmatos, Franceia, Fragilaria, Gloeothamnion, Haematococcus, Halocafeteria, Heterosigma, Hymenomonas, Isochrysis, Lepocinclis, Micractinium, Monoraphidium, Nannochloris, Nannochloropsis, Navicula, Neochloris, Nephrochloris, Nephroselmis, Nitzschia, Ochromonas, Oedogonium, Oocystis, Ostreococcus, Pavlova, Parachlorella, Pascheria, Pelagomonas, Phaeodactylum, Phagus, Picochlorum, Platymonas, Pleurochrysis, Pleurococcus, Prototheca, Pseudochlorella, Pseudoneochloris, Pseudostaurastrum, Pyramimonas, Pyrobotrys, Scenedesmus, Schizochlamydella, Skeletonema, Spyrogyra, Stichococcus, Tetrachlorella, Tetraselmis, Thalassiosira, Tribonema, Vaucheria, Viridiella, Vischeria, or Volvox genus.
    
    


Embodiment 10. A recombinant microorganism according to any of Embodiments 7-9, wherein the recombinant eukaryotic microorganism produces a greater amount of a lipid than a control recombinant eukaryotic identical in all respects to the recombinant eukaryotic microorganism, but lacking the recombinant nucleic acid molecule.
Embodiment 11. A recombinant microorganism according to Embodiment 10, wherein the lipid is a triglyceride.
Embodiment 12. A recombinant microorganism comprising a non-native DGAT1 gene that includes a PH domain, wherein the non-native DGAT1 gene encodes a polypeptide that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to a DGAT1 gene of a microalga of a species of the Bacillariophyceae, Prasinophyceae, or Trebouxiophyceae class, or a functional fragment thereof, wherein the recombinant microalga produces a greater amount of a lipid than a control recombinant eukaryotic identical in all respects to the recombinant eukaryotic microorganism, but lacking the non-native DGAT1 gene that includes a PH domain.
Embodiment 13. A recombinant microorganism according to Embodiment 12, wherein the recombinant DGAT1 gene encodes a polypeptide having at least 80% identity to SEQ ID NO:2 or a functional fragment thereof; at least 80% identity to SEQ ID NO:8 or a functional fragment thereof; at least 80% identity to SEQ ID NO:14 or a functional fragment thereof; at least 80% identity to SEQ ID NO:20 or a functional fragment thereof; at least 80% identity to SEQ ID NO:26 or a functional fragment thereof; or at least 80% identity to SEQ ID NO:32 or a functional fragment thereof.
Embodiment 14. recombinant microorganism according to Embodiment 12, wherein the recombinant DGAT1 gene encodes a polypeptide having:

    
    
        at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to a SEQ ID NO:2;
        at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO:8;
        at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO:14;
        at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO:20;
        at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO:26; or
        at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO:32.
    
    


Embodiment 15. A method for producing TAG, the method comprising culturing a recombinant microorganism according to any of Embodiments 7-14 under conditions in which the acyltransferase is expressed to produce TAG.
Embodiment 16. The method of Embodiment 15, wherein the recombinant microorganism is cultured under nitrogen replete conditions.
Embodiment 17. The method of Embodiment 15 or 16, wherein the microorganism is a microalga, optionally a species of Achnanthes, Amphiprora, Amphora, Ankistrodesmus, Asteromonas, Boekelovia, Bolidomonas, Borodinella, Botrydium, Botryococcus, Bracteococcus, Chaetoceros, Carteria, Chlamydomonas, Chlorococcum, Chlorogonium, Chlorella, Chroomonas, Chrysosphaera, Cricosphaera, Crypthecodinium, Cryptomonas, Cyclotella, Dunaliella, Ellipsoidon, Emiliania, Eremosphaera, Ernodesmius, Euglena, Eustigmatos, Franceia, Fragilaria, Gloeothamnion, Haematococcus, Halocafeteria, Heterosigma, Hymenomonas, Isochrysis, Lepocinclis, Micractinium, Monoraphidium, Nannochloris, Nannochloropsis, Navicula, Neochloris, Nephrochloris, Nephroselmis, Nitzschia, Ochromonas, Oedogonium, Oocystis, Ostreococcus, Pavlova, Parachlorella, Pascheria, Pelagomonas, Phaeodactylum, Phagus, Picochlorum, Platymonas, Pleurochrysis, Pleurococcus, Prototheca, Pseudochlorella, Pseudoneochloris, Pseudostaurastrum, Pyramimonas, Pyrobotrys, Scenedesmus, Schizochlamydella, Skeletonema, Spyrogyra, Stichococcus, Tetrachlorella, Tetraselmis, Thalassiosira, Tribonema, Vaucheria, Viridiella, Vischeria, or Volvox. 
Embodiment 18. The method of Embodiment 17, wherein the recombinant microorganism is cultured under photoautotrophic conditions.
EXAMPLES
The invention as described above can be readily understood by reference to the following examples, which are included for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.
Example 1
Identification of a Pleckstrin Homology Domain in an Algal DGAT1 Gene
Based on sequencing of the transcriptome of a proprietary Cyclotella strain, a DGAT1 gene (SEQ ID NO:1) was identified that included an N-terminal region not seen in previously identified DGAT1 genes. While DGAT1 genes are known to include a membrane bound O-acyltransferase (MBOAT) domain (occurring at approximately amino acid 413 to amino acid 699 of SEQ ID NO:2), DGAT1 genes were not previously known to include the Pleckstrin Homology (PH) domain, which was identified as occurring between about amino acid 52 and about amino acid 205 of SEQ ID NO:2. This protein domain is found N-terminal of the MBOAT domain in the Cyclotella DGAT1 (FIG. 3).
Example 2
Expression of a Full Length Cyclotella DGAT1 and a Truncated Cyclotella DGAT1 Having a Deleted PH Domain
To determine the functional significance of the PH domain in an algal DGAT1 gene, constructs encoding the Cyclotella full-length DGAT1 and N-terminally truncated DGAT1 were expressed in a Sacharomyces cerevisiae strain lacking endogenous DGAT1 genes. The quadruple yeast knock out mutant—having the DGA1, LRO1, ARE1 and ARE2 genes disrupted using a kanomycin resistance gene, a his gene for auxotrophy complementation, a hygromycin resistance gene, and a leu gene for auxotrophy complementation, respectively—was used as a host. The full length DGAT1 gene from Cyclotella (“DGAT1-452”; SEQ ID NO:1, encoding the polypeptide of SEQ ID NO:2 that includes the PH domain) and the truncated version containing the MBOAT domain but lacking the PH domain; (“DGAT1-452T”, SEQ ID NO:3, encoding the N-terminally truncated polypeptide of SEQ ID NO:4) were cloned into the p416TEF vector (Mumberg et al. (1995) Gene 156:119-22) for expression in yeast. DGAT1-452 was amplified from cDNA with forward primer DGAT1-452F (SEQ ID NO:49) and reverse primer DGAT1-452R (SEQ ID NO:50). DGAT1-452T was amplified from DGAT1-452 with forward primer DGAT1-452TF (SEQ ID NO:51) and the same reverse primer used for DGAT1-452. Genes were cloned into a BamHI linearized p416TEF vector via homologous recombination in E. coli alpha gold cells (Bioline, Boston, Mass.). The vector and PCR fragment were added to competent cells, followed by transformation and plating on LB-carbenicillin. Primers used in the cloning procedures are listed in Table 1 below, with the nucleotide residues homologous to yeast plasmid p416TEF shown in uppercase.







TABLE 1







Primers for cloning DGAT genes into p416TEF









Name
Primer sequence
SEQ ID NO:





DGAT1-452F
TAGAACTAGTGGATCCatggagaccgaggaggaattac
49


 


DGAT1-452R
GCTTGATATCGAATTCtcaaagctcaggagaagcac
50


 


DGAT1-452TF
TAGAACTAGTGGATCCatgttgaaacaacaacaacgacaac
51


 


Phaeo-TEFF
TAGAACTAGTGGATCCatgaccacgcctgtatcttc
52


 


Phaeo-TEFR
GCTTGATATCGAATTCtcaacgaatcaagcaggaatt
53


 


Phaeo-TEFTF
TAGAACTAGTGGATCCatggcccaagccatgcctg
54


 


Thala-TEFF
TAGAACTAGTGGATCCatggactctacccccagcgag
55


 


Thala-TEFR
GCTTGATATCGAATTCttataactcggaatgggcac
56


 


Thala-TEFTF
TAGAACTAGTGGATCCATGaagcaacaagaacaacaaattc
57









Genes were sequence-confirmed, and transformed into the yeast DGAT1 quadruple knockout mutant BY4741:MATαhis1Δleu2Δmet15Δura3Δ0 using the Zymo frozen EZ yeast transformation II KitTM, followed by plating onto SD-URA media, where colonies appeared two days post transformation. The presence of the transformation constructs in colonies was verified with gene specific primers.
Verified colonies were picked into 10 mL aliquots of SD-URA in 50 mL filtered cap tubes. As a negative control, the wild-type (WT) strain was grown in SD media. Cultures were grown for 24 hrs at 30° C. with shaking at 230 rpm. For HPLC analysis of lipids, 2 mL samples of each culture were spun down at maximum speed for 5 minutes, the supernatants were removed, and pellets were re-suspended in 400 μL of H2O.
The cell suspensions (approximately 500 μL) were transferred to 4 mL glass vials with Teflon lined caps. 500 μL of glass beads (212-300 μm diameter) were added to each of the cell suspensions, after which 50 μL of 50% H2SO4 and 100 μL of 5M NaCl were added. Bead beating was performed for 5 minutes at 1 krpm, then 2 mL of hexane was added to each sample, and bead beating was repeated for 5 minutes at 1 krpm. The samples were loaded onto a multi-tube vortexer and shaken for 30 minutes at 1 krpm, and then vortexed for 30 seconds at 2.5 krpm. 500 μL of the organic layer was transferred to an HPLC vial, and 50 μL of internal standard solution (1 mg/mL 6-ketocholestanol in toluene) was added to each vial. Standards were from NuCheck, Sigma-Aldrich, or Supelco. The vials were capped and vortexed briefly (5 seconds at 2.5 krpm) prior to HPLC analysis. The HPLC was run at a flow rate of 2 mL/minute on a Chromegasphere SI-60 150 mm×4.6 mm×10 μm column (ES Industries), with a column compartment set at 40° C. The injection volume was 25 μL with a draw and eject speed of 200 μL/minute. Eluent A was hexane and Eluent B was a 80:10:10:1 mixture of hexane, isopropanol, ethyl acetate, and 10% formic acid in isopropanaol, run as a gradient program as follows: 2% B at 0.0 min; 2% B at 1.0 min; 35% B at 8.0 min; 98% B at 8.5 min; 98% B at 11.5 min; 2% B at 11.6 min; stop time: 11.6 minutes; 5 minutes post time. The detector was ELSD at 30° C. and 3.5 bar N2, with a gain of 5.
The results of TAG biosynthesis are shown in FIG. 4. The results show that the presence of the PH domain greatly increases the amount of TAG produced by the mutant yeast strain, with the strain that included the PH domain-containing DGAT1 producing at least seven-fold the amount of TAG produced by the strain expressing the DGAT1 lacking the PH domain. The results are striking as yeast DGATs do not include the PH domain. Thus, the presence of the PH domain in a DGAT improves lipid yields dramatically even in strains in which the PH domain does not occur in the strain's native DGAT1.
FAME analysis was also performed to detect both storage lipid (TAG) and membrane lipids of the transformed yeast cells. 1 mL samples, measured in triplicate for fatty acid methyl ester analysis, were dried using an HT-4X GeneVac. To the dried pellets the following was added: 500 μL of 500 mM KOH in methanol, 200 μL of tetrahydrofuran containing 0.05% butylated hydroxyl toluene, 40 μL of a C11:0 free fatty acid/C13:0 triglyceride/C23:0 fatty acid methyt1 ester internal standard mix and 500 μL of glass beads (425-600 μm diameter). The vials were capped with open top PTFE septa-lined caps and placed in an SPEX GenoGrinder at 1.75 krpm for 7.5 minutes. The samples were then heated at 80° C. for five minutes and allowed to cool. For derivatization, 500 μL of 10% boron trifluoride in methanol was added to the samples prior to heating at 80° C. for 30 minutes. The tubes were allowed to cool prior to adding 2 mL of heptane and 500 μL of 5 M NaCl. The samples were vortexed for five minutes at 2 krpm and finally centrifuged for three minutes at 1 krpm. The heptane layer was sampled using a Gerstel MPS Autosampler.
The samples were run on an Agilent 7890A gas chromatography system using a J&W Scientific 127-3212 DB-FFAP, 10 m×100 μm×100 nm column and an FID detector at 260° C. The flow rate was 500 μL/minute using H2 as a carrier with constant flow control. The oven was set at 100° C. for 0.98 min, then 15.301° C./minute to 230° C. and held for 1.66 min. The inlet contained a 4 mm glass wool packed liner (Agilent P/N 5183-4647), and was set at 250° C. and used a split ratio of 40:1. The injection volume was 900 nL.
The results of the FAME analysis show that lipid production was not simply re-directed toward TAG production, but was increased overall, both on a per unit volume basis (FIG. 4) and on a per cell basis (FIG. 5).
Example 3
Bioinformatic Analysis of Algal DGAT1s
Bioinformatic analysis of the genomes of several other algal species demonstrated that the PH domain occurs in DGAT1 genes of not only diatoms but also of some green algal species. For example, manual curation of the DGAT1 genes of other algal genomes revealed the PH domain occurs in the DGAT1 genes of species of the diatoms Navicula, Phaeodactylum, Thalassiosira, and Fragilariopsis (members of the Bacillariophyceae class and Heterokontophyta division), as well as in the green algae Botryococcus and Chlorella (members of the Trebouxiophyceae class of the Chlorophyta division) as well as Tetraselmis, (a member of the Chlorophyceae class of the Chlorophyta division). Alignment of the DGAT1-encoding sequences determined from bioinformatics analysis of the genomes sequences of these algal species with higher plant DGAT1 sequences (FIG. 2E-F) demonstrates the presence of a conserved region occurring approximately from amino acid 52 to amino acid 205 of the Cyclotella DGAT1 (SEQ ID NO:2) that is not found in the higher plant DGAT1 enzymes.
Genomic sequence analysis of a proprietary Navicula species resulted in the identification of the DGAT1 gene provided as SEQ ID NO:3, encoding the DGAT1 of SEQ ID NO: 8, which includes a PH domain extending from approximately amino acid 104 to amino acid 238 of SEQ ID NO:8. SEQ ID NO:43 represents a predicted protein coding region of the Phaeodactylum tricornutum DGAT1 as determined Bolwer analysis methods (Bowler et al. (2008) Nature 456:239-44; genome available at http://ncbi.nlm.nih.gov/genome/418). SEQ ID NO:43 encodes the DGAT1 of SEQ ID NO:44 having a PH domain extending from approximately amino acid 71 to amino acid 200. The protein coding region of the Thalassiosira pseudonana DGAT1 as determined from the public genome sequences (Armbrust et al. (2004) Science 306:79-86; genome available at http://ncbi.nlm.nih.gov/genome/54) is provided as SEQ ID NO:37, encoding the DGAT1 of SEQ ID NO:38 which has a PH domain extending from approximately amino acid 53 to amino acid 186. The protein coding region of the Fragilariopsis cylindrus DGAT1 (determined from the genome sequence available at http://genome.jgi-psf.org/Fracy1/Fracy1.home.html) is provided as SEQ ID NO:13, encoding the DGAT1 of SEQ ID NO:14 having a PH domain extending from approximately amino acid 44 to amino acid 180. The protein coding region of a DGAT1 gene of a proprietary Botryococcus strain (SEQ ID NO:19) encodes the DGAT1 of SEQ ID NO:20, having a PH domain extending from approximately amino acid 28 to amino acid 153. The protein coding region of DGAT1 gene of a proprietary Tetraselmis strain (SEQ ID NO:25) encodes the DGAT1 of SEQ ID NO:26 which includes a PH domain extending from approximately amino acid 59 to amino acid 170. The protein coding region of a DGAT1 gene determined from the genome sequence of a proprietary Chlorella strain is provided as SEQ ID NO:31, encoding the DGAT1 of SEQ ID NO:32 having a PH domain extending from approximately amino acid 40 to amino acid 166.
Example 4
Comparison of Effects on Lipid Production of Expressing Additional Algal DGAT1 Genes with and without a PH Domain in Saccharomyces cerevisiae 
The full-length DGAT1 gene of Phaeodactylum tricornutum (SEQ ID NO:43), encoding the PH domain-containing DGAT1 (SEQ ID NO:44); a truncated gene (SEQ ID NO:45) encoding an N-terminally truncated Phaeodactylum DGAT1 lacking the PH domain (SEQ ID NO:46); the full-length Thalassiosira pseudonana DGAT1 gene (SEQ ID NO:37), encoding the PH domain-containing DGAT1 (SEQ ID NO:38); and a truncated gene (SEQ ID NO:39) encoding an N-terminally truncated Thalassiosira pseudonana DGAT1 lacking the PH domain (SEQ ID NO:40); were also cloned into the p416TEF vector (Mumberg et al. (1995) Gene 156:119-22) for expression in yeast using gene-specific primers that include vector-homologus sequences using the strategy provided in Example 2 and Table 1, supra. The full-length Phaaeodactylum DGAT1 was amplified from cDNA with forward primer Phaeo-TEFF (SEQ ID NO:52) and reverse primer Phaeo-TEFR (SEQ ID NO:53). The truncated Phaeodactylum DGAT1 was amplified with forward primer Phaeo-TEFTF (SEQ ID NO:54) and the same reverse primer used for the full length Phaeodactylum DGAT1 construct. The full-length Thalassiosira DGAT1 was amplified from cDNA with forward primer Thala-TEFF (SEQ ID NO:55) and reverse primer Thala-TEFR (SEQ ID NO:56). The truncated Phaeodactylum DGAT1 was amplified with forward primer Thala-TEFTF (SEQ ID NO:57) and the same reverse primer used for the full length Thalassiosira DGAT1 construct. The constructs were transformed into the quadruple knock-out as provided above.
Transformants were selected and yeast cultures of clones containing each DGAT1 gene were grown and processed for lipid analysis, as provided in Example 1, supra.
The results for TAG analysis, provided in FIG. 6, demonstrate that expression of the full-length Phaeodactylum DGAT1 gene results in the production of detectable amounts of TAG in the quadruple knockout TAG-deficient yeast strain, whereas expression of the truncated Phaeodactylum gene that lacks the PH domain does not. Further, expression of the full-length Thalassiosira DGAT1 gene results in production of at least 40% more TAG than does expression of the Thalassiosira DGAT1 gene encoding an N-terminally truncated DGAT1 that lacks the PH domain.
The amount of lipid produced by the full-length DGAT1 expressing yeast clones and the yeast clones expressing algal DGAT1 genes lacking the PH domain as determined by FAME analysis show a similar pattern (FIG. 7), with the strain expressing the full-length Phaeodactylum DGAT1 producing more FAME than the strain expressing the Phaeodactylum DGAT1 lacking the PH domain, and the strain expressing the full-length Thalassiosira DGAT1 producing more FAME than the strain expressing the Thalassiosira DGAT1 lacking the PH domain.
Example 5
Transformation of Nannochloropsis with Algal DGAT1 Genes
Media used for the growth of Nannochloropsis included the following:
PM024: 35 g/L Instant Ocean Salts, 10× Guillard's F/2 marine water enrichment solution (50× stock from Sigma-Aldrich, St. Louis, Mo., cat. No. G0154; final concentrations of components in media: 8.825 mM NaNO3; 320 μM NaH2PO4.2H2O; 205 nM Biotin; 420 nM CoCl2.6H2O; 400 nM CuSO4.5H2O; 117.13 μM Na2EDTA.2H2O; 9.095 μM MnCl2.4H2O; 248 nM Na2MoO4.2H2O; 2.965 μM Thiamine.HCl; 37 nM Vitamin B12; 765 nM ZnSO4.7H2O).
PM068: 35 g/L Instant Ocean Salts, 500 mg/L NaHCO3, 17.5 mM NaNO3, 770 μM NaH2PO4.2H2O, 9.0 μM MnCl2.4H2O, 760 nM ZnSO4.7H2O, 420 nM CoCl2.6H2O, 390 nM CuSO4.5H2O, 260 nM Na2MoO4.2H2O, 58 μM FeCl3.6H2O, 58 μM Na2EDTA.2H2O, 1.2 μM Thiamine.HCl, 8 nM biotin, 1.5 nM cyanocobalamine.
All transformants were grown in the presence of ZeocinTM (5 μg/mL) unless noted otherwise.
An E. coli/Nannochloropsis shuttle vector referred to as p5416 was used for cloning of algal DGAT1 genes. The vector contained 1) a chloramphenicol resistance gene for antibiotic selection of E. coli transformants; 2) a Bleomycin® (ble) selectable marker codon-optimized for Nannochloropsis under the control of the SV40 promoter for selection of algal transformants, as well as 3) the Nannochloropsis eIF3 promoter (SEQ ID NO:58) for operable linkage to a gene of interest. DGAT1 genes were amplified by PCR with primers having regions of homology to the shuttle vector. The Phaeodactylum DGAT1 gene was amplified with Phaeo-DGAT1F (SEQ ID NO:59) and Phaeo-DGAT1R (SEQ ID NO:60) primers. The Thalassiosira DGAT1 gene was amplified with the Thala-DGAT1F (SEQ ID NO:61) and Thala-DGAT1R (SEQ ID NO:62) primers. The amplified gene fragments and linearized p5416 were transformed together into E. coli as described in Example 2 for the yeast shuttle vector constructs. Clones were screened for inserts by PCR. Primers used in the cloning procedures are listed in Table 2 below, with the nucleotide residues homologous to p5416 shown in uppercase.







TABLE 2







Primers for cloning DGAT genes into p5416









Name
Primer sequence
SEQ ID NO:





Phaeo-DGAT1F
CAGACAGAGACACACAGGGATCatgaccacgcctgtatcttc
59


 


Phaeo-DGAT1R
GAGCGGAACCGGGGTTACAGTGCCtcaacgaatcaagcaggaatt
60


 


Thala-DGAT1F
CAGACAGAGACACACAGGGATCatggactctacccccagcgag
61


 


Thala-DGAT1R
GAGCGGAACCGGGGTTACAGTGCCttataactcggaatgggcac
62









Vectors having inserts that included the full length Cyclotella DGAT1 gene (SEQ ID NO:1); the full length Phaeodactylum DGAT1 gene (SEQ ID NO:43), and the full length Thalasiossira DGAT1 gene (SEQ ID NO:37) were linearized and introduced by electroporation into a Nannochloropsis gaditana wild-type strain obtained from the Center for Culture of Marine Phytoplankton (CCMP catalog number 1894). Briefly, two 2 L shake flasks each containing 500 mL of N. gaditiana culture were grown to a cell density of 9×106 cells/mL. The cells were centrifuged for 10 minutes at 25° C. and 2500×g and resuspended in 385 mM sorbitol. This process was repeated three times to wash cells, before resuspending in 385 mM sorbitol at a concentration of 1.1×1010 cells/mL. Approximately 1 μg of linearized DNA was mixed into 100 μL of cell suspension and transferred to a 2 mm gap cuvette (BioRad). Electroporation was performed on a BioRad GenePulser set to 50 μF capacitance, 500 ohms resistance, and 2.2 kV. Immediately after the electroporation, 1 mL of 385 mM sorbitol was gently mixed into the transformed cell suspension and the cells were allowed to sit at room temperature for a few minutes. The cell mixture was then transferred to 10 mL of PM024 liquid media and allowed to recover overnight at room temperature at 25° C. in dim light (5 μE, m−2 s−1). The cell mixture was then centrifuged at 2500×g at 25° C. for 10 minutes, decanted, and resuspended in PM024 to a final volume of approximately 600 μL. Resuspended cells (300 μL) were spread using sterile 4 mm glass beads onto PM024 plates containing 5 μg/mL ZeocinTM to select for transformants. Colonies were observed after 24 days of selection. Colonies were picked from each transformation to test for the presence of the respective DGAT1 gene. The colonies were patched onto fresh PM024+ZeocinTM plates and cultured in liquid media under selection. gDNA was prepared from the three transgenic clones as well as the wild-type. The presence of a DNA fragment containing the DGAT1 gene was verified using PCR with transgenic cells as the template.
Example 6
Lipid Production by Nannochloropsis Transformants
To determine the effect of the DGAT1 transgenes on TAG production, eight Nannochloropsis transformants that included the Cyclotella DGAT1 gene (SEQ ID NO:1) (clones designated P1A1, P1A2, P1A3, P1A6, P1B3, P1C2, P1C5, and P1D3) three Nannochloropsis transformants that included the Phaeodactylum DGAT1 gene (SEQ ID NO:43) (clones designated P2C4, P2D1, and P2D2) and one Nannochloropsis transformant that included the Thalasiossira DGAT1 gene (SEQ ID NO:37) (clone designated P4C5) were grown in 100 mL cultures in T75 tissue culture flasks under a 16 hour light: 8 hour dark diel cycle, with light provided at 90 μE, m−2 s−1 shaking at 115 rpm and 25° C. in the presence of ambient 1% CO2. The cells were inoculated from a 25 mL starter culture with and amount of cells equivalent to 0.40 OD600, and were grown in PM068 nitrogen-replete media, a condition that does not normally lead to TAG production during the exponential growth phase. After two days, at the end of the dark period, 2 mL aliquots of each culture were removed for determining Total Organic Carbon (TOC), TAG content, and total lipids as assessed by FAME analysis.
TAG was measured as in Example 2, supra. To determine FAME content of the cultures, FAME analysis was performed as described in Example 2, supra, except that 2 mL of culture was dried and the SPEX GenoGrinder was run at only 1.65 krpm for 7.5 minutes.
Total organic carbon (TOC) for the cultures was determined by centrifuging 2 mL cell culture aliquots to remove media and resuspending the cells in 1 mL water. Three cell samples per measurement were injected into a Shimadzy TOC-Vcsj Analyzer for determination of Total Carbon (TC) and Total Inorganic Carbon (TIC). The combustion furnace was set to 720° C., and TOC was determined by subtracting TIC from TC. The calibration range was from 2 ppm to 200 ppm. The correlation coefficient requirement was r2>0.999.
The results of the lipid analysis are presented on a per total organic carbon basis in FIGS. 8 and 9. FIG. 8 shows that nearly all Nannochloropsis strains expressing algal DGAT1 genes comprising the PH domain produced more TAG per TOC than the negative controls (wild type strain WT 3730 and strains that were transformed with an empty vector JC861 EV). Of the eight Cyclotella DGAT1 PH domain-containing isolates, seven produced more TAG/TOC than the wild type strain; the highest producing full-length clone, P1B3, produced approximately seven-fold the amount of TAG/TOC as the wild-type. All three PH domain-DGAT1-expressing Phaeodactylum DGAT1 transgenic algal strains (clones P2C4, P2D1, and P2D2) produced more TAG/TOC than did the wild type algal strain, as did the transgenic algal strain expressing the Thalassiosira PH domain containing DGAT1 (clone P4C5). FIG. 9 shows that the same pattern is also true for these same transformants with regard to FAME/TOC output.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.







TABLE 3







Index of Sequence Listings








SEQ ID



NO:
Description











1
Cyclotella full length DGAT1 DNA


2
Cyclotella full length DGAT1 protein


3
Cyclotella truncated DGAT1 DNA


4
Cyclotella truncated DGAT1 protein


5
Cyclotella PH domain DNA


6
Cyclotella PH domain protein


7
Navicula DGAT1 DNA


8
Navicula DGAT1 protein


9
Navicula truncated DGAT1 DNA


10
Navicula truncated DGAT1 protein


11
Navicula PH domain DNA


12
Navicula PH domain protein


13
Fragilariopsis DGAT1 DNA


14
Fragilariopsis DGAT1 protein


15
Fragilariopsis truncated DGAT1 DNA


16
Fragilariopsis truncated DGAT1 protein


17
Fragilariopsis PH domain DNA


18
Fragilariopsis PH domain protein


19
Botryococcus DGAT1 DNA


20
Botryococcus DGAT1 protein


21
Botryococcus truncated DGAT1 DNA


22
Botryococcus truncated DGAT1 protein


23
Botryococcus PH domain DNA


24
Botryococcus PH domain protein


25
Tetraselmis DGAT1 DNA


26
Tetraselmis DGAT1 protein


27
Tetraselmis truncated DGAT1 DNA


28
Tetraselmis truncated DGAT1 protein


29
Tetraselmis PH domain DNA


30
Tetraselmis PH domain protein


31
Chlorella DGAT1 DNA


32
Chlorella DGAT1 protein


33
Chlorella truncated DGAT1 DNA


34
Chlorella truncated DGAT1 protein


35
Chlorella PH domain DNA


36
Chlorella PH domain protein


37
Thalassiosira DGAT1 DNA


38
Thalassiosira DGAT1 protein


39
Thalassiosira truncated DGAT1 DNA


40
Thalassiosira truncated DGAT1 protein


41
Thalassiosira PH domain DNA


42
Thalassiosira PH domain protein


43
Phaeodactylum DGAT1 DNA


44
Phaeodactylum DGAT1 protein


45
Phaeodactylum truncated DGAT1 DNA


46
Phaeodactylum truncated DGAT1 protein


47
Phaeodactylum PH domain DNA


48
Phaeodactylum PH domain protein


49
DGAT1-452F primer


50
DGAT1-452R primer


51
DGAT1-452TF primer


52
Phaeo-TEFF primer


53
Phaeo-TEFR primer


54
Phaeo-TEFTF primer


55
Thala-TEFF primer


56
Thala-TEFR primer


57
Thala-TEFTF primer


58
Nannochloropsis eIF3 promoter


59
Phaeo-DGAT1F primer


60
Phaeo-DGAT1R primer


61
Thala-DGAT1F primer


62
Thala-DGAT1R primer


63
Eukaryotic DGAT1 consensus motif

## Claims

1. An expression cassette comprising a heterologous promoter operably linked to an isolated or recombinant nucleic acid molecule encoding a DGAT1 polypeptide having DGAT activity comprising a sequence selected from the group consisting of: an amino acid sequence having at least 85% identity to SEQ ID NO:2; an amino acid sequence having at least 85% identity to SEQ ID NO:8; an amino acid sequence having at least 85% identity to SEQ ID NO:14; an amino acid sequence having at least 85% identity to SEQ ID NO:20; an amino acid sequence having at least 85% identity to SEQ ID NO:26; and an amino acid sequence having at least 85% identity to SEQ ID NO:32.

2. An expression cassette according to claim 1, wherein the isolated or recombinant nucleic acid molecule encodes a DGAT1 polypeptide having DGAT activity comprising a sequence selected from the group consisting of: an amino acid sequence having at least 95% identity to SEQ ID NO:2; an amino acid sequence having at least 95% identity to SEQ ID NO:8; an amino acid sequence having at least 95% identity to SEQ ID NO:14; an amino acid sequence having at least 95% identity to SEQ ID NO:20; an amino acid sequence having at least 95% identity to SEQ ID NO:26; and an amino acid sequence having at least 95% identity to SEQ ID NO:32.

3. An expression cassette comprising a heterologous promoter operably linked to an isolated or recombinant nucleic acid molecule encoding a Pleckstrin Homology (PH) domain, wherein the PH domain comprises a sequence selected from the group consisting of: an amino acid sequence having at least 85% identity to SEQ ID NO:6; an amino acid sequence having at least 85% identity to SEQ ID NO:12; an amino acid sequence having at least 85% identity to SEQ ID NO:18; an amino acid sequence having at least 85% identity to SEQ ID NO:24; an amino acid sequence having at least 85% identity to SEQ ID NO:30; and an amino acid sequence having at least 85% identity to SEQ ID NO:36.

4. An expression cassette according to claim 3, wherein the PH domain comprises a sequence selected from the group consisting of: an amino acid sequence having at least 95% identity to SEQ ID NO:6; an amino acid sequence having at least 95% identity to SEQ ID NO:12; an amino acid sequence having at least 95% identity to SEQ ID NO:18; an amino acid sequence having at least 95% identity to SEQ ID NO:24; an amino acid sequence having at least 95% identity to SEQ ID NO:30; and an amino acid sequence having at least 95% identity to SEQ ID NO:36.

5. A recombinant eukaryotic cell comprising an expression cassette according to claim 1.

6. A method for producing triacylglycerol (TAG), the method comprising culturing a recombinant microorganism that comprises a recombinant nucleic acid molecule under conditions in which the recombinant nucleic acid molecule is expressed, to produce TAG, wherein the recombinant nucleic acid molecule encodes a DGAT1 polypeptide having DGAT activity comprising a sequence selected from the group consisting of: an amino acid sequence having at least 85% identity to SEQ ID NO:2; an amino acid sequence having at least 85% identity to SEQ ID NO:8; an amino acid sequence having at least 85% identity to SEQ ID NO:14; an amino acid sequence having at least 85% identity to SEQ ID NO:20; an amino acid sequence having at least 85% identity to SEQ ID NO:26; and an amino acid sequence having at least 85% identity to SEQ ID NO:32.

7. The method of claim 6, wherein the recombinant nucleic acid molecule encodes a DGAT1 polypeptide having DGAT activity comprising a sequence selected from the group consisting of: an amino acid sequence having at least 95% identity to SEQ ID NO:2; an amino acid sequence having at least 95% identity to SEQ ID NO:8; an amino acid sequence having at least 95% identity to SEQ ID NO:14; an amino acid sequence having at least 95% identity to SEQ ID NO:20; an amino acid sequence having at least 95% identity to SEQ ID NO:26; and an amino acid sequence having at least 95% identity to SEQ ID NO:32.

8. The method of claim 6, wherein the recombinant microorganism is cultured under nitrogen replete conditions.

9. The method of claim 6, wherein the microorganism is a microalga.

10. The method of claim 9, wherein the microalga is a species of Achnanthes, Amphiprora, Amphora, Ankistrodesmus, Asteromonas, Boekelovia, Bolidomonas, Borodinella, Botrydium, Botryococcus, Bracteococcus, Chaetoceros, Carteria, Chlamydomonas, Chlorococcum, Chlorogonium, Chlorella, Chroomonas, Chrysosphaera, Cricosphaera, Crypthecodinium, Cryptomonas, Cyclotella, Dunaliella, Ellipsoidon, Emiliania, Eremosphaera, Ernodesmius, Euglena, Eustigmatos, Franceia, Fragilaria, Gloeothamnion, Haematococcus, Halocafeteria, Heterosigma, Hymenomonas, Isochrysis, Lepocinclis, Micractinium, Monoraphidium, Nannochloris, Nannochloropsis, Navicula, Neochloris, Nephrochloris, Nephroselmis, Nitzschia, Ochromonas, Oedogonium, Oocystis, Ostreococcus, Pavlova, Parachlorella, Pascheria, Pelagomonas, Phaeodactylum, Phagus, Picochlorum, Platymonas, Pleurochrysis, Pleurococcus, Prototheca, Pseudochlorella, Pseudoneochloris, Pseudostaurastrum, Pyramimonas, Pyrobotrys, Scenedesmus, Schizochlamydella, Skeletonema, Spyrogyra, Stichococcus, Tetrachlorella, Tetraselmis, Thalassiosira, Tribonema, Vaucheria, Viridiella, Vischeria, or Volvox.

11. The method of claim 9, wherein the microalga is a Nannochloropsis cell.

12. The method of claim 9, wherein the recombinant microorganism is cultured under photoautotrophic conditions.

13. The method of claim 8, wherein the recombinant microorganism generates TAG under nitrogen replete conditions at levels at least two fold the amount produced by a control microorganism substantially identical to the recombinant microorganism except that the control microorganism lacks the recombinant nucleic acid molecule having DGAT activity.

14. The method of claim 8, wherein the recombinant microorganism generates TAG under nitrogen replete conditions at levels at least five fold the amount produced by a control microorganism substantially identical to the recombinant microorganism except that the control microorganism lacks the recombinant nucleic acid molecule having DGAT activity.

15. The method of claim 8, wherein the recombinant microorganism generates TAG under nitrogen replete conditions at levels at least ten fold the amount produced by a control microorganism substantially identical to the recombinant microorganism except that the control microorganism lacks the recombinant nucleic acid molecule having DGAT activity.

16. The recombinant eukaryotic cell of claim 5, wherein the expression cassette comprises a nucleic acid molecule encoding a PH domain comprising a sequence selected from the group consisting of: an amino acid sequence having at least 95% identity to SEQ ID NO:2; an amino acid sequence having at least 95% identity to SEQ ID NO:8; an amino acid sequence having at least 95% identity to SEQ ID NO:14; an amino acid sequence having at least 95% identity to SEQ ID NO:20; an amino acid sequence having at least 95% identity to SEQ ID NO:26; and an amino acid sequence having at least 95% identity to SEQ ID NO:32.

17. The recombinant eukaryotic cell of claim 5, wherein the expression cassette comprises a nucleic acid molecule encoding a PH domain comprising a sequence selected from the group consisting of: an amino acid sequence having at least 85% identity to SEQ ID NO:2; an amino acid sequence having at least 85% identity to SEQ ID NO:8; an amino acid sequence having at least 85% identity to SEQ ID NO:14; an amino acid sequence having at least 85% identity to SEQ ID NO:20; an amino acid sequence having at least 85% identity to SEQ ID NO:26; and an amino acid sequence having at least 85% identity to SEQ ID NO:32.

18. The recombinant eukaryotic cell of claim 5, wherein the recombinant eukaryotic cell is a recombinant microorganism.

19. The recombinant microorganism of claim 18, wherein the recombinant microorganism produces a greater amount of triglyceride than is produced by a control microorganism substantially identical to the recombinant microorganism, but wherein the control microorganism lacks the isolated or recombinant nucleic acid molecule encoding the DGAT1 polypeptide having DGAT activity.

20. The recombinant microorganism of claim 18, wherein the recombinant microorganism is a photosynthetic microorganism.