# Patent

## Title
Modified photosynthetic microorganisms for producing triglycerides

## Abstract

This disclosure describes genetically modified photosynthetic microorganisms, including Cyanobacteria, that contain one or more exogenous genes encoding a diacyglycerol acyltransferase, a phosphatidate phosphatase, and/or an acetyl-CoA carboxylase, and which are capable of producing increased amounts of fatty acids and/or synthesizing triglycerides.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application is a continuation of U.S. Utility patent application Ser. No. 12/605,204 filed Oct. 23, 2009, where this utility application is incorporated herein by reference in its entirety and claims the benefit 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 61/107,979 filed Oct. 23, 2008
This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 61/107,979 filed Oct. 23, 2008, where this provisional application is incorporated herein by reference in its entirety.


STATEMENT REGARDING SEQUENCE LISTING
The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 890071—401_SEQUENCE_LISTING.txt. The text file is 115 KB, was created on Oct. 23, 2009 and is being submitted electronically via EFS-Web.
BACKGROUND
1. Technical Field
The present invention relates generally to genetically modified photosynthetic microorganisms, including Cyanobacteria, capable of synthesizing triglycerides, which may be used as a feedstock for producing biofuels and other specialty chemicals.
2. Description of the Related Art
Triglycerides are neutral polar molecules consisting of glycerol esterified with three fatty acid molecules. Triglycerides are utilized as carbon and energy storage molecules by most eukaryotic organisms, including plants and algae, and by certain prokaryotic organisms, including certain species of actinomycetes and members of the genus Acinetobacter. 
Triglycerides may also be utilized as a feedstock in the production of biofuels and/or various specialty chemicals. For example, triglycerides may be subject to a transesterification reaction, in which an alcohol reacts with triglyceride oils, such as those contained in vegetable oils, animal fats, recycled greases, to produce biodiesels such as fatty acid alkyl esters. Such reactions also produce glycerin as a by-product, which can be purified for use in the pharmaceutical and cosmetic industries
Certain organisms can be utilized as a source of triglycerides in the production of biofuels. For example, algae naturally produce triglycerides as energy storage molecules, and certain biofuel-related technologies are presently focused on the use of algae as a feedstock for biofuels. Algae are photosynthetic organisms, and the use of triglyceride-producing organisms such as algae provides the ability to produce biodiesel from sunlight, water, CO2, macronutrients, and micronutrients. Algae, however, cannot be readily genetically manipulated, and produce much less oil (i.e., triglycerides) under culture conditions than in the wild.
Like algae, Cyanobacteria obtain energy from photosynthesis, utilizing chlorophyll A and water to reduce CO2. Certain Cyanobacteria can produce metabolites, such as carbohydrates, proteins, and fatty acids, from just sunlight, water CO2, water, and inorganic salts. Unlike algae, Cyanobacteria can be genetically manipulated. For example, S. elongatus PCC 7942 (hereafter referred to as “S. elongatus PCC 7942”) is a genetically manipulable, oligotrophic Cyanobacterium that thrives in low nutrient level conditions, and in the wild accumulates fatty acids in the form of lipid membranes to about 4 to 8% by dry weight. Cyanobacteria such as Synechococcus, however, produce no triglyceride energy storage molecules, since Cyanobacteria typically lack the essential enzymes involved in triglyceride synthesis.
Clearly, therefore, there is a need in the art for modified photosynthetic microorganisms, including Cyanobacteria, capable of producing triglycerides, e.g., to be used as feedstock in the production of biofuels and/or various specialty chemicals.
BRIEF SUMMARY
Embodiments of the present invention relate to the demonstration that photosynthetic microorganisms, including Cyanobacteria, can be genetically modified to increase fatty acid biosynthesis, and to produce triglycerides from their natural-occurring fatty acids. Generally, the modified photosynthetic microorganisms, e.g., Cyanobacteria, of the present invention comprise one or more polynucleotides encoding one or more enzymes associated with triglyceride biosynthesis and/or fatty acid synthesis.
Embodiments of the present invention include methods of producing triglycerides in a photosynthetic microorganism, e.g., a Cyanobacterium, comprising introducing one or more polynucleotides encoding one or more enzymes associated with triglyceride biosynthesis into a photosynthetic microorganism, e.g., a Cyanobacterium. In certain aspects, the one or more enzymes comprise diacylglycerol acyltransferase (DGAT) and/or phosphatidate phosphatase.
In certain embodiments of the methods and compositions of the present invention, the DGAT is an Acinetobacter DGAT or a variant thereof. In certain embodiments, the Acinetobacter DGAT is Acinetobacter baylii ADP1 diacylglycerol acyltransferase (AtfA). Other DGATs that may be used according to the present invention include, but are not limited to, Streptomyces coelicolor DGAT, Alcanivorax borkumensis DGAT, or the modified DGATs described herein.
In certain embodiments of the methods and compositions of the present invention, said phosphatidate phosphatase is a yeast phosphatidate phosphatase. In certain aspects, said yeast phosphatidate phosphatase is Saccharomyces cerevisiae phosphatidate phosphatase (yPah1). However, other phosphatidate phosphatases, including but not limited to those described herein, may also be used.
In certain embodiments of the methods and compositions of the present invention, said one or more enzymes comprise acetyl-CoA carboxylase (ACCase), optionally in combination with diacylglycerol acyltransferase (DGAT) and/or phosphatidate phosphatase. In particular embodiments, the ACCase is a Saccharomyces cerevisiae ACCase, a Triticum aestivum ACCase, or a Synechococcus sp. PCC 7002 ACCAse, including, but not limited to, any of those described herein.
In various embodiments of the present invention, said one or more polynucleotides are codon-optimized for expression in a photosynthetic microorganism, e.g., a Cyanobacterium.
Certain embodiments include methods of increasing fatty acid production by a photosynthetic microorganism, e.g., a Cyanobacterium, comprising introducing one or more polynucleotides encoding one or more enzymes associated with fatty acid biosynthesis into a photosynthetic microorganism, e.g., a Cyanobacterium. In certain aspects, said one or more enzymes comprise acetyl-CoA carboxylase (ACCase). In certain aspects, said ACCase is a yeast ACCase or a derivative thereof. In certain aspects, said ACCase is Saccharomyces cerevisiae acetyl-CoA carboxylase (yACC1), a Triticum aestivum ACCase, or a Synechococcus sp. PCC 7002 ACCAse, including, but not limited to, any of those described herein. In certain aspects, said one or more enzymes further comprise diacylglycerol acyltransferase (DGAT) and phosphatidate phosphatase. In certain embodiments, the DGAT is an Acinetobacter DGAT or a variant thereof, including wherein said DGAT is Acinetobacter baylii ADP1 diacylglycerol acyltransferase (AtfA). Other DGATs that may be used according to the present invention include, but are not limited to, Streptomyces coelicolor DGAT, Alcanivorax borkumensis DGAT, or the modified DGATs described herein. In certain aspects, said phosphatidate phosphatase is a yeast phosphatidate phosphatase, such as a Saccharomyces cerevisiae phosphatidate phosphatase (yPah1). In certain embodiments, said one or more enzymes comprise acetyl-CoA carboxylase (ACCase) and diacylglycerol acyltransferase (DGAT). In certain embodiments, said one or more enzymes comprise acetyl-CoA carboxylase (ACCase) and phosphatidate phosphatase. In certain embodiments, said one or more enzymes comprise acetyl-CoA carboxylase (ACCase), diacylglycerol acyltransferase (DGAT), and phosphatidate phosphatase. In certain aspects, the one or more polynucleotides are codon-optimized for expression in a photosynthetic microorganism, e.g., a Cyanobacterium.
Certain embodiments include methods of producing a triglyceride in a photosynthetic microorganism, e.g., a Cyanobacterium, comprising culturing a Cyanobacterium comprising one or more polynucleotides encoding one or more enzymes associated with triglyceride biosynthesis. In certain embodiments, said one or more enzymes comprise diacylglycerol acyltransferase (DGAT) and/or phosphatidate phosphatase. In certain aspects, said DGAT is an Acinetobacter DGAT or a variant thereof, including wherein said Acinetobacter DGAT is Acinetobacter baylii ADP1 diacylglycerol acyltransferase (AtfA). Other DGATs that may be used according to the present invention include, but are not limited to, Streptomyces coelicolor DGAT, Alcanovorax borkumensis DGAT, or the modified DGATs described herein. In certain aspects, said phosphatidate phosphatase is a yeast phosphatidate phosphatase, including wherein said yeast phosphatidate phosphatase is Saccharomyces cerevisiae phosphatidate phosphatase (yPah1). In certain embodiments, said one or more enzymes comprise acetyl-CoA carboxylase (ACCase), alone or in combination with diacylglycerol acyltransferase (DGAT) and/or phosphatidate phosphatase.
Certain embodiments of the present invention include methods of producing an increased amount of fatty acid in a photosynthetic microorganism, e.g., a Cyanobacterium, comprising culturing a photosynthetic microorganism, e.g., Cyanobacterium comprising one or more polynucleotides encoding one or more enzymes associated with fatty acid biosynthesis, wherein said polynucleotides are exogenous to the photosynthetic microorganism's native genome. In certain embodiments, said one or more enzymes comprise acetyl-CoA carboxylase (ACCase). In certain aspects, said ACCase is a yeast ACCase or a variant thereof, such as wherein said yeast ACCase is Saccharomyces cerevisiae acetyl-CoA carboxylase (yACC1). In certain embodiments, said one or more enzymes comprise diacylglycerol acyltransferase (DGAT) and/or phosphatidate phosphatase. In certain aspects, said DGAT is an Acinetobacter DGAT or a variant thereof, including wherein said Acinetobacter DGAT is Acinetobacter baylii ADP1 diacylglycerol acyltransferase (AtfA). In certain aspects, said phosphatidate phosphatase is a yeast phosphatidate phosphatase, including wherein said yeast phosphatidate phosphatase is Saccharomyces cerevisiae phosphatidate phosphatase (yPah1). In other embodiments, any other DGAT, ACCase, or phosphatidate phosphate, including but not limited to those described herein, may be used.
In certain embodiments of the methods provided herein, said one or more polynucleotides are exogenous to the photosynthetic microorganism's, e.g., Cyanobacterium's, native genome. The one or more polynucleotides may also be present in one or more expression constructs, which may be stably integrated into the photosynthetic microorganism's genome, such as by recombination. Certain expression constructs comprise a constitutive promoter, and certain expression constructs comprise an inducible promoter. In certain aspects, said one or more polynucleotides are codon-optimized for expression in a photosynthetic microorganism, e.g., a Cyanobacterium.
The present invention also includes modified photosynthetic microorganisms, e.g., Cyanobacteria, comprising one or more polynucleotides encoding one or more enzymes associated with triglyceride biosynthesis, or variants or fragments thereof. In certain embodiments, said one or more enzymes comprise diacylglycerol acyltransferase (DGAT) and/or phosphatidate phosphatase. In certain aspects, said DGAT is an Acinetobacter DGAT or a variant thereof, including wherein said Acinetobacter DGAT is Acinetobacter baylii ADP1 diacylglycerol acyltransferase (AtfA). Other DGATs that may be used according to the present invention include, but are not limited to, Streptomyces coelicolor DGAT, Alcanivorax borkumensis DGAT, or the modified DGATs described herein. In certain aspects, said phosphatidate phosphatase is a yeast phosphatidate phosphatase, including wherein said yeast phosphatidate phosphatase is Saccharomyces cerevisiae phosphatidate phosphatase (yPah1).
Embodiments of the present invention also include modified photosynthetic microorganisms, e.g., Cyanobacteria, comprising one or more polynucleotides encoding one or more enzymes associated with fatty acid biosynthesis. In particular embodiments, said polynucleotides are exogenous to the Cyanobacterium's native genome. In certain embodiments, said one or more enzymes comprise diacylglycerol acyltransferase (DGAT) and/or phosphatidate phosphatase. In certain aspects, said DGAT is an Acinetobacter DGAT or a variant thereof, including wherein said Acinetobacter DGAT is Acinetobacter baylii ADP1 diacylglycerol acyltransferase (AtfA). Other DGATs that may be used according to the present invention include, but are not limited to, Streptomyces coelicolor DGAT, Alcanivorax borkumensis DGAT, or the modified DGATs described herein. In certain aspects, said phosphatidate phosphatase is a yeast phosphatidate phosphatase, including wherein said yeast phosphatidate phosphatase is Saccharomyces cerevisiae phosphatidate phosphatase (yPah1).
In certain embodiments, said one or more enzymes comprise acetyl-CoA carboxylase (ACCase). In certain aspects, said ACCase is a yeast ACCase or a variant thereof. In certain aspects, said ACCase is Saccharomyces cerevisiae acetyl-CoA carboxylase (yACC1), a Triticum aestivum ACCase, or a Synechococcus sp. PCC 7002 ACCAse. In certain embodiments, the one or more enzymes of a modified Cyanobacterium comprise acetyl-CoA carboxylase (ACCase), in combination with diacylglycerol acyltransferase (DGAT) and/or phosphatidate phosphatase.
In certain aspects, the one or more polynucleotides are exogenous to the photosynthetic microorganism's, e.g., Cyanobacterium's, native genome. The one or more polynucleotide sequences may be present in one or more expression constructs, which may be stably integrated into the photosynthetic microorganism's genome. In certain aspects, the one or more expression constructs comprise a constitutive promoter. In certain aspects, the one or more expression constructs comprise an inducible promoter. The one or more polynucleotides of the modified photosynthetic microorganism may be codon-optimized for expression in the photosynthetic microorganism, e.g., a Cyanobacteria. In certain aspects, a modified Cyanobacteria is S. elongatus PCC7942.
Certain embodiments contemplate modified photosynthetic microorganisms, e.g., Cyanobacteria, comprising one or more polynucleotides encoding one or more enzymes associated with fatty acid biosynthesis, wherein said one or more enzymes comprise Saccharomyces cerevisiae acetyl-CoA carboxylase (yAcc1), Acinetobacter baylii ADP1 diacylglycerol acyltransferase (AtfA), and Saccharomyces cerevisiae phosphatidate phosphatase (yPah1), wherein said one or more polynucleotides are codon-optimized for expression in Cyanobacterium, wherein expression of said one or more enzymes in regulated by one or more inducible promoters, and wherein said Cyanobacterium is S. elongatus PCC7942.
Particular embodiments of the various compositions and methods of the present invention contemplate the use of modified photosynthetic microorganisms, e.g., Cyanobacteria, comprising two or more different exogenous DGAT polynucleotides, or fragments or variants thereof, alone or in combination with one or more phosphatidate phosphatase and/or acetyl-CoA carboxylase polynucleotides, or fragments or variants thereof.
In various embodiments of the methods and compositions of the present invention, the modified photosynthetic microorganism is a Cyanobacteria selected from S. elongatus PCC 7942, a salt tolerant variant of S. elongatus PCC 7942, Synechococcus PCC 7002, and Synechocystis PCC 6803.



BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
FIG. 1 shows the lipid content as measured by gas chromatography (GC) of S. elongatus PCC 7942 strain transformed with a diacylglycerol acyltransferase (ADP1-DGAT) gene from Acinetobacter baylii as compared to an empty vector control. Expression of the DGAT gene was under the control of an IPTG inducible promoter.
FIG. 2 shows a thin layer chromatography assay of triacylglceride (TAG) and fatty acids present in extracts obtained from S. elongatus PCC 7942 strains that carried one of four different DGATs (ADGATd, ADGATn, ScoDGAT, or AboDGAT) or a vector control, either uninduced or induced with IPTG. Control TAG (C16TAG) and fatty acid (palmitate) standards are also shown.
FIG. 3 is a graph showing the results of HPLC analysis of lipid extracts from S. elongatus PCC7942 expressing ADGATd (dashed line) as compared to wild type S. elongatus (solid line) following induction. The y-axis indicates the intensity of the peaks for the different lipid species, free fatty acids (FFAs), phospholipids, and TAGs, and the x-axis indicates the corresponding retention time.
FIGS. 4A-4B provide graphs showing the acyl chain composition of TAGs produced by S. elongatus PCC7942 expressing ADP1-DGAT or ScoDGAT following induction, as determined by gas chromatography of TAGs isolated by TLC. FIG. 4A shows the amount of various acyl chains in TAGs from cells expressing ADGATd, and FIG. 4B shows the amount of various acyl chains in TAGs from cells expressing ScoGAT.
FIGS. 5A-5B show thin layer chromatography assays of triacylglceride (TAG) obtained from two different strains that carried ADP1-DGAT. FIG. 5A shows the TAGs expressed by a Synechocystis sp. strain PCC 6803 that carried ADP1-DGAT (+) or a vector control (−), following induction. FIG. 5B shows the TAGs expressed by a salt tolerant S. elongatus PCC 7942 that carried ADP1-DGAT, when grown in salt water, either uninduced (−) or induced (+) with IPTG. Control TAG (C16TAG) and fatty acid (palmitate) standards are also shown.
FIG. 6 shows a thin layer chromatography assay of triacylglceride (TAG) present in extracts obtained from S. elongatus PCC 7942 strains that over-expressed either Adp1-DGAT or Sco-DGAT, alone or in combination with a Synechococcus sp. PCC 7002 Accase. A control TAG standard is shown.



DETAILED DESCRIPTION
The present invention relates, in part, to the demonstration that photosynthetic organisms, including but not limited to Cyanobacteria, such as Synechococcus, which do not naturally produce triglycerides, can be genetically modified to synthesize triglycerides. In particular, as shown in the accompanying Examples, the addition of one or more polynucleotide sequences that encode one or more enzymes associated with triglyceride synthesis renders Cyanobacteria capable of converting their naturally-occurring fatty acids into triglyceride energy storage molecules. Examples of enzymes associated with triglyceride synthesis include enzymes having a phosphatidate phosphatase activity and enzymes having a diacylglycerol acyltransferase activity (DGAT). Specifically, phosphatidate phosphatase enzymes catalyze the production of diacylglycerol molecules, an immediate pre-cursor to triglycerides, and DGAT enzymes catalyze the final step of triglyceride synthesis by converting the diacylglycerol precursors to triglycerides.
The present invention also relates, in part, to the demonstration that Cyanobacteria can be genetically modified to increase the production of fatty acids. Since fatty acids provide the starting material for triglycerides, increasing the production of fatty acids in genetically modified Cyanobacteria may be utilized to increase the production of triglycerides. As shown in the accompanying Examples, Cyanobacteria can be modified to increase the production of fatty acids by introducing one or more exogenous polynucleotide sequences that encode one or more enzymes associated with fatty acid synthesis. In certain aspects, the exogenous polynucleotide sequence encodes an enzyme that comprises an acyl-CoA carboxylase (ACCase) activity, typically allowing increased ACCase expression, and, thus, increased intracellular ACCase activity. Increased intracellular ACCase activity contributes to the increased production of fatty acids because this enzyme catalyzes the “commitment step” of fatty acid synthesis. Specifically, ACCase catalyzes the production of a fatty acid synthesis precursor molecule, malonyl-CoA. In certain embodiments, the polynucleotide sequence encoding the ACCase is not native the Cyanobacterium's genome.
DEFINITIONS
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
The term “biologically active fragment”, as applied to fragments of a reference polynucleotide or polypeptide sequence, refers to a fragment that has at least about 0.1, 0.5, 1, 2, 5, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100, 110, 120, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000% or more of the activity of a reference sequence. The term “reference sequence” refers generally to a nucleic acid coding sequence, or amino acid sequence, of any enzyme having a diacylglycerol acyltransferase activity, a phosphatidate phosphatase activity, and/or an acetyl-CoA carboxylase activity, as described herein (see, e.g., SEQ ID NOS:1-9).
Included within the scope of the present invention are biologically active fragments of at least about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 500, 600 or more contiguous nucleotides or amino acid residues in length, including all integers in between, which comprise or encode a polypeptide having an enzymatic activity of a reference polynucleotide or polypeptide. Representative biologically active fragments generally participate in an interaction, e.g., an intra-molecular or an inter-molecular interaction. An inter-molecular interaction can be a specific binding interaction or an enzymatic interaction. Examples of enzymatic interactions or activities include diacylglycerol acyltransferase activity, phosphatidate phosphatase activity, and/or acetyl-CoA carboxylase activity, as described herein.
By “coding sequence” is meant any nucleic acid sequence that contributes to the code for the polypeptide product of a gene. By contrast, the term “non-coding sequence” refers to any nucleic acid sequence that does not contribute to the code for the polypeptide product of a gene.
Throughout this specification, unless the context requires otherwise, the words “comprise”, “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.
By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present.
By “consisting essentially of” is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.
The terms “complementary” and “complementarity” refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence “A-G-T,” is complementary to the sequence “T-C-A.” Complementarity may be “partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.
By “corresponds to” or “corresponding to” is meant (a) a polynucleotide having a nucleotide sequence that is substantially identical or complementary to all or a portion of a reference polynucleotide sequence or encoding an amino acid sequence identical to an amino acid sequence in a peptide or protein; or (b) a peptide or polypeptide having an amino acid sequence that is substantially identical to a sequence of amino acids in a reference peptide or protein.
By “derivative” is meant a polypeptide that has been derived from the basic sequence by modification, for example by conjugation or complexing with other chemical moieties (e.g., pegylation) or by post-translational modification techniques as would be understood in the art. The term “derivative” also includes within its scope alterations that have been made to a parent sequence including additions or deletions that provide for functionally equivalent molecules.
By “enzyme reactive conditions” it is meant that any necessary conditions are available in an environment (i.e., such factors as temperature, pH, lack of inhibiting substances) which will permit the enzyme to function. Enzyme reactive conditions can be either in vitro, such as in a test tube, or in vivo, such as within a cell.
As used herein, the terms “function” and “functional” and the like refer to a biological, enzymatic, or therapeutic function.
By “gene” is meant a unit of inheritance that occupies a specific locus on a chromosome and consists of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e., introns, 5′ and 3′ untranslated sequences).
“Homology” refers to the percentage number of amino acids that are identical or constitute conservative substitutions. Homology may be determined using sequence comparison programs such as GAP (Deveraux et al., 1984, Nucleic Acids Research 12, 387-395) which is incorporated herein by reference. In this way sequences of a similar or substantially different length to those cited herein could be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.
The term “host cell” includes an individual cell or cell culture which can be or has been a recipient of any recombinant vector(s) or isolated polynucleotide of the invention. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A host cell includes cells transfected or infected in vivo or in vitro with a recombinant vector or a polynucleotide of the invention. A host cell which comprises a recombinant vector of the invention is a recombinant host cell.
By “isolated” is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, an “isolated polynucleotide”, as used herein, refers to a polynucleotide, which has been purified from the sequences which flank it in a naturally-occurring state, e.g., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment. Alternatively, an “isolated peptide” or an “isolated polypeptide” and the like, as used herein, refer to in vitro isolation and/or purification of a peptide or polypeptide molecule from its natural cellular environment, and from association with other components of the cell.
By “increased” or “increasing” is meant the ability of one or more modified photosynthetic microorganisms, e.g., Cyanobacteria, to produce a greater amount of a given fatty acid, lipid molecule, or triglyceride as compared to a control Cyanobacteria, such as an unmodified Cyanobacteria or a differently modified Cyanobacteria. Production of fatty acids can be measured according to techniques known in the art, such as Nile Red staining and gas chromatography. Production of triglycerides can be measured, for example, using commercially available enzymatic tests, including colorimetric enzymatic tests using glycerol-3-phosphate-oxidase.
By “obtained from” is meant that a sample such as, for example, a polynucleotide extract or polypeptide extract is isolated from, or derived from, a particular source, such as a desired organism or a specific tissue within a desired organism. “Obtained from” can also refer to the situation in which a polynucleotide or polypeptide sequence is isolated from, or derived from, a particular organism or tissue within an organism. For example, a polynucleotide sequence encoding a diacylglycerol acyltransferase, phosphatidate phosphatase, and/or acetyl-CoA carboxylase enzyme may be isolated from a variety of prokaryotic or eukaryotic organisms, or from particular tissues or cells within certain eukaryotic organism.
The term “operably linked” as used herein means placing a gene under the regulatory control of a promoter, which then controls the transcription and optionally the translation of the gene. In the construction of heterologous promoter/structural gene combinations, it is generally preferred to position the genetic sequence or promoter at a distance from the gene transcription start site that is approximately the same as the distance between that genetic sequence or promoter and the gene it controls in its natural setting; i.e. the gene from which the genetic sequence or promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of function. Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting; i.e., the genes from which it is derived. “Constitutive promoters” are typically active, i.e., promote transcription, under most conditions. “Inducible promoters” are typically active only under certain conditions, such as in the presence of a given molecule factor (e.g., IPTG) or a given environmental condition (e.g., particular CO2 concentration, nutrient levels, light, heat). In the absence of that condition, inducible promoters typically do not allow significant or measurable levels of transcriptional activity. For example, inducible promoters may be induced according to temperature, pH, a hormone, a metabolite (e.g., lactose, mannitol, an amino acid), light (e.g., wavelength specific), osmotic potential (e.g., salt induced), a heavy metal, or an antibiotic. Numerous standard inducible promoters will be known to one of skill in the art.
The recitation “polynucleotide” or “nucleic acid” as used herein designates mRNA, RNA, cRNA, rRNA, cDNA or DNA. The term typically refers to polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.
The terms “polynucleotide variant” and “variant” and the like refer to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions that are defined hereinafter. These terms also encompass polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion or substitution of at least one nucleotide. Accordingly, the terms “polynucleotide variant” and “variant” include polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides. In this regard, it is well understood in the art that certain alterations inclusive of mutations, additions, deletions and substitutions can be made to a reference polynucleotide whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide, or has increased activity in relation to the reference polynucleotide (i.e., optimized). Polynucleotide variants include, for example, polynucleotides having at least 50% (and at least 51% to at least 99% and all integer percentages in between) sequence identity with a reference polynucleotide sequence that encodes a diacylglycerol acyltransferase, a phosphatidate phosphatase, and/or an acetyl-CoA carboxylase enzyme. The terms “polynucleotide variant” and “variant” also include naturally-occurring allelic variants and orthologs that encode these enzymes.
With regard to polynucleotides, the term “exogenous” refers to a polynucleotide sequence that does not naturally occur in a wild-type cell or organism, but is typically introduced into the cell by molecular biological techniques. Examples of exogenous polynucleotides include vectors, plasmids, and/or man-made nucleic acid constructs encoding a desired protein. With regard to polynucleotides, the term “endogenous” or “native” refers to naturally occurring polynucleotide sequences that may be found in a given wild-type cell or organism. For example, certain cyanobacterial species do not typically contain a DGAT gene, and, therefore, do not comprise an “endogenous” polynucleotide sequence that encodes a DGAT polypeptide. Also, a particular polynucleotide sequence that is isolated from a first organism and transferred to second organism by molecular biological techniques is typically considered an “exogenous” polynucleotide with respect to the second organism.
“Polypeptide,” “polypeptide fragment,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers. In certain aspects, polypeptides may include enzymatic polypeptides, or “enzymes,” which typically catalyze (i.e., increase the rate of) various chemical reactions.
The recitation polypeptide “variant” refers to polypeptides that are distinguished from a reference polypeptide sequence by the addition, deletion or substitution of at least one amino acid residue. In certain embodiments, a polypeptide variant is distinguished from a reference polypeptide by one or more substitutions, which may be conservative or non-conservative. In certain embodiments, the polypeptide variant comprises conservative substitutions and, in this regard, it is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide. Polypeptide variants also encompass polypeptides in which one or more amino acids have been added or deleted, or replaced with different amino acid residues.
The present invention contemplates the use in the methods described herein of variants of full-length enzymes having diacylglycerol acyltransferase activity, phosphatidate phosphatase activity, and/or acetyl-CoA carboxylase activity, truncated fragments of these full-length polypeptides, variants of truncated fragments, as well as their related biologically active fragments. Typically, biologically active fragments of a polypeptide may participate in an interaction, for example, an intra-molecular or an inter-molecular interaction. An inter-molecular interaction can be a specific binding interaction or an enzymatic interaction (e.g., the interaction can be transient and a covalent bond is formed or broken). Biologically active fragments of a polypeptide/enzyme having a diacylglycerol acyltransferase activity, a phosphatidate phosphatase activity, and/or acetyl-CoA carboxylase activity include peptides comprising amino acid sequences sufficiently similar to, or derived from, the amino acid sequences of a (putative) full-length reference polypeptide sequence. Typically, biologically active fragments comprise a domain or motif with at least one activity of a diacylglycerol acyltransferase polypeptide, phosphatidate phosphatase polypeptide, and/or acetyl-coA carboxylase polypeptide, and may include one or more (and in some cases all) of the various active domains. A biologically active fragment of a diacylglycerol acyltransferase, phosphatidate phosphatase, and/or acetyl-CoA carboxylase polypeptide can be a polypeptide fragment which is, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 450, 500, 600 or more contiguous amino acids, including all integers in between, of a reference polypeptide sequence. In certain embodiments, a biologically active fragment comprises a conserved enzymatic sequence, domain, or motif, as described elsewhere herein and known in the art. Suitably, the biologically-active fragment has no less than about 1%, 10%, 25%, 50% of an activity of the wild-type polypeptide from which it is derived.
A “reference sequence,” as used herein, refers to a wild-type polynucleotide or polypeptide sequence from any organism, e.g., wherein the polynucleotide encodes a polypeptide having a diacylglycerol acyltransferase enzymatic activity, a phosphatidate phosphatase enzymatic activity, or an acetyl-CoA carboxylase enzymatic activity, as described herein and known in the art. Exemplary polypeptide “reference sequences” are provided herein, including the amino acid sequence of a diacylglycerol acyltransferase polypeptide from Acinetobacter baylii (SEQ ID NO:1), the amino acid sequence of a phosphatidate phosphatase polypeptide (yPah1) from Saccharomyces cerevisiae (SEQ ID NO:2), and the amino acid sequence of an acyl-CoA carboxylase (yAcc1) from Saccharomyces cerevisiae (SEQ ID NO:3), among others (see, e.g., SEQ ID NOS:4-9, among others known to a person skilled in the art).
The recitations “sequence identity” or, for example, comprising a “sequence 50% identical to,” as used herein, refer to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence”, “comparison window”, “sequence identity”, “percentage of sequence identity” and “substantial identity”. A “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids Res. 25:3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley & Sons Inc, 1994-1998, Chapter 15.
As used herein, the term “triglyceride” (triacylglycerol or neutral fat) refers to a fatty acid triester of glycerol. Triglycerides are typically non-polar and water-insoluble. Phosphoglycerides (or glycerophospholipids) are major lipid components of biological membranes.
“Transformation” refers to the permanent, heritable alteration in a cell resulting from the uptake and incorporation of foreign DNA into the host-cell genome; also, the transfer of an exogenous gene from one organism into the genome of another organism.
By “vector” is meant a polynucleotide molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, yeast or virus, into which a polynucleotide can be inserted or cloned. A vector preferably contains one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector can be an autonomously replicating vector, i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extra-chromosomal element, a mini-chromosome, or an artificial chromosome. The vector can contain any means for assuring self-replication. Alternatively, the vector can be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Such a vector may comprise specific sequences that allow recombination into a particular, desired site of the host chromosome. A vector system can comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. In the present case, the vector is preferably one which is operably functional in a bacterial cell, such as a cyanobacterial cell. The vector can include a reporter gene, such as a green fluorescent protein (GFP), which can be either fused in frame to one or more of the encoded polypeptides, or expressed separately. The vector can also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants.
The terms “wild-type” and “naturally occurring” are used interchangeably to refer to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild type gene or gene product (e.g., a polypeptide) is that which is most frequently observed in a population and is thus arbitrarily designed the “normal” or “wild-type” form of the gene.
Modified Phoosynthetic Microorganisms and Methods of Producing Triglycerides and Fatty Acids
Certain embodiments of the present invention relate to modified photosynthetic microorganisms, e.g., Cyanobacteria, and methods of use thereof, wherein the modified photosynthetic microorganism comprises one or more polynucleotides encoding one or more enzymes associated with triglyceride biosynthesis, such as wherein the enzymes comprise a diacylglycerol acyltransferase (DGAT) activity and/or a phosphatidate phosphatase activity. The present invention contemplates the use of naturally-occurring and non-naturally-occurring variants of these DGAT and phosphatidate phosphatase enzymes, as well as variants of their encoding polynucleotides. In certain aspects, the DGAT encoding polynucleotide sequence is derived from Acinetobacter baylii (ADP1-DGAT), and the phosphatidate phosphatase encoding polynucleotide sequence is from Saccharomyces cerevisiae (yPah1). These enzyme encoding sequences, however, may be derived from any organism having a suitable DGAT or phosphatidate phosphatase enzyme, and may also include any man-made variants thereof, such as any optimized coding sequences (i.e., codon-optimized polynucleotides) or optimized polypeptide sequences. Exemplary polypeptide and polynucleotide sequences are described infra.
In certain embodiments, the modified photosynthetic microorganisms of the present invention may comprise two or more polynucleotides that encode DGAT or a variant or fragment thereof. In particular embodiments, the two or more polynucletoides are identical or express the same DGAT. In certain embodiments, these two or more polynucletoides may be different or may encode two different DGAT polypeptides. For example, in one embodiment, one of the polynucleotides may encode ADGATd, while another polynucleotide may encode ScoDGAT. In particular embodiments, the following DGATs are coexpressed in modified photosynthetic microorganisms, e.g., Cyanobacteria, using one of the following double DGAT strains: ADGATd(NS1)::ADGATd(NS2); ADGATn(NS1)::ADGATn(NS2); ADGATn(NS1)::SDGAT(NS2); SDGAT(NS1)::ADGATn(NS2); SDGAT(NS1)::SDGAT(NS2). For the NS1 vector, pAM2291, EcoRI follows ATG and is part of the open reading frame (ORF). For the NS2 vector, pAM1579, EcoRI follows ATG and is part of the ORF. A DGAT having EcoRI nucleotides following ATG may be cloned in either pAM2291 or pAM1579; such a DGAT is referred to as ADGATd. Other embodiments utilize the vector, pAM2314FTrc3, which is an NS1 vector with Nde/BgIII sites, or the vector, pAM1579FTrc3, which is the NS2 vector with Nde/BgIII sites. A DGAT without EcoRI nucleotides may be cloned into either of these last two vectors. Such a DGAT is referred to as ADGATn. As shown in the accompanying Examples, modified photosynthetic microorganisms expressing different DGATs express TAGs having different fatty acid compositions. Accordingly, certain embodiments of the present invention contemplate expressing two or more different DGATs, in order to produce DAGs having varied fatty acid compositions.
Related embodiments contemplate expressing two or more different phosphatidate phosphatase and/or two or more different acetyl-CoA carboxylases.
Embodiments of the present invention also relate to modified photosynthetic microorganisms, e.g., Cyanobacteria, and methods of use thereof, wherein the modified Cyanobacteria comprise one or more polynucleotides encoding enzymes associated with fatty acid biosynthesis, such as wherein said polynucleotides are exogenous to the Cyanobacterium's native genome. In certain aspects, the enzymes associated with fatty acid synthesis comprise an acetyl-CoA carboxylase (ACCase) activity, including naturally-occurring and non-naturally-occurring functional variants of such enzymes and their encoding polynucleotides. In certain embodiments, the polynucleotide sequence encoding the ACCase enzyme is derived from Saccharomyces cerevisiae (yAcc1). As above, however, these ACCase enzyme encoding sequences may be derived from any organism having a suitable ACCase enzyme, and may also include any man-made variants thereof, such as any optimized coding sequences (i.e., codon-optimized polynucleotides) or optimized polypeptide sequences.
Since, as noted above, fatty acids provide the starting material for triglyceride production, genetically modified Cyanobacteria having increased fatty acid production may by utilized to improve the overall production of triglycerides. Accordingly, certain embodiments relate to modified Cyanobacteria, and methods of use thereof, wherein the Cyanobacteria comprise one or more polynucleotides encoding enzymes associated with fatty acid synthesis and triglyceride synthesis. As such, in certain embodiments, the modified Cyanobacteria of the present invention comprise one or more polynucleotides encoding enzymes that comprise a DGAT activity and/or a phosphatidate phosphatase activity, as well as an exogenous enzyme comprising an ACCase activity.
Embodiments of the present invention also include methods of producing triglyceride in a photosynthetic microorganism, e.g., a Cyanobacterium, comprising introducing one or more polynucleotides encoding one or more enzymes associated with triglyceride biosynthesis into a photosynthetic microorganism, incubating the photosynthetic microorganism for a time and under conditions sufficient to allow triglyceride production, thereby producing triglyceride in the photosynthetic microorganism. Also contemplated are methods of producing a triglyceride in a photosynthetic microorganism, e.g., a Cyanobacterium, comprising culturing a photosynthetic microorganism comprising one or more polynucleotides encoding one or more enzymes associated with triglyceride biosynthesis, for a time and under conditions sufficient to allow triglyceride production. In certain embodiments, the one or more enzymes comprise a diacylglycerol acyltransferase (DGAT) enzymatic activity and/or a phosphatidate phosphatase enzymatic activity. In particular embodiments, the one or more enzymes comprise both a DGAT enzymatic activity and a phosphotidate phosphatase enzymatic activity. In certain embodiments the one or more enzymes comprise an acyl-CoA carboxylase (ACCase) enzymatic activity, as well as a diacylglycerol DGAT enzymatic activity and/or a phosphatidate phosphatase enzymatic activity. In one embodiment, the one or more enzymes comprise a DGAT enzymatic activity, a phosphotidate phosphatase enzymatic activity, and an acyl-CoA carboxylase (ACCase) enzymatic activity. In particular embodiments, one or more of the polynucleotides are exogenous to the photosynthetic microorganism's native genome.
The present invention also relates to methods of producing an increased amount of fatty acid, e.g., a free fatty acid, in a photosynthetic microorganism, e.g., a Cyanobacterium, comprising introducing one or more polynucleotides encoding one or more enzymes associated with fatty acid biosynthesis into a photosynthetic microorganism, culturing the Cyanobacterium for a time and under conditions sufficient to allow increased fatty acid production, thereby producing an increased amount of fatty acid in the Cyanobacterium. Also contemplated are methods of producing an increased amount of fatty acid in a photosynthetic microorganism, e.g., a Cyanobacterium, comprising culturing a photosynthetic microorganism comprising one or more polynucleotides encoding one or more enzymes associated with fatty acid biosynthesis. In certain embodiments, one or more of the polynucleotides are exogenous to the photosynthetic microorganism's native genome. In certain embodiments, the one or more enzymes comprise an ACCase enzymatic activity. In producing triglycerides, the modified Cyanobacteria of the present invention may be cultured according to routine techniques known in the art and exemplified herein, such as photobioreactor based culture techniques.
The present invention also relates to methods of preparing a modified photosynthetic microorganism, e.g., a modified Cyanobacterium, such as by genetic modification, to increase production of naturally-occurring fatty acids, e.g., free fatty acids, and/or to produce triglycerides. Photosynthetic microorganisms, such as Cyanobacteria, can be genetically modified according to routine techniques known in the art, such as by transformation of vectors suitable for use in Cyanobacteria. In certain aspects, genetic manipulation in Cyanobacteria can be performed by the introduction of non-replicating vectors which contain native Cyanobacterial sequences, exogenous genes of interest, and drug resistance genes. Upon introduction into the Cyanobacterial cell, the vectors may be integrated into the Cyanobacterial genome through homologous recombination. In this way, the exogenous gene of interest and the drug resistance gene are stably integrated into the Cyanobacterial genome. Such recombinants cells can then be isolated from non-recombinant cells by drug selection. Examples of suitable vectors are provided herein.
Embodiments of the present invention include methods of producing triglycerides in a Cyanobacterium, comprising introducing one or more polynucleotides encoding one or more enzymes associated with triglyceride biosynthesis into a Cyanobacterium, such as an enzyme having a phosphatidate phosphatase activity and/or an enzyme having a diacylglycerol transferase activity.
In certain aspects, genetically modified photosynthetic microorganisms, e.g., Cyanobacteria, may be prepared by (i) introducing one or more desired polynucleotides encoding one or more enzymes associated with triglyceride biosynthesis into a photosynthetic microorganism, and (ii) selecting for, and/or isolating, photosynthetic microorganisms that comprise the one or more desired polynucleotides. As one example, selection and isolation may include the use of antibiotic resistant markers known in the art (e.g., kanamycin, spectinomycin, and streptomycin) In certain embodiments, genetically modified photosynthetic microorganisms, e.g., Cyanobacteria, may be prepared by (i) introducing one or more desired, exogenous polynucleotides encoding one or more enzymes associated with fatty acid biosynthesis into a photosynthetic microorganism, e.g., a Cyanobacteria, and (ii) selecting for, and/or isolating, photosynthetic microorganisms that comprise one or more desired, exogenous polynucleotides. In certain embodiments, genetically modified photosynthetic microorganisms may be prepared by (i) introducing one or more desired polynucleotides encoding one or more enzymes associated with fatty acid biosynthesis and triglyceride synthesis into a photosynthetic microorganism, and (ii) selecting for, and/or isolating, photosynthetic microorganisms that comprise the one or more desired polynucleotides.
In certain embodiments, the one or more enzymes associated with triglyceride synthesis comprise a diacylglycerol acyltransferase (DGAT) enzymatic activity or a phosphatidate phosphatase enzymatic activity. In some embodiments, the one or more enzymes associated with triglyceride synthesis comprise both a DGAT enzymatic activity and a phosphatidate phosphatase enzymatic activity. In certain embodiments the one or more enzymes associated fatty acid biosynthesis comprise an acyl-CoA carboxylase (ACCase) enzymatic activity. In particular embodiments, the one or more enzymes associated with triglyceride synthesis comprise an acyl-CoA carboxylase (ACCase) enzymatic activity and either a DGAT enzymatic activity or a phosphatidate phosphatase enzymatic activity. In one embodiment, the one or more enzymes associated with triglyceride synthesis comprise an acyl-CoA carboxylase (ACCase) enzymatic activity, a DGAT enzymatic activity, and a phosphatidate phosphatase enzymatic activity.
Photosynthetic Microorganisms
Modified photosynthetic microorganisms of the present invention may be any type of photosynthetic microorganism. These include, but are not limited to photosynthetic bacteria, green algae, and cyanobacteria. The photosynthetic microorganism can be, for example, a naturally photosynthetic microorganism, such as a cyanobacterium, or an engineered photosynthetic microorganism, such as an artificially photosynthetic bacterium. Exemplary microorganisms that are either naturally photosynthetic or can be engineered to be photosynthetic include, but are not limited to, bacteria; fungi; archaea; protists; eukaryotes, such as a green algae; and animals such as plankton, planarian, and amoeba. Examples of naturally occurring photosynthetic microorganisms include, but are not limited to, Spirulina maximum, Spirulina platensis, Dunaliella salina, Botrycoccus braunii, Chlorella vulgaris, Chlorella pyrenoidosa, Serenastrum capricomutum, Scenedesmus auadricauda, Porphyridium cruentum, Scenedesmus acutus, Dunaliella sp., Scenedesmus obliquus, Anabaenopsis, Aulosira, Cylindrospermum, Synechoccus sp., Synechocystis sp., and/or Tolypothrix. 
A modified Cyanobacteria of the present invention may be from any genera or species of Cyanobacteria that is genetically manipulable, i.e., permissible to the introduction and expression of exogenous genetic material. Examples of Cyanobacteria that can be engineered according to the methods of the present invention include, but are not limited to, the genus Synechocystis, Synechococcus, Thermosynechococcus, Nostoc, Prochlorococcu, Microcystis, Anabaena, Spirulina, and Gloeobacter. 
Cyanobacteria, also known as blue-green algae, blue-green bacteria, or Cyanophyta, is a phylum of bacteria that obtain their energy through photosynthesis. Cyanobacteria can produce metabolites, such as carbohydrates, proteins, lipids and nucleic acids, from CO2, water, inorganic salts and light. Any Cyanobacteria may be used according to the present invention.
Cyanobacteria include both unicellular and colonial species. Colonies may form filaments, sheets or even hollow balls. Some filamentous colonies show the ability to differentiate into several different cell types, such as vegetative cells, the normal, photosynthetic cells that are formed under favorable growing conditions; akinetes, the climate-resistant spores that may form when environmental conditions become harsh; and thick-walled heterocysts, which contain the enzyme nitrogenase, vital for nitrogen fixation.
Heterocysts may also form under the appropriate environmental conditions (e.g., anoxic) whenever nitrogen is necessary. Heterocyst-forming species are specialized for nitrogen fixation and are able to fix nitrogen gas, which cannot be used by plants, into ammonia (NH3), nitrites (NO2−), or nitrates (NO3−), which can be absorbed by plants and converted to protein and nucleic acids.
Many Cyanobacteria also form motile filaments, called hormogonia, which travel away from the main biomass to bud and form new colonies elsewhere. The cells in a hormogonium are often thinner than in the vegetative state, and the cells on either end of the motile chain may be tapered. In order to break away from the parent colony, a hormogonium often must tear apart a weaker cell in a filament, called a necridium.
Each individual cyanobacterial cell typically has a thick, gelatinous cell wall. Cyanobacteria differ from other gram-negative bacteria in that the quorum sensing molecules autoinducer-2 and acyl-homoserine lactones are absent. They lack flagella, but hormogonia and some unicellular species may move about by gliding along surfaces. In water columns some Cyanobacteria float by forming gas vesicles, like in archaea.
Cyanobacteria have an elaborate and highly organized system of internal membranes that function in photosynthesis. Photosynthesis in Cyanobacteria generally uses water as an electron donor and produces oxygen as a by-product, though some Cyanobacteria may also use hydrogen sulfide, similar to other photosynthetic bacteria. Carbon dioxide is reduced to form carbohydrates via the Calvin cycle. In most forms the photosynthetic machinery is embedded into folds of the cell membrane, called thylakoids. Due to their ability to fix nitrogen in aerobic conditions, Cyanobacteria are often found as symbionts with a number of other groups of organisms such as fungi (e.g., lichens), corals, pteridophytes (e.g., Azolla), and angiosperms (e.g., Gunnera), among others.
Cyanobacteria are the only group of organisms that are able to reduce nitrogen and carbon in aerobic conditions. The water-oxidizing photosynthesis is accomplished by coupling the activity of photosystem (PS) II and I (Z-scheme). In anaerobic conditions, Cyanobacteria are also able to use only PS I (i.e., cyclic photophosphorylation) with electron donors other than water (e.g., hydrogen sulfide, thiosulphate, or molecular hydrogen), similar to purple photosynthetic bacteria. Furthermore, Cyanobacteria share an archaeal property; the ability to reduce elemental sulfur by anaerobic respiration in the dark. The Cyanobacterial photosynthetic electron transport system shares the same compartment as the components of respiratory electron transport. Typically, the plasma membrane contains only components of the respiratory chain, while the thylakoid membrane hosts both respiratory and photosynthetic electron transport.
Phycobilisomes, attached to the thylakoid membrane, act as light harvesting antennae for the photosystems of Cyanobacteria. The phycobilisome components (phycobiliproteins) are responsible for the blue-green pigmentation of most Cyanobacteria. Color variations are mainly due to carotenoids and phycoerythrins, which may provide the cells with a red-brownish coloration. In some Cyanobacteria, the color of light influences the composition of phycobilisomes. In green light, the cells accumulate more phycoerythrin, whereas in red light they produce more phycocyanin. Thus, the bacteria appear green in red light and red in green light. This process is known as complementary chromatic adaptation and represents a way for the cells to maximize the use of available light for photosynthesis.
In particular embodiments, the Cyanobacteria may be, e.g., a marine form of Cyanobacteria or a fresh water form of Cyanobacteria. Examples of marine forms of Cynobacteria include, but are not limited to Synechococcus WH8102, Synechococcus RCC307, Synechococcus NKBG 15041c, and Trichodesmium. Examples of fresh water forms of Cyaobacteria include, but are not limited to, S. elongatus PCC 7942, Synechocystis PCC6803, Plectonema boryanum, and Anabaena sp. Exogenous genetic material encoding the desired enzymes may be introduced either transiently, such as in certain self-replicating vectors, or stably, such as by integration (e.g., recombination) into the Cyanobacterium's native genome.
In other embodiments, a genetically modified Cyanobacteria of the present invention may be capable of growing in brackish or salt water. When using a fresh water form of Cyanobacteria, the overall net cost for production of triglycerides will depend on both the nutrients required to grow the culture and the price for freshwater. One can foresee freshwater being a limited resource in the future, and in that case it would be more cost effective to find an alternative to freshwater. Two such alternatives include: (1) the use of waste water from treatment plants; and (2) the use of salt or brackish water.
Salt water in the oceans can range in salinity between 3.1% and 3.8%, the average being 3.5%, and this is mostly, but not entirely, made up of sodium chloride (NaCl) ions. Brackish water, on the other hand, has more salinity than freshwater, but not as much as seawater. Brackish water contains between 0.5% and 3% salinity, and thus includes a large range of salinity regimes and is therefore not precisely defined. Waste water is any water that has undergone human influence. It consists of liquid waste released from domestic and commercial properties, industry, and/or agriculture and can encompass a wide range of possible contaminants at varying concentrations.
There is a broad distribution of Cyanobacteria in the oceans, with Synechococcus filling just one niche. Specifically, Synechococcus sp. PCC 7002 (formerly known as Agmenellum quadruplicatum strain PR-6) grows in brackish water, is unicellular and has an optimal growing temperature of 38° C. While this strain is well suited to grow in conditions of high salt, it will grow slowly in freshwater. In particular embodiments, the present invention contemplates the use of a Cyanobacteria PCC 7942, altered in a way that allows for growth in either waste water or salt/brackish water. A Synechococcus elongatus PCC 7942 mutant resistant to sodium chloride stress has been described (Bagchi, S. N. et al., Photosynth Res. 2007, 92:87-101), and a genetically modified S. elongatus PCC 7942 tolerant of growth in salt water has been described (Waditee, R. et al., PNAS 2002, 99:4109-4114). Salt water tolerant Cyanobacteria may also be prepared as described in the accompanying Examples. According to the present invention a salt water tolerant strain is capable of growing in water or media having a salinity in the range of 0.5% to 4.0% salinity, although it is not necessarily capable of growing in all salinities encompassed by this range. In one embodiment, a salt tolerant strain is capable of growth in water or media having a salinity in the range of 1.0% to 2.0% salinity. In another embodiment, a salt water tolerant strain is capable of growth in water or media having a salinity in the range of 2.0% to 3.0% salinity.
Examples of cyanobacteria that may be utilized and/or genetically modified according to the methods described herein include, but are not limited to, Chroococcales cyanobacteria from the genera Aphanocapsa, Aphanothece, Chamaesiphon, Chroococcus, Chroogloeocystis, Coelosphaerium, Crocosphaera, Cyanobacterium, Cyanobium, Cyanodictyon, Cyanosarcina, Cyanothece, Dactylococcopsis, Gloecapsa, Gloeothece, Merismopedia, Microcystis, Radiocystis, Rhabdoderma, Snowella, Synychococcus, Synechocystis, Thermosenechococcus, and Woronichinia; Nostacales cyanobacteria from the genera Anabaena, Anabaenopsis, Aphanizomenon, Aulosira, Calothrix, Coleodesmium, Cyanospira, Cylindrospermosis, Cylindrospermum, Fremyella, Gleotrichia, Microchaete, Nodularia, Nostoc, Rexia, Richelia, Scytonema, Sprirestis, and Toypothrix; Oscillatoriales cyanobacteria from the genera Arthrospira, Geitlennema, Halomicronema, Halospirulina, Katagnymene, Leptolyngbya, Limnothrix, Lyngbya, Microcoleus, Oscillatoria, Phormidium, Planktothricoides, Planktothrix, Plectonema, Pseudoanabaena/Limnothrix, Schizothrix, Spirulina, Symploca, Trichodesmium, Tychonema; Pleurocapsales cyanobacterium from the genera Chroococcidiopsis, Dermocarpa, Dermocarpella, Myxosarcina, Pleurocapsa, Stanieria, Xenococcus; Prochlorophytes cyanobacterium from the genera Prochloron, Prochlorococcus, Prochlorothrix; and Stigonematales cyanobacterium from the genera Capsosira, Chlorogeoepsis, Fischerella, Hapalosiphon, Mastigocladopsis, Nostochopsis, Stigonema, Symphyonema, Symphonemopsis, Umezakia, and Westiellopsis. In certain embodiments, the cyanobacterium is from the genus Synechococcus, including, but not limited to Synechococcus bigranulatus, Synechococcus elongatus, Synechococcus leopoliensis, Synechococcus lividus, Synechococcus nidulans, and Synechococcus rubescens. 
In certain embodiments, the cyanobacterium is Anabaena sp. strain PCC 7120, Synechocystis sp. strain PCC 6803, Nostoc muscorum, Nostoc ellipsosporum, or Nostoc sp. strain PCC 7120. In certain preferred embodiments, the cyanobacterium is Synechococcus elongatus sp. strain PCC 7942. Additional examples of Cyanobacteria that may utilized in the methods provided herein include, but are not limited to, Synechococcus sp. strains WH7803, WH8102, WH8103 (typically genetically modified by conjugation), Baeocyte-forming Chroococcidiopsis spp. (typically modified by conjugation/electroporation), non-heterocyst-forming filamentous strains Planktothrix sp., Plectonema boryanum M101 (typically modified by electroporation), and Heterocyst-forming strains Anabaena sp. strains ATCC 29413 (typically modified by conjugation), Tolypothrix sp. strain PCC 7601 (typically modified by conjugation/electroporation) and Nostoc punctiforme strain ATCC 29133 (typically modified by conjugation/electroporation).
In particular embodiments, the genetically modified, photosynthetic microorganism, e.g., Cyanobacteria, of the present invention may be used to produce triglycerides from just sunlight, water, air, and minimal nutrients, using routine culture techniques of any reasonably desired scale. In particular embodiments, the present invention contemplates using spontaneous mutants of photosynthetic microorganisms that demonstrate a growth advantage under a defined growth condition. Among other benefits, the ability to produce large amounts of triglycerides from minimal energy and nutrient input makes the modified photosynthetic microorganism, e.g., Cyanobacteria, of the present invention a readily manageable and efficient source of feedstock in the subsequent production of both biofuels, such as biodiesel, as well as specialty chemicals, such as glycerin.
Methods of Producing and Culturing Modified Photosynthetic Microorganisms
Photosynthetic microorganisms, e.g., Cyanobacteria, may be genetically modified according to techniques known in the art. As noted above, in certain aspects, genetic manipulation in Cyanobacteria can be performed by the introduction of non-replicating vectors that contain native Cyanobacterial sequences, exogenous genes of interest and drug resistance genes. Upon introduction into the Cyanobacterial cell, the vectors may be integrated into the Cyanobacterial genome through homologous recombination. In this way, the exogenous gene of interest and the drug resistance gene are stably integrated into the Cyanobacterial genome. Such recombinants cells can then be isolated from non-recombinant cells by drug selection. Cell transformation methods and selectable markers for Cyanobacteria are also well known in the art (see, e.g., Wirth, Mol Gen Genet 216:175-7, 1989; and Koksharova, Appl Microbiol Biotechnol 58:123-37, 2002).
Cyanobacteria may be cultured or cultivated according to techniques known in the art, such as those described in Acreman et al. (Journal of Industrial Microbiology and Biotechnology 13:193-194, 1994), in addition to photobioreactor based techniques, such as those described in Nedbal et al. (Biotechnol Bioeng. 100:902-10, 2008). One example of typical laboratory culture conditions for Cyanobacterium is growth in BG-11 medium (ATCC Medium 616) at 30° C. in a vented culture flask with constant agitation and constant illumination at 30-100 μmole photons m−2 sec−1.
A wide variety of mediums are available for culturing Cyanobacteria, including, for example, Aiba and Ogawa (AO) Medium, Allen and Amon Medium plus Nitrate (ATCC Medium 1142), Antia's (ANT) Medium, Aquil Medium, Ashbey's Nitrogen-free Agar, ASN-III Medium, ASP 2 Medium, ASW Medium (Artificial Seawater and derivatives), ATCC Medium 617 (BG-11 for Marine Blue-Green Algae; Modified ATCC Medium 616 [BG-11 medium]), ATCC Medium 819 (Blue-green Nitrogen-fixing Medium; ATCC Medium 616 [BG-11 medium] without NO3), ATCC Medium 854 (ATCC Medium 616 [BG-11 medium] with Vitamin B12), ATCC Medium 1047 (ATCC Medium 957 [MN marine medium] with Vitamin B12), ATCC Medium 1077 (Nitrogen-fixing marine medium; ATCC Medium 957 [MN marine medium] without NO3), ATCC Medium 1234 (BG-11 Uracil medium; ATCC Medium 616 [BG-11 medium] with uracil), Beggiatoa Medium (ATCC Medium 138), Beggiatoa Medium 2 (ATCC Medium 1193), BG-11 Medium for Blue Green Algae (ATCC Medium 616), Blue-Green (BG) Medium, Bold's Basal (BB) Medium, Castenholtz D Medium, Castenholtz D Medium Modified (Halophilic cyanobacteria), Castenholtz DG Medium, Castenholtz DGN Medium, Castenholtz ND Medium, Chloroflexus Broth, Chloroflexus Medium (ATCC Medium 920), Chu's #10 Medium (ATCC Medium 341), Chu's #10 Medium Modified, Chu's #11 Medium Modified, DCM Medium, DYIV Medium, E27 Medium, E31 Medium and Derivatives, f/2 Medium, f/2 Medium Derivatives, Fraquil Medium (Freshwater Trace Metal-Buffered Medium), Gorham's Medium for Algae (ATCC Medium 625), h/2 Medium, Jaworski's (JM) Medium, K Medium, L1 Medium and Derivatives, MN Marine Medium (ATCC Medium 957), Plymouth Erdschreiber (PE) Medium, Prochlorococcus PC Medium, Proteose Peptone (PP) Medium, Prov Medium, Prov Medium Derivatives, S77 plus Vitamins Medium, S88 plus Vitamins Medium, Saltwater Nutrient Agar (SNA) Medium and Derivatives, SES Medium, SN Medium, Modified SN Medium, SNAX Medium, Soil/Water Biphasic (S/W) Medium and Derivatives, SOT Medium for Spirulina: ATCC Medium 1679, Spirulina (SP) Medium, van Rijn and Cohen (RC) Medium, Walsby's Medium, Yopp Medium, and Z8 Medium, among others.
In certain embodiments, modified photosynthetic microorganisms, e.g., Cyanobacteria, are grown under conditions favorable for producing triglycerides and/or fatty acids. In particular embodiments, light intensity is between 100 and 2000 uE/m2/s, or between 200 and 1000 uE/m2/s. In particular embodiments, the pH range of culture media is between 7.0 and 10.0. In certain embodiments, CO2 is injected into the culture apparatus to a level in the range of 1% to 10%. In particular embodiments, the range of CO2 is between 2.5% and 5%. In certain embodiments, nutrient supplementation is performed during the linear phase of growth. Each of these conditions are desirable for triglyceride production.
Nucleic Acids and Polypeptides
In various embodiments, modified photosynthetic microorganisms, e.g., Cyanobacteria, of the present invention comprise one or more exogenous or introduced nucleic acids that encode a polypeptide having an activity associated with triglyceride or fatty acid biosynthesis, including but not limited to any of those described herein. In particular embodiments, the exogenous nucleic acid does not comprises a nucleic acid sequence that is native to the microorganism's genome. In particular embodiments, the exogenous nucleic acid comprises a nucleic acid sequence that is native to the microorganism's genome, but it has been introduced into the microorganism, e.g., in a vector or by molecular biology techniques, for example, to increase expression of the nucleic acid and/or its encoded polypeptide in the microorganism.
Triglyceride Biosynthesis
Triglycerides, or triacylglycerols (TAG), consist primarily of glycerol esterified with three fatty acids, and yield more energy upon oxidation than either carbohydrates or proteins. Triglycerides provide an important mechanism of energy storage for most eukaryotic organisms. In mammals, TAGs are synthesized and stored in several cell types, including adipocytes and hepatocytes (Bell et al. Annu. Rev. Biochem. 49:459-487, 1980) (herein incorporated by reference). In plants, TAG production is mainly important for the generation of seed oils.
In contrast to eukaryotes, the observation of triglyceride production in prokaryotes has been limited to certain actinomycetes, such as members of the genera Mycobacterium, Nocardia, Rhodococcus and Streptomyces, in addition to certain members of the genus Acinetobacter. In certain actinomycetes species, triglycerides may accumulate to nearly 80% of the dry cell weight, but accumulate to only about 15% of the dry cell weight in Acinetobacter. In general, triglycerides are stored in spherical lipid bodies, with quantities and diameters depending on the respective species, growth stage, and cultivation conditions. For example, cells of Rhodococcus opacus and Streptomyces lividans contain only few TAGs when cultivated in complex media with a high content of carbon and nitrogen; however, the lipid content and the number of TAG bodies increase drastically when the cells are cultivated in mineral salt medium with a low nitrogen-to-carbon ratio, yielding a maximum in the late stationary growth phase. At this stage, cells can be almost completely filled with lipid bodies exhibiting diameters ranging from 50 to 400 nm. One example is R. opacus PD630, in which lipids can reach more than 70% of the total cellular dry weight.
In bacteria, TAG formation typically starts with the docking of a diacylglycerol acyltransferase enzyme to the plasma membrane, followed by formation of small lipid droplets (SLDs). These SLDs are only some nanometers in diameter and remain associated with the membrane-docked enzyme. In this phase of lipid accumulation, SLDs typically form an emulsive, oleogenous layer at the plasma membrane. During prolonged lipid synthesis, SLDs leave the membrane-associated acyltransferase and conglomerate to membrane-bound lipid prebodies. These lipid prebodies reach distinct sizes, e.g., about 200 nm in A. calcoaceticus and about 300 nm in R. opacus, before they lose contact with the membrane and are released into the cytoplasm. Free and membrane-bound lipid prebodies correspond to the lipid domains occurring in the cytoplasm and at the cell wall, as observed in M. smegmatis during fluorescence microscopy and also confirmed in R. opacus PD630 and A. calcoaceticus ADP1 (see, e.g., Christensen et al., Mol. Microbiol. 31:1561-1572, 1999); and Wältermann et al., Mol. Microbiol. 55:750-763, 2005). Inside the lipid prebodies, SLDs coalesce with each other to form the homogenous lipid core found in mature lipid bodies, which often appear opaque in electron microscopy.
The compositions and structures of bacterial TAGs vary considerably depending on the microorganism and on the carbon source. In addition, unusual acyl moieties, such as phenyldecanoic acid and 4,8,12 trimethyl tridecanoic acid, may also contribute to the structural diversity of bacterial TAGs. (see, e.g., Alvarez et al., Appl Microbiol Biotechnol. 60:367-76, 2002).
As with eukaryotes, the main function of TAGs in prokaryotes is to serve as a storage compound for energy and carbon. TAGs, however, may provide other functions in prokaryotes. For example, lipid bodies may act as a deposit for toxic or useless fatty acids formed during growth on recalcitrant carbon sources, which must be excluded from the plasma membrane and phospholipid (PL) biosynthesis. Furthermore, many TAG-accumulating bacteria are ubiquitous in soil, and in this habitat, water deficiency causing dehydration is a frequent environmental stress. Storage of evaporation-resistant lipids might be a strategy to maintain a basic water supply, since oxidation of the hydrocarbon chains of the lipids under conditions of dehydration would generate considerable amounts of water. Cyanobacteria such as Synechococcus, however, do not produce triglycerides, because these organisms lack the enzymes necessary for triglyceride biosynthesis.
Triglycerides are synthesized from fatty acids and glycerol. As one mechanism of triglyceride (TAG) synthesis, sequential acylation of glycerol-3-phosphate via the “Kennedy Pathway” leads to the formation of phosphatidate. Phosphatidate is then dephosphorylated by the enzyme phosphatidate phosphatase to yield 1,2 diacylglycerol (DAG). Using DAG as a substrate, at least three different classes of enzymes are capable of mediating TAG formation. As one example, an enzyme having diacylglycerol transferase (DGAT) activity catalyzes the acylation of DAG using acyl-CoA as a substrate. Essentially, DGAT enzymes combine acyl-CoA with 1,2 diacylglycerol molecule to form a TAG. As an alternative, Acyl-CoA-independent TAG synthesis may be mediated by a phospholipid:DAG acyltransferase found in yeast and plants, which uses phospholipids as acyl donors for DAG esterification. Third, TAG synthesis in animals and plants may be mediated by a DAG-DAG-transacylase, which uses DAG as both an acyl donor and acceptor, yielding TAG and monoacylglycerol.
Modified photosynthetic microorganisms, e.g., Cyanobacteria, of the present invention may comprise one or more exogenous polynucleotides encoding polypeptides comprising one or more of the polypeptides and enzymes described above. In particular embodiments, the one or more exogenous polynucleotides encode a diacylglycerol transferase and/or a phosphatidate phosphatase, or a variant or function fragment thereof.
Since wild-type Cyanobacteria do not typically encode the enzymes necessary for triglyceride synthesis, such as the enzymes having phosphatidate phosphatase activity and diacylglycerol transferase activity, embodiments of the present invention include genetically modified Cyanobacteria that comprise polynucleotides encoding one or more enzymes having a phosphatidate phosphatase activity and/or one or more enzymes having a diacylglycerol transferase activity.
Moreover, since triglycerides are typically formed from fatty acids, the level of fatty acid biosynthesis in a cell may limit the production of triglycerides. Increasing the level of fatty acid biosynthesis may, therefore, allow increased production of triglycerides. As discussed below, Acetyl-CoA carboxylase catalyzes the commitment step to fatty acid biosynthesis. Thus, certain embodiments of the present invention include Cyanobacterium, and methods of use thereof, comprising polynucleotides that encode one or more enzymes having Acetyl-CoA carboxylase activity to increase fatty acid biosynthesis and lipid production, in addition to one or more enzymes having phosphatidate phosphatase and/or diacylglycerol transferase activity to catalyze triglyceride production.
As used herein, a “phosphatidate phosphatase” gene of the present invention includes any polynucleotide sequence encoding amino acids, such as protein, polypeptide or peptide, obtainable from any cell source, which demonstrates the ability to catalyze the dephosphorylation of phosphatidate (PtdOH) under enzyme reactive conditions, yielding diacylglycerol (DAG) and inorganic phosphate, and further includes any naturally-occurring or non-naturally occurring variants of a phosphatidate phosphatase sequence having such ability.
Phosphatidate phosphatases (PAP, 3-sn-phosphatidate phosphohydrolase) catalyze the dephosphorylation of phosphatidate (PtdOH), yielding diacylglycerol (DAG) and inorganic phosphate. This enzyme belongs to the family of hydrolases, specifically those acting on phosphoric monoester bonds. The systematic name of this enzyme class is 3-sn-phosphatidate phosphohydrolase. Other names in common use include phosphatic acid phosphatase, acid phosphatidyl phosphatase, and phosphatic acid phosphohydrolase. This enzyme participates in at least 4 metabolic pathways: glycerolipid metabolism, glycerophospholipid metabolism, ether lipid metabolism, and sphingolipid metabolism.
PAP enzymes have roles in both the synthesis of phospholipids and triacylglycerol through its product diacylglycerol, as well as the generation or degradation of lipid-signaling molecules in eukaryotic cells. PAP enzymes are typically classified as either Mg2+-dependent (referred to as PAP1 enzymes) or Mg2+-independent (PAP2 or lipid phosphate phosphatase (LPP) enzymes) with respect to their cofactor requirement for catalytic activity. In both yeast and mammalian systems, PAP2 enzymes are known to be involved in lipid signaling. By contrast, PAP1 enzymes, such as those found in Saccharomyces cerevisiae, play a role in de novo lipid synthesis (Han, et al. J Biol Chem. 281:9210-9218, 2006), thereby revealing that the two types of PAP are responsible for different physiological functions.
In both yeast and higher eukaryotic cells, the PAP reaction is the committed step in the synthesis of the storage lipid triacylglycerol (TAG), which is formed from PtdOH through the intermediate DAG. The reaction product DAG is also used in the synthesis of the membrane phospholipids phosphatidylcholine (PtdCho) and phosphatidylethanolamine. The substrate PtdOH is used for the synthesis of all membrane phospholipids (and the derivative inositol-containing sphingolipids) through the intermediate CDP-DAG. Thus, regulation of PAP activity might govern whether cells make storage lipids and phospholipids through DAG or phospholipids through CDP-DAG. In addition, PAP is involved in the transcriptional regulation of phospholipid synthesis.
PAP1 enzymes have been purified and characterized from the membrane and cytosolic fractions of yeast, including a gene (Pah1, formerly known as Smp2) been identified to encode a PAP1 enzyme in S. cerevisiae. The Pah1-encoded PAP1 enzyme is found in the cytosolic and membrane fractions of the cell, and its association with the membrane is peripheral in nature. As expected from the multiple forms of PAP1 that have been purified from yeast, pah1Δ mutants still contain PAP1 activity, indicating the presence of an additional gene or genes encoding enzymes having PAP1 activity.
Analysis of mutants lacking the Pah1-encoded PAP1 has provided evidence that this enzyme generates the DAG used for lipid synthesis. Cells containing the pah1Δ mutation accumulate PtdOH and have reduced amounts of DAG and its acylated derivative TAG. Phospholipid synthesis predominates over the synthesis of TAG in exponentially growing yeast, whereas TAG synthesis predominates over the synthesis of phospholipids in the stationary phase of growth. The effects of the pah1Δ mutation on TAG content are most evident in the stationary phase. For example, stationary phase cells devoid of the Pah1 gene show a reduction of >90% in TAG content. Likewise, the pah1Δ mutation shows the most marked effects on phospholipid composition (e.g. the consequent reduction in PtdCho content) in the exponential phase of growth. The importance of the Pah1-encoded PAP1 enzyme to cell physiology is further emphasized because of its role in the transcriptional regulation of phospholipid synthesis.
The requirement of Mg2+ ions as a cofactor for PAP enzymes is correlated with the catalytic motifs that govern the phosphatase reactions of these enzymes. For example, the Pah1-encoded PAP1 enzyme has a DxDxT (SEQ ID NO:30) catalytic motif within a haloacid dehalogenase (HAD)-like domain (“x” is any amino acid). This motif is found in a superfamily of Mg2+-dependent phosphatase enzymes, and its first aspartate residue is responsible for binding the phosphate moiety in the phosphatase reaction. By contrast, the DPP1- and LPP1-encoded PAP2 enzymes contain a three-domain lipid phosphatase motif that is localized to the hydrophilic surface of the membrane. This catalytic motif, which comprises the consensus sequences KxxxxxxRP (domain 1) (SEQ ID NO:10), PSGH (domain 2) (SEQ ID NO:11), and SRxxxxxHxxxD (domain 3) (SEQ ID NO:12), is shared by a superfamily of lipid phosphatases that do not require Mg2+ ions for activity. The conserved arginine residue in domain 1 and the conserved histidine residues in domains 2 and 3 may be essential for the catalytic activity of PAP2 enzymes. Accordingly, a phosphatide phosphatase polypeptide may comprise one or more of the above-described catalytic motifs.
A polynucleotide encoding a polypeptide having a phosphatidate phosphatase enzymatic activity may be obtained from any organism having a suitable, endogenous phosphatidate phosphatase gene. Examples of organisms that may be used to obtain a phosphatidate phosphatase encoding polynucleotide sequence include, but are not limited to, Homo sapiens, Mus musculus, Rattus norvegicus, Bos taurus, Drosophila melanogaster, Arabidopsis thaliana, Magnaporthe grisea, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Cryptococcus neoformans, and Bacillus pumilus, among others. As used herein, a “diacylglycerol acyltransferase” (DGAT) gene of the present invention includes any polynucleotide sequence encoding amino acids, such as protein, polypeptide or peptide, obtainable from any cell source, which demonstrates the ability to catalyze the production of triacylglycerol from 1,2-diacylglycerol and fatty acyl substrates under enzyme reactive conditions, in addition to any naturally-occurring (e.g., allelic variants, orthologs) or non-naturally occurring variants of a diacylglycerol acyltransferase sequence having such ability. DGAT genes of the present invention also polynucleotide sequences that encode bi-functional proteins, such as those bi-functional proteins that exhibit a DGAT activity as well as a CoA:fatty alcohol acyltransferase activity, i.e., a wax ester synthesis (WS) activity, as often found in many TAG producing bacteria.
Diacylglycerol acyltransferases (DGATs) are members of the O-acyltransferase superfamily, which esterify either sterols or diacyglycerols in an oleoyl-CoA-dependent manner. DGAT in particular esterifies diacylglycerols, which reaction represents the final enzymatic step in the production of triacylglycerols in plants, fungi and mammals. Specifically, DGAT is responsible for transferring an acyl group from acyl-coenzyme-A to the sn-3 position of 1,2-diacylglycerol (DAG) to form triacylglycerol (TAG). DGAT is an integral membrane protein that has been generally described in Harwood (Biochem. Biophysics. Acta, 1301:7-56, 1996), Daum et al. (Yeast 16:1471-1510, 1998), and Coleman et al. (Annu. Rev. Nutr. 20:77-103, 2000) (each of which are herein incorporated by reference).
In plants and fungi, DGAT is associated with the membrane and lipid body fractions. In catalyzing TAGs, DGAT contributes mainly to the storage of carbon used as energy reserves. In animals, however, the role of DGAT is more complex. DGAT not only plays a role in lipoprotein assembly and the regulation of plasma triacylglycerol concentration (Bell, R. M., et al.), but participates as well in the regulation of diacylglycerol levels (Brindley, Biochemistry of Lipids, Lipoproteins and Membranes, eds. Vance, D. E. & Vance, J. E. (Elsevier, Amsterdam), 171-203; and Nishizuka, Science 258:607-614 (1992) (each of which are herein incorporated by reference)).
In eukaryotes, at least three independent DGAT gene families (DGAT1, DGAT2, and PDAT) have been described that encode proteins with the capacity to form TAG. Yeast contain all three of DGAT1, DGAT2, and PDAT, but the expression levels of these gene families varies during different phases of the life cycle (Dahlqvst, A., et al. Proc. Natl. Acad. Sci. USA 97:6487-6492 (2000) (herein incorporated by reference).
In prokaryotes, WS/DGAT from Acinetobacter calcoaceticus ADP1 represents the first identified member of a widespread class of bacterial wax ester and TAG biosynthesis enzymes. This enzyme comprises a putative membrane-spanning region but shows no sequence homology to the DGAT1 and DGAT2 families from eukaryotes. Under in vitro conditions, WS/DGAT shows a broad capability of utilizing a large variety of fatty alcohols, and even thiols as acceptors of the acyl moieties of various acyl-CoA thioesters. WS/DGAT acylatransferase enzymes exhibit extraordinarily broad substrate specificity. Genes for homologous acyltransferases have been found in almost all bacteria capable of accumulating neutral lipids, including, for example, Acinetobacter baylii, A. baumanii, and M. avium, and M. tuberculosis CDC1551, in which about 15 functional homologues are present (see, e.g., Daniel et al., J. Bacteriol. 186:5017-5030, 2004; and Kalscheuer et al., J. Biol. Chem. 287:8075-8082, 2003).
DGAT proteins may utilize a variety of acyl substrates in a host cell, including fatty acyl-CoA and fatty acyl-ACP molecules. In addition, the acyl substrates acted upon by DGAT enzymes may have varying carbon chain lengths and degrees of saturation, although DGAT may demonstrate preferential activity towards certain molecules.
Like other members of the eukaryotic β-acyltransferase superfamily, eukaryotic DGAT polypeptides typically contain a FYxDWWN (SEQ ID NO:13) heptapeptide retention motif, as well as a histidine (or tyrosine)-serine-phenylalanine (H/YSF) tripeptide motif, as described in Zhongmin et al. (Journal of Lipid Research, 42:1282-1291, 2001) (herein incorporated by reference). The highly conserved FYxDWWN (SEQ ID NO:13) is believed to be involved in fatty Acyl-CoA binding.
DGAT enzymes utilized according to the present invention may be isolated from any organism, including eukaryotic and prokaryotic organisms. Eukaryotic organisms having a DGAT 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 S. 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 a DGAT activity include, for example, Mycobacterium tuberculosis, M. avium, M. smegmatis, Micromonospora echinospora, Rhodococcus opacus, R. ruber, and Streptomyces lividans. Additional 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).
Fatty Acid Biosynthesis
Fatty acids are a group of negatively charged, linear hydrocarbon chains of various length and various degrees of oxidation states. The negative charge is located at a carboxyl end group and is typically deprotonated at physiological pH values (pK ̃2-3). The length of the fatty acid ‘tail’ determines its water solubility (or rather insolubility) and amphipathic characteristics. Fatty acids are components of phospholipids and sphingolipids, which form part of biological membranes, as well as triglycerides, which are primarily used as energy storage molecules inside cells
Fatty acids are formed from acetyl-CoA and malonyl-CoA precursors. Malonyl-CoA is a carboxylated form of acetyl-CoA, and contains a 3-carbon dicarboxylic acid, malonate, bound to Coenzyme A. Acetyl-CoA carboxylase catalyzes the 2-step reaction by which acetyl-CoA is carboxylated to form malonyl-CoA. In particular, malonate is formed from acetyl-CoA by the addition of CO2 using the biotin cofactor of the enzyme acetyl-CoA carboxylase.
Fally acid synthase (FAS) carries out the chain elongation steps of fatty acid biosynthesis. FAS is a large multienzyme complex. In mammals, FAS contains two subunits, each containing multiple enzyme activities. In bacteria and plants, individual proteins, which associate into a large complex, catalyze the individual steps of the synthesis scheme. For example, in bacteria and plants, the acyl carrier protein is a smaller, independent protein.
Fatty acid synthesis starts with acetyl-CoA, and the chain grows from the “tail end” so that carbon 1 and the alpha-carbon of the complete fatty acid are added last. The first reaction is the transfer of an acetyl group to a pantothenate group of acyl carrier protein (ACP), a region of the large mammalian fatty acid synthase (FAS) protein. In this reaction, acetyl CoA is added to a cysteine —SH group of the condensing enzyme (CE) domain: acetyl CoA+CE-cys-SH→acetyl-cys-CE+CoASH. Mechanistically, this is a two step process, in which the group is first transferred to the ACP (acyl carrier peptide), and then to the cysteine —SH group of the condensing enzyme domain.
In the second reaction, malonyl CoA is added to the ACP sulfhydryl group: malonyl CoA+ACP-SH→malonyl ACP+CoASH. This —SH group is part of a phosphopantethenic acid prosthetic group of the ACP.
In the third reaction, the acetyl group is transferred to the malonyl group with the release of carbon dioxide: malonyl ACP+acetyl-cys-CE→beta-ketobutyryl-ACP+CO2.
In the fourth reaction, the keto group is reduced to a hydroxyl group by the beta-ketoacyl reductase activity: beta-ketobutyryl-ACP+NADPH+H+→beta-hydroxybutyryl-ACP+NAD+.
In the fifth reaction, the beta-hydroxybutyryl-ACP is dehydrated to form a trans-monounsaturated fatty acyl group by the beta-hydroxyacyl dehydratase activity: beta-hydroxybutyryl-ACP→2-butenoyl-ACP+H2O.
In the sixth reaction, the double bond is reduced by NADPH, yielding a saturated fatty acyl group two carbons longer than the initial one (an acetyl group was converted to a butyryl group in this case): 2-butenoyl-ACP+NADPH+H+→butyryl-ACP+NADP+. The butyryl group is then transferred from the ACP sulfhydryl group to the CE sulfhydryl: butyryl-ACP+CE-cys-SH→ACP-SH+butyryl-cys-CE. This step is catalyzed by the same transferase activity utilized previously for the original acetyl group. The butyryl group is now ready to condense with a new malonyl group (third reaction above) to repeat the process. When the fatty acyl group becomes 16 carbons long, a thioesterase activity hydrolyses it, forming free palmitate: palmitoyl-ACP+H2O→palmitate+ACP-SH. Fatty acid molecules can undergo further modification, such as elongation and/or desaturation.
Modified photosynthetic microorganisms, e.g., Cyanobacteria, may comprise one or more exogenous polynucleotides encoding any of the above polypeptides or enzymes involved in fatty acid synthesis. In particular embodiments, the enzyme is an acetyl-CoA carboxylase or a variant or functional fragment thereof.
As used herein, an “acetyl CoA carboxylase” gene of the present invention includes any polynucleotide sequence encoding amino acids, such as protein, polypeptide or peptide, obtainable from any cell source, which demonstrates the ability to catalyze the carboxylation of acetyl-CoA to produce malonyl-CoA under enzyme reactive conditions, and further includes any naturally-occurring or non-naturally occurring variants of an acetyl CoA carboxylase sequence having such ability.
Acetyl-CoA carboxylase (ACCase) is a biotin-dependent enzyme that catalyses the irreversible carboxylation of acetyl-CoA to produce malonyl-CoA through its two catalytic activities, biotin carboxylase (BC) and carboxyltransferase (CT). The biotin carboxylase (BC) domain catalyzes the first step of the reaction: the carboxylation of the biotin prosthetic group that is covalently linked to the biotin carboxyl carrier protein (BCCP) domain. In the second step of the reaction, the carboxyltransferase (CT) domain catalyzes the transfer of the carboxyl group from (carboxy) biotin to acetyl-CoA. Formation of malonyl-CoA by Acetyl-CoA carboxylase (ACCase) represents the commitment step for fatty acid synthesis, because malonyl-CoA has no metabolic role other than serving as a precursor to fatty acids. Because of this reason, acetyl-CoA Carboxylase represents a pivotal enzyme in the synthesis of fatty acids.
In most prokaryotes, ACCase is a multi-subunit enzyme, whereas in most eukaryotes it is a large, multi-domain enzyme. In yeast, the crystal structure of the CT domain of yeast ACCase has been determined at 2.7 A resolution (Zhang et al., Science, 299:2064-2067 (2003). This structure contains two domains, which share the same backbone fold. This fold belongs to the crotonase/ClpP family of proteins, with a b-b-a superhelix. The CT domain contains many insertions on its surface, which are important for the dimerization of ACCase. The active site of the enzyme is located at the dimer interface.
Although Cyanobacteria, such as Synechococcus, express a native ACCase enzyme, these bacteria typically do not produce or accumulate significant amounts of fatty acids. For example, Synechococcus in the wild accumulates fatty acids in the form of lipid membranes to a total of about 4% by dry weight.
Given the role of ACCase in the commitment step of fatty acid biosynthesis, embodiments of the present invention include methods of increasing the production of fatty acid biosynthesis, and, thus, lipid production, in Cyanobacteria by introducing one or more polynucleotides that encode an ACC enzyme that is exogenous to the Cyanobacterium's native genome. Embodiments of the present invention also include a modified Cyanobacterium, and compositions comprising said Cyanobacterium, comprising one or more polynucleotides that encode an ACCase enzyme that is exogenous to the Cyanobacterium's native genome.
A polynucleotide encoding an ACCase enzyme may be isolated or obtained from any organism, such as any prokaryotic or eukaryotic organism that contains an endogenous ACCase gene. Examples of eukaryotic organisms having an ACCase 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 S. cerevisiae and Schizosaccharomyces pombe). In certain embodiments, the ACCase encoding polynucleotide sequence is obtained from Saccharomyces cerevisiae. 
Examples of prokaryotic organisms that may be utilized to obtain a polynucleotide encoding an enzyme having ACCase activity include, but are not limited to, Escherichia coli, Legionella pneumophila, Listeria monocytogenes, Streptococcus pneumoniae, Bacillus subtilis, Ruminococcus obeum ATCC 29174, marine gamma proteobacterium HTCC2080, Roseovarius sp. HTCC2601, Oceanicola granulosus HTCC2516, Bacteroides caccae ATCC 43185, Vibrio alginolyticus 12G01, Pseudoalteromonas tunicata D2, Marinobacter sp. ELB17, marine gamma proteobacterium HTCC2143, Roseobacter sp. SK209-2-6, Oceanicola batsensis HTCC2597, Rhizobium leguminosarum bv. trifolii WSM1325, Nitrobacter sp. Nb-311A, Chloroflexus aggregans DSM 9485, Chlorobaculum parvum, Chloroherpeton thalassium, Acinetobacter baumannii, Geobacillus, and Stenotrophomonas maltophilia, among others.
Polynucleotides and Vectors
The present invention includes modified photosynthetic microorganisms comprising one or more exogenous polynucleotides encoding a polypeptide associated with triglyceride or fatty acid biosynthesis, or a variant or a functional fragment thereof. Accordingly, the present invention utilizes isolated polynucleotides that encode the various triglyceride and lipid biosynthesis enzymes utilized herein, such as diacylglycerol acyltransferase, phosphatidate phosphatase, and acetyl-CoA carboxylase, in addition to nucleotide sequences that encode any functional naturally-occurring variants or fragments (i.e., allelic variants, orthologs, splice variants) or non-naturally occurring variants or fragments of these native enzymes (i.e., optimized by engineering), as well as compositions comprising such polynucleotides, including, e.g., cloning and expression vectors.
As used herein, the terms “DNA” and “polynucleotide” and “nucleic acid” refer to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment encoding a polypeptide refers to a DNA segment that contains one or more coding sequences yet is substantially isolated away from, or purified free from, total genomic DNA of the species from which the DNA segment is obtained. Included within the terms “DNA segment” and “polynucleotide” are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phagemids, phage, viruses, and the like.
As will be understood by those skilled in the art, the polynucleotide sequences of this invention can include genomic sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, peptides and the like. Such segments may be naturally isolated, or modified synthetically by the hand of man.
As will be recognized by the skilled artisan, polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present invention, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.
Polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes a diacylglycerol acyltransferase, a phosphatidate phosphatase, an acetyl-CoA carboxylase, or a portion thereof) or may comprise a variant, or a biological functional equivalent of such a sequence. Polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions, as further described below, preferably such that the enzymatic activity of the encoded polypeptide is not substantially diminished relative to the unmodified polypeptide. The effect on the enzymatic activity of the encoded polypeptide may generally be assessed as described herein.
In certain embodiments of the present invention, a polynucleotide encodes a DGAT comprising of consisting of a polypeptide sequence set forth in any one of SEQ ID NOs:1, 14, 15, or 18, or a fragment or variant thereof. SEQ ID NO:1 is the sequence of DGATn; SEQ ID NO: 14 is the sequence of Streptomyces coelicolor DGAT (ScoDGAT or SDGAT); SEQ ID NO:15 is the sequence of Alcanivorax borkumensis DGAT (AboDGAT); and SEQ ID NO:18 is the sequence of DGATd. In certain embodiments of the present invention, a DGAT polynucleotide comprises or consists of a polynucleotide sequence set forth in any one of SEQ ID NOs:4, 7, 16, 17, or 19, or a fragment or variant thereof. SEQ ID NO:4 is a codon-optimized for expression in Cyanbacteria sequence that encodes DGATn; SEQ ID NO: 7 has homology to SEQ ID NO:4; SEQ ID NO:16 is a codon-optimized for expression in Cyanobacteria sequence that encodes ScoDGAT; SEQ ID NO:17 is a codon-optimized for expression in Cyanobacteria sequence that encodes AboDGAT; and SEQ ID NO:19 is a codon-optimized for expression in Cyanobacteria sequence that encodes DGATd. DGATn and DGATd correspond to Acinetobacter baylii DGAT and a modified form thereof, which includes two additional amino acid residues immediately following the initiator methionine.
In certain embodiments of the present invention, a polynucleotide encodes a phosphatidate phosphatase comprising or consisting of a polypeptide sequence set forth in SEQ ID NO:2, or a fragment or variant thereof. In particular embodiments, a phosphatidate phosphatase polynucleotide comprises or consists of a polynucleotide sequence set forth in SEQ ID NO:5 or SEQ ID NO:8, or a fragment or variant thereof. SEQ ID NO:2 is the sequence of Saccharomyces cerevisiae phosphatidate phosphatase (yPah1), and SEQ ID NO:5 is a codon-optimized for expression in Cyanobacteria sequence that encodes yPAH1.
In certain embodiments of the present invention, a polynucleotide encodes an acetyl-CoA carboxylase (ACCase) comprising or consisting of a polypeptide sequence set forth in any of SEQ ID NOs:3, 20, 21, 22, 23, or 28, or a fragment or variant thereof. In particular embodiments, a ACCase polynucleotide comprises or consists of a polynucleotide sequence set forth in any of SEQ ID NOs:6, 9, 24, 25, 26, 27, or 29, or a fragment or variant thereof. SEQ ID NO:3 is the sequence of Saccharomyces cerevisiae acetyl-CoA carboxylase (yAcc1); and SEQ ID NO:6 is a codon-optimized for expression in Cyanobacteria sequence that encodes yACC1. SEQ ID NO:20 is Synechococcus sp. PCC 7002 AccA; SEQ ID NO:21 is Synechococcus sp. PCC 7002 AccB; SEQ ID NO:22 is Synechococcus sp. PCC 7002 AccC; and SEQ ID NO:23 is Synechococcus sp. PCC 7002 AccD. SEQ ID NO:24 encodes Synechococcus sp. PCC 7002 AccA; SEQ ID NO:25 encodes Synechococcus sp. PCC 7002 AccB; SEQ ID NO:26 encodes Synechococcus sp. PCC 7002 AccC; and SEQ ID NO:27 encodes Synechococcus sp. PCC 7002 AccD. SEQ ID NO:28 is a Triticum aestivum ACCase; and SEQ ID NO:29 encodes this Triticum aestivum ACCase.
In certain embodiments, the present invention provides isolated polynucleotides comprising various lengths of contiguous stretches of sequence identical to or complementary to a diacylglycerol acyltransferase, a phosphatidate phosphatase, or an acetyl-CoA carboxylase, wherein the isolated polynucleotides encode a biologically active, truncated enzyme.
Exemplary nucleotide sequences that encode the enzymes of the application encompass full-length diacylglycerol acyltransferases, phosphatidate phosphatases, and/or acetyl-CoA carboxylases, as well as portions of the full-length or substantially full-length nucleotide sequences of these genes or their transcripts or DNA copies of these transcripts. Portions of a nucleotide sequence may encode polypeptide portions or segments that retain the biological activity of the reference polypeptide. A portion of a nucleotide sequence that encodes a biologically active fragment of an enzyme provided herein may encode at least about 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 300, 400, 500, 600, or more contiguous amino acid residues, almost up to the total number of amino acids present in a full-length enzyme. It will be readily understood that “intermediate lengths,” in this context and in all other contexts used herein, means any length between the quoted values, such as 101, 102, 103, etc.; 151, 152, 153, etc.; 201, 202, 203, etc.
The polynucleotides of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a polynucleotide fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
The invention also contemplates variants of the nucleotide sequences of the diacylglycerol acyltransferases, phosphatidate phosphatases, and acetyl-CoA carboxylases utilized according to methods and compositions provided herein. Nucleic acid variants can be naturally-occurring, such as allelic variants (same locus), homologs (different locus), and orthologs (different organism) or can be non naturally-occurring. Naturally occurring variants such as these can be identified and isolated using well-known molecular biology techniques including, for example, various polymerase chain reaction (PCR) and hybridization-based techniques as known in the art. Naturally occurring variants can be isolated from any organism that encodes one or more genes having a diacylglycerol acyltransferase activity, a phosphatidate phosphatase activity, and/or a acetyl-CoA carboxylase activity. Embodiments of the present invention, therefore, encompass cyanobacteria comprising such naturally occurring polynucleotide variants.
Non-naturally occurring variants can be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms. The variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. In certain aspects, non-naturally occurring variants may have been optimized for use in Cyanobacteria, such as by engineering and screening the enzymes for increased activity, stability, or any other desirable feature. The variations can produce both conservative and non-conservative amino acid substitutions (as compared to the originally encoded product). For nucleotide sequences, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of a reference polypeptide. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis but which still encode a biologically active polypeptide, such as a polypeptide having either a diacylglycerol acyltransferase activity, a phosphatidate phosphatase activity, or a acetyl-CoA carboxylase activity. Generally, variants of a particular reference nucleotide sequence will have at least about 30%, 40% 50%, 55%, 60%, 65%, 70%, generally at least about 75%, 80%, 85%, desirably about 90% to 95% or more, and more suitably about 98% or more sequence identity to that particular nucleotide sequence as determined by sequence alignment programs described elsewhere herein using default parameters.
Known diacylglycerol acyltransferase, phosphatidate phosphatase, and/or acetyl-CoA carboxylase nucleotide sequences can be used to isolate corresponding sequences and alleles from other organisms, particularly other microorganisms. Methods are readily available in the art for the hybridization of nucleic acid sequences. Coding sequences from other organisms may be isolated according to well known techniques based on their sequence identity with the coding sequences set forth herein. In these techniques all or part of the known coding sequence is used as a probe which selectively hybridizes to other reference coding sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism.
Accordingly, the present invention also contemplates polynucleotides that hybridize to reference diacylglycerol acyltransferase, phosphatidate phosphatase, or a acetyl-CoA carboxylase nucleotide sequences, or to their complements, under stringency conditions described below. As used herein, the term “hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions” describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Ausubel et al., (1998, supra), Sections 6.3.1-6.3.6. Aqueous and non-aqueous methods are described in that reference and either can be used.
Reference herein to “low stringency” conditions include and encompass from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization at 42° C., and at least about 1 M to at least about 2 M salt for washing at 42° C. Low stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% SDS for hybridization at 65° C., and (i) 2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 5% SDS for washing at room temperature. One embodiment of low stringency conditions includes hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes can be increased to 55° C. for low stringency conditions).
“Medium stringency” conditions include and encompass from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization at 42° C., and at least about 0.1 M to at least about 0.2 M salt for washing at 55° C. Medium stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% SDS for hybridization at 65° C., and (i) 2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 5% SDS for washing at 60-65° C. One embodiment of medium stringency conditions includes hybridizing in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.
“High stringency” conditions include and encompass from at least about 31% v/v to at least about 50% v/v formamide and from about 0.01 M to about 0.15 M salt for hybridization at 42° C., and about 0.01 M to about 0.02 M salt for washing at 55° C. High stringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% SDS for hybridization at 65° C., and (i) 0.2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 1% SDS for washing at a temperature in excess of 65° C. One embodiment of high stringency conditions includes hybridizing in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.
In certain embodiments, a diacylglycerol acyltransferase enzyme, a phosphatidate phosphatase enzyme, or a acetyl-CoA carboxylase enzyme is encoded by a polynucleotide that hybridizes to a disclosed nucleotide sequence under very high stringency conditions. One embodiment of very high stringency conditions includes hybridizing in 0.5 M sodium phosphate, 7% SDS at 65° C., followed by one or more washes in 0.2×SSC, 1% SDS at 65° C.
Other stringency conditions are well known in the art and a skilled addressee will recognize that various factors can be manipulated to optimize the specificity of the hybridization. Optimization of the stringency of the final washes can serve to ensure a high degree of hybridization. For detailed examples, see Ausubel et al., supra at pages 2.10.1 to 2.10.16 and Sambrook et al. (1989, supra) at sections 1.101 to 1.104.
While stringent washes are typically carried out at temperatures from about 42° C. to 68° C., one skilled in the art will appreciate that other temperatures may be suitable for stringent conditions. Maximum hybridization rate typically occurs at about 20° C. to 25° C. below the Tm for formation of a DNA-DNA hybrid. It is well known in the art that the Tm is the melting temperature, or temperature at which two complementary polynucleotide sequences dissociate. Methods for estimating Tm are well known in the art (see Ausubel et al., supra at page 2.10.8).
In general, the Tm of a perfectly matched duplex of DNA may be predicted as an approximation by the formula: Tm=81.5+16.6 (log10 M)+0.41 (% G+C)−0.63 (% formamide)−(600/length) wherein: M is the concentration of Na+, preferably in the range of 0.01 molar to 0.4 molar; % G+C is the sum of guano sine and cytosine bases as a percentage of the total number of bases, within the range between 30% and 75% G+C; % formamide is the percent formamide concentration by volume; length is the number of base pairs in the DNA duplex. The Tm of a duplex DNA decreases by approximately 1° C. with every increase of 1% in the number of randomly mismatched base pairs. Washing is generally carried out at Tm−15° C. for high stringency, or Tm−30° C. for moderate stringency.
In one example of a hybridization procedure, a membrane (e.g., a nitrocellulose membrane or a nylon membrane) containing immobilized DNA is hybridized overnight at 42° C. in a hybridization buffer (50% deionizer formamide, 5×SSC, 5× Reinhardt's solution (0.1% fecal, 0.1% polyvinylpyrollidone and 0.1% bovine serum albumin), 0.1% SDS and 200 mg/mL denatured salmon sperm DNA) containing a labeled probe. The membrane is then subjected to two sequential medium stringency washes (i.e., 2×SSC, 0.1% SDS for 15 min at 45° C., followed by 2×SSC, 0.1% SDS for 15 min at 50° C.), followed by two sequential higher stringency washes (i.e., 0.2×SSC, 0.1% SDS for 12 min at 55° C. followed by 0.2×SSC and 0.1% SDS solution for 12 min at 65-68° C.
Polynucleotides and fusions thereof may be prepared, manipulated and/or expressed using any of a variety of well established techniques known and available in the art. For example, polynucleotide sequences which encode polypeptides of the invention, or fusion proteins or functional equivalents thereof, may be used in recombinant DNA molecules to direct expression of an triglyceride or lipid biosynthesis enzyme in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences that encode substantially the same or a functionally equivalent amino acid sequence may be produced and these sequences may be used to clone and express a given polypeptide.
As will be understood by those of skill in the art, it may be advantageous in some instances to produce polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce a recombinant RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence. Such nucleotides are typically referred to as “codon-optimized.”
Moreover, the polynucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter polypeptide encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, expression and/or activity of the gene product.
In order to express a desired polypeptide, a nucleotide sequence encoding the polypeptide, or a functional equivalent, may be inserted into appropriate expression vector, i.e., a vector that contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding a polypeptide of interest and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook et al., Molecular Cloning, A Laboratory Manual (1989), and Ausubel et al., Current Protocols in Molecular Biology (1989).
A variety of expression vector/host systems are known and may be utilized to contain and express polynucleotide sequences. The polynucleotides of the present invention will typically be introduced and expressed in cyanobacterial systems. As such, the present invention contemplates the use of vector and plasmid systems having regulatory sequences (e.g., promoters and enhancers) that are suitable for use in various cyanobacteria (see, e.g., Koksharova et al. Applied Microbiol Biotechnol 58:123-37, 2002). For example, the promiscuous RSF1010 plasmid provides autonomous replication in several cyanobacteria of the genera Synechocystis and Synechococcus (see, e.g., Mermet-Bouvier et al., Curr Microbiol 26:323-327, 1993). As another example, the pFC1 expression vector is based on the promiscuous plasmid RSF1010. pFC1 harbors the lambda c1857 repressor-encoding gene and pR promoter, followed by the lambda cro ribosome-binding site and ATG translation initiation codon (see, e.g., Mermet-Bouvier et al., Curr Microbiol 28:145-148, 1994). The latter is located within the unique NdeI restriction site (CATATG) of pFC1 and can be exposed after cleavage with this enzyme for in-frame fusion with the protein-coding sequence to be expressed.
The “control elements” or “regulatory sequences” present in an expression vector are those non-translated regions of the vector—enhancers, promoters, 5′ and 3′ untranslated regions—which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. Generally, it is well-known that strong E. coli promoters work well in Cyanobacteria. Also, when cloning in cyanobacterial systems, inducible promoters such as the hybrid lacZ promoter of the PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid (Gibco BRL, Gaithersburg, Md.) and the like may be used. Other vectors containing IPTG inducible promoters, such as pAM1579 and pAM2991trc, may be utilized according to the present invention.
Certain embodiments may employ a temperature inducible system. As one example, an operon with the bacterial phage left-ward promoter (PL) and a temperature sensitive repressor gene CI857 may be employed to produce a temperature inducible system for producing fatty acids and/or triglycerides in Cyanobacteria (see, e.g., U.S. Pat. No. 6,306,639, herein incorporated by reference). It is believed that at a non-permissible temperature (low temperature, 30 degrees Celsius), the repressor binds to the operator sequence, and thus prevents RNA polymerase from initiating transcription at the PL promoter. Therefore, the expression of encoded gene or genes is repressed. When the cell culture is transferred to a permissible temperature (37-42 degrees Celsius), the repressor can not bind to the operator. Under these conditions, RNA polymerase can initiate the transcription of the encoded gene or genes.
In cyanobacterial systems, a number of expression vectors may be selected depending upon the use intended for the expressed polypeptide. When large quantities are needed, vectors which direct high level expression of encoded proteins may be used. For example, overexpression of ACCase enzymes may be utilized to increase fatty acid biosynthesis. Such vectors include, but are not limited to, the multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the sequence encoding the polypeptide of interest may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of β-galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke & Schuster, J. Biol. Chem. 264:5503 5509 (1989)); and the like. pGEX Vectors (Promega, Madison, Wis.) may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
Certain embodiments may employ Cyanobacterial promoters or regulatory operons. In certain embodiments, a promoter may comprise an rbcLS operon of Synechococcus, as described, for example, in Ronen-Tarazi et al. (Plant Physiology 18:1461-1469, 1995), or a cpc operon of Synechocystis sp. strain PCC 6714, as described, for example, in Imashimizu et al. (J Bacteriol. 185:6477-80, 2003). In certain embodiments, the tRNApro gene from Synechococcus may also be utilized as a promoter, as described in Chungjatupornchai et al. (Curr Microbiol. 38:210-216, 1999). Certain embodiments may employ the nirA promoter from Synechococcus sp. strain PCC 7942, which is repressed by ammonium and induced by nitrite (see, e.g., Maeda et al., J. Bacteriol. 180:4080-4088, 1998; and Qi et al., Applied and Environmental Microbiology 71:5678-5684, 2005). The efficiency of expression may be enhanced by the inclusion of enhancers which are appropriate for the particular cyanobacterial cell system which is used, such as those described in the literature.
Specific initiation signals may also be used to achieve more efficient translation of sequences encoding a polypeptide of interest. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding the polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic.
A variety of protocols for detecting and measuring the expression of polynucleotide-encoded products, using either polyclonal or monoclonal antibodies specific for the product are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). These and other assays are described, among other places, in Hampton et al., Serological Methods, a Laboratory Manual (1990) and Maddox et al., J. Exp. Med. 158:1211-1216 (1983). The presence of a desired polynucleotide, such as a diacylglycerol acyltransferase, phosphatidate phosphatase, and/or an acetyl-CoA carboxylase encoding polypeptide, may also be confirmed by PCR.
A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide. Alternatively, the sequences, or any portions thereof may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits. Suitable reporter molecules or labels, which may be used include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
Cyanobacterial host cells transformed with a polynucleotide sequence of interest may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a recombinant cell may be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides of the invention may be designed to contain signal sequences which direct localization of the encoded polypeptide to a desired site within the cell. Other recombinant constructions may be used to join sequences encoding a polypeptide of interest to nucleotide sequence encoding a polypeptide domain which will direct secretion of the encoded protein.
Polypeptides
Embodiments of the present invention contemplate the use of modified photosynthetic microorganisms, e.g., Cyanobacteria, comprising polypeptides having a diacylglycerol acyltransferase activity, a phosphatidate phosphatase activity, and/or an acetyl-CoA carboxylase activity, including truncated, variant and/or modified polypeptides thereof, for increasing lipid production and/or producing triglycerides said Cyanobacteria.
In certain embodiments of the present invention, a DGAT polypeptide comprises or consists of a polypeptide sequence set forth in any one of SEQ ID NOs:1, 14, 15, or 18, or a fragment or variant thereof. SEQ ID NO:1 is the sequence of DGATn; SEQ ID NO: 14 is the sequence of Streptomyces coelicolor DGAT (ScoDGAT or SDGAT); SEQ ID NO:15 is the sequence of Alcanivorax borkumensis DGAT (AboDGAT); and SEQ ID NO:18 is the sequence of DGATd. In certain embodiments of the present invention, a DGAT polypeptide is encoded by a polynucleotide sequence set forth in any one of SEQ ID NOs:4, 7, 16, 17, or 19, or a fragment or variant thereof. SEQ ID NO:4 is a codon-optimized for expression in Cyanbacteria sequence that encodes DGATn; SEQ ID NO: 7 has homology to SEQ ID NO:4; SEQ ID NO:16 is a codon-optimized for expression in Cyanobacteria sequence that encodes ScoDGAT; SEQ ID NO:17 is a codon-optimized for expression in Cyanobacteria sequence that encodes AboDGAT; and SEQ ID NO:19 is a codon-optimized for expression in Cyanobacteria sequence that encodes DGATd.
In certain embodiments of the present invention, a phosphatidate phosphatase polypeptide comprises or consists of a polypeptide sequence set forth in SEQ ID NO:2, or a fragment or variant thereof. In particular embodiments, a phosphatidate phosphatase is encoded by a polynucleotide sequence set forth in SEQ ID NO:5 or SEQ ID NO:8, or a fragment or variant thereof. SEQ ID NO:2 is the sequence of Saccharomyces cerevisiae phosphatidate phosphatase (yPah1), and SEQ ID NO:5 is a codon-optimized for expression in Cyanobacteria sequence that encodes yPAH1.
In certain embodiments of the present invention, an acetyl-CoA carboxylase (ACCase) polypeptide comprises or consists of a polypeptide sequence set forth in any of SEQ ID NOs:3, 20, 21, 22, 23, or 28, or a fragment or variant thereof. In particular embodiments, an ACCase polypeptide is encoded by a polynucleotide sequence set forth in any of SEQ ID NOs:6, 9, 24, 25, 26, 27, or 29, or a fragment or variant thereof. SEQ ID NO:3 is the sequence of Saccharomyces cerevisiae acetyl-CoA carboxylase (yAcc1); and SEQ ID NO:6 is a codon-optimized for expression in Cyanobacteria sequence that encodes yAcc1. SEQ ID NO:20 is Synechococcus sp. PCC 7002 AccA; SEQ ID NO:21 is Synechococcus sp. PCC 7002 AccB; SEQ ID NO:22 is Synechococcus sp. PCC 7002 AccC; and SEQ ID NO:23 is Synechococcus sp. PCC 7002 AccD. SEQ ID NO:24 encodes Synechococcus sp. PCC 7002 AccA; SEQ ID NO:25 encodes Synechococcus sp. PCC 7002 AccB; SEQ ID NO:26 encodes Synechococcus sp. PCC 7002 AccC; and SEQ ID NO:27 encodes Synechococcus sp. PCC 7002 AccD. SEQ ID NO:28 is a T. aestivum ACCase; and SEQ ID NO:29 encodes this Triticum aestivum ACCase.
Variant proteins encompassed by the present application are biologically active, that is, they continue to possess the enzymatic activity of a reference polypeptide. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of a reference diacylglycerol acyltransferase, phosphatidate phosphatase, and/or acetyl-CoA carboxylase polypeptide fragment will have at least 40%, 50%, 60%, 70%, generally at least 75%, 80%, 85%, usually about 90% to 95% or more, and typically about 98% or more sequence similarity or identity with the amino acid sequence for a reference protein as determined by sequence alignment programs described elsewhere herein using default parameters. A biologically active variant of a reference polypeptide may differ from that protein generally by as much 200, 100, 50 or 20 amino acid residues or suitably by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue. In some embodiments, a variant polypeptide differs from the reference sequences in SEQ ID NOS: 1, 2, 3, 6, 8, 10, 12, and 14 by at least one but by less than 15, 10 or 5 amino acid residues. In other embodiments, it differs from the reference sequences by at least one residue but less than 20%, 15%, 10% or 5% of the residues.
A diacylglycerol acyltransferase, phosphatidate phosphatase, or acetyl-CoA carboxylase polypeptide may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82: 488-492), Kunkel et al., (1987, Methods in Enzymol, 154: 367-382), U.S. Pat. No. 4,873,192, Watson, J. D. et al., (“Molecular Biology of the Gene”, Fourth Edition, Benjamin/Cummings, Menlo Park, Calif., 1987) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.).
Methods for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property are known in the art. Such methods are adaptable for rapid screening of the gene libraries generated by combinatorial mutagenesis of diacylglycerol acyltransferase, phosphatidate phosphatase, and/or acetyl-CoA carboxylase polypeptides. Recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify polypeptide variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89: 7811-7815; Delgrave et al., (1993) Protein Engineering, 6: 327-331). Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be desirable as discussed in more detail below.
Polypeptide variants may contain conservative amino acid substitutions at various locations along their sequence, as compared to a reference amino acid sequence. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub-classified as follows:
Acidic: The residue has a negative charge due to loss of H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having an acidic side chain include glutamic acid and aspartic acid.
Basic: The residue has a positive charge due to association with H ion at physiological pH or within one or two pH units thereof (e.g., histidine) and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having a basic side chain include arginine, lysine and histidine.
Charged: The residues are charged at physiological pH and, therefore, include amino acids having acidic or basic side chains (i.e., glutamic acid, aspartic acid, arginine, lysine and histidine).
Hydrophobic: The residues are not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a hydrophobic side chain include tyrosine, valine, isoleucine, leucine, methionine, phenylalanine and tryptophan.
Neutral/polar: The residues are not charged at physiological pH, but the residue is not sufficiently repelled by aqueous solutions so that it would seek inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a neutral/polar side chain include asparagine, glutamine, cysteine, histidine, serine and threonine.
This description also characterizes certain amino acids as “small” since their side chains are not sufficiently large, even if polar groups are lacking, to confer hydrophobicity. With the exception of proline, “small” amino acids are those with four carbons or less when at least one polar group is on the side chain and three carbons or less when not. Amino acids having a small side chain include glycine, serine, alanine and threonine. The gene-encoded secondary amino acid proline is a special case due to its known effects on the secondary conformation of peptide chains. The structure of proline differs from all the other naturally-occurring amino acids in that its side chain is bonded to the nitrogen of the α-amino group, as well as the α-carbon. Several amino acid similarity matrices (e.g., PAM120 matrix and PAM250 matrix as disclosed for example by Dayhoff et al., (1978), A model of evolutionary change in proteins. Matrices for determining distance relationships In M. O. Dayhoff, (ed.), Atlas of protein sequence and structure, Vol. 5, pp. 345-358, National Biomedical Research Foundation, Washington D.C.; and by Gonnet et al., (Science, 256: 14430-1445, 1992), however, include proline in the same group as glycine, serine, alanine and threonine. Accordingly, for the purposes of the present invention, proline is classified as a “small” amino acid.
The degree of attraction or repulsion required for classification as polar or nonpolar is arbitrary and, therefore, amino acids specifically contemplated by the invention have been classified as one or the other. Most amino acids not specifically named can be classified on the basis of known behaviour.
Amino acid residues can be further sub-classified as cyclic or non-cyclic, and aromatic or non-aromatic, self-explanatory classifications with respect to the side-chain substituent groups of the residues, and as small or large. The residue is considered small if it contains a total of four carbon atoms or less, inclusive of the carboxyl carbon, provided an additional polar substituent is present; three or less if not. Small residues are, of course, always non-aromatic. Dependent on their structural properties, amino acid residues may fall in two or more classes. For the naturally-occurring protein amino acids, sub-classification according to this scheme is presented in Table A.







TABLE A







Amino acid sub-classification








Sub-classes
Amino acids





Acidic
Aspartic acid, Glutamic acid


Basic
Noncyclic: Arginine, Lysine; Cyclic: Histidine


Charged
Aspartic acid, Glutamic acid, Arginine, Lysine,



Histidine


Small
Glycine, Serine, Alanine, Threonine, Proline


Polar/neutral
Asparagine, Histidine, Glutamine, Cysteine, Serine,



Threonine


Polar/large
Asparagine, Glutamine


Hydrophobic
Tyrosine, Valine, Isoleucine, Leucine, Methionine,



Phenylalanine, Tryptophan


Aromatic
Tryptophan, Tyrosine, Phenylalanine


Residues that
Glycine and Proline


influence chain


orientation









Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional truncated and/or variant polypeptide can readily be determined by assaying its enzymatic activity, as described herein (see, e.g., Example 3). Conservative substitutions are shown in Table B under the heading of exemplary substitutions. Amino acid substitutions falling within the scope of the invention, are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. After the substitutions are introduced, the variants are screened for biological activity.







TABLE B







Exemplary Amino Acid Substitutions









Original Residue
Exemplary Substitutions
Preferred Substitutions





Ala
Val, Leu, Ile
Val


Arg
Lys, Gln, Asn
Lys


Asn
Gln, His, Lys, Arg
Gln


Asp
Glu
Glu


Cys
Ser
Ser


Gln
Asn, His, Lys,
Asn


Glu
Asp, Lys
Asp


Gly
Pro
Pro


His
Asn, Gln, Lys, Arg
Arg


Ile
Leu, Val, Met, Ala, Phe,
Leu



Norleu


Leu
Norleu, Ile, Val, Met, Ala,
Ile



Phe


Lys
Arg, Gln, Asn
Arg


Met
Leu, Ile, Phe
Leu


Phe
Leu, Val, Ile, Ala
Leu


Pro
Gly
Gly


Ser
Thr
Thr


Thr
Ser
Ser


Trp
Tyr
Tyr


Tyr
Trp, Phe, Thr, Ser
Phe


Val
Ile, Leu, Met, Phe, Ala,
Leu



Norleu









Alternatively, similar amino acids for making conservative substitutions can be grouped into three categories based on the identity of the side chains. The first group includes glutamic acid, aspartic acid, arginine, lysine, histidine, which all have charged side chains; the second group includes glycine, serine, threonine, cysteine, tyrosine, glutamine, asparagine; and the third group includes leucine, isoleucine, valine, alanine, proline, phenylalanine, tryptophan, methionine, as described in Zubay, G., Biochemistry, third edition, Wm. C. Brown Publishers (1993).
Thus, a predicted non-essential amino acid residue in a diacylglycerol acyltransferase, phosphatidate phosphatase, or acetyl-CoA carboxylase polypeptide is typically replaced with another amino acid residue from the same side chain family. Alternatively, mutations can be introduced randomly along all or part of a coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for an activity of the parent polypeptide to identify mutants which retain that activity. Following mutagenesis of the coding sequences, the encoded peptide can be expressed recombinantly and the activity of the peptide can be determined. A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of an embodiment polypeptide without abolishing or substantially altering one or more of its activities. Suitably, the alteration does not substantially abolish one of these activities, for example, the activity is at least 20%, 40%, 60%, 70% or 80% 100%, 500%, 1000% or more of wild-type. An “essential” amino acid residue is a residue that, when altered from the wild-type sequence of a reference polypeptide, results in abolition of an activity of the parent molecule such that less than 20% of the wild-type activity is present. For example, such essential amino acid residues include those that are conserved in diacylglycerol acyltransferase, phosphatidate phosphatase, or acetyl-CoA carboxylase polypeptides across different species, including those sequences that are conserved in the enzymatic sites of polypeptides from various sources.
Accordingly, the present invention also contemplates variants of the naturally-occurring diacylglycerol acyltransferase, phosphatidate phosphatase, or acetyl-CoA carboxylase polypeptide sequences or their biologically-active fragments, wherein the variants are distinguished from the naturally-occurring sequence by the addition, deletion, or substitution of one or more amino acid residues. In general, variants will display at least about 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% similarity or sequence identity to a reference polypeptide sequence. Moreover, sequences differing from the native or parent sequences by the addition, deletion, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids but which retain the properties of a parent or reference polypeptide sequence are contemplated.
In some embodiments, variant polypeptides differ from a reference diacylglycerol acyltransferase, phosphatidate phosphatase, or acetyl-CoA carboxylase polypeptide sequence by at least one but by less than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3 or 2 amino acid residue(s). In other embodiments, variant polypeptides differ from a reference by at least 1% but less than 20%, 15%, 10% or 5% of the residues. (If this comparison requires alignment, the sequences should be aligned for maximum similarity. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.)
In certain embodiments, a variant polypeptide includes an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98% or more sequence identity or similarity to a corresponding sequence of a diacylglycerol acyltransferase, phosphatidate phosphatase, or acetyl-CoA carboxylase reference polypeptide, and retains the enzymatic activity of that reference polypeptide.
Calculations of sequence similarity or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In certain embodiments, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch, (1970, J. Mol. Biol. 48: 444-453) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller (1989, Cabios, 4: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
The nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al., (1990, J. Mol. Biol, 215: 403-10). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997, Nucleic Acids Res, 25: 3389-3402). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
Variants of a diacylglycerol acyltransferase, phosphatidate phosphatase, or acetyl-CoA carboxylase reference polypeptide can be identified by screening combinatorial libraries of mutants of a reference polypeptide. Libraries or fragments e.g., N terminal, C terminal, or internal fragments, of protein coding sequence can be used to generate a variegated population of fragments for screening and subsequent selection of variants of a reference polypeptide.
Methods for screening gene products of combinatorial libraries made by point mutation or truncation, and for screening cDNA libraries for gene products having a selected property are known in the art. Such methods are adaptable for rapid screening of the gene libraries generated by combinatorial mutagenesis of polypeptides.
The present invention also contemplates the use of chimeric or fusion proteins for increasing lipid production and/or producing triglycerides. As used herein, a “chimeric protein” or “fusion protein” includes a diacylglycerol acyltransferase, phosphatidate phosphatase, or acetyl-CoA carboxylase reference polypeptide or polypeptide fragment linked to either another reference polypeptide (e.g., to create multiple fragments), to a non-reference polypeptide, or to both. A “non-reference polypeptide” refers to a “heterologous polypeptide” having an amino acid sequence corresponding to a protein which is different from the diacylglycerol acyltransferase, phosphatidate phosphatase, or acetyl-CoA carboxylase protein sequence, and which is derived from the same or a different organism. The reference polypeptide of the fusion protein can correspond to all or a portion of a biologically active amino acid sequence. In certain embodiments, a fusion protein includes at least one (or two) biologically active portion of an diacylglycerol acyltransferase, phosphatidate phosphatase, or acetyl-CoA carboxylase protein. The polypeptides forming the fusion protein are typically linked C-terminus to N-terminus, although they can also be linked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. The polypeptides of the fusion protein can be in any order.
The fusion partner may be designed and included for essentially any desired purpose provided they do not adversely affect the enzymatic activity of the polypeptide. For example, in one embodiment, a fusion partner may comprise a sequence that assists in expressing the protein (an expression enhancer) at higher yields than the native recombinant protein. Other fusion partners may be selected so as to increase the solubility or stability of the protein or to enable the protein to be targeted to desired intracellular compartments.
The fusion protein can include a moiety which has a high affinity for a ligand. For example, the fusion protein can be a GST-fusion protein in which the diacylglycerol acyltransferase, phosphatidate phosphatase, or acetyl-CoA carboxylase sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification and/or identification of the resulting polypeptide. Alternatively, the fusion protein can be a diacylglycerol acyltransferase, phosphatidate phosphatase, or acetyl-CoA carboxylase protein containing a heterologous signal sequence at its N-terminus. In certain host cells, expression and/or secretion of such proteins can be increased through use of a heterologous signal sequence.
Fusion proteins may generally be prepared using standard techniques. For example, DNA sequences encoding the polypeptide components of a desired fusion may be assembled separately, and ligated into an appropriate expression vector. The 3′ end of the DNA sequence encoding one polypeptide component is ligated, with or without a peptide linker, to the 5′ end of a DNA sequence encoding the second polypeptide component so that the reading frames of the sequences are in phase. This permits translation into a single fusion protein that retains the biological activity of both component polypeptides.
A peptide linker sequence may be employed to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures, if desired. Such a peptide linker sequence is incorporated into the fusion protein using standard techniques well known in the art. Certain peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39 46 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258 8262 (1986); U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. The linker sequence may generally be from 1 to about 50 amino acids in length. Linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
The ligated DNA sequences may be operably linked to suitable transcriptional or translational regulatory elements. The regulatory elements responsible for expression of DNA are located 5′ to the DNA sequence encoding the first polypeptides. Similarly, stop codons required to end translation and transcription termination signals are present 3′ to the DNA sequence encoding the second polypeptide.
In general, polypeptides and fusion polypeptides (as well as their encoding polynucleotides) are isolated. An “isolated” polypeptide or polynucleotide is one that is removed from its original environment. For example, a naturally-occurring protein is isolated if it is separated from some or all of the coexisting materials in the natural system. Preferably, such polypeptides are at least about 90% pure, more preferably at least about 95% pure and most preferably at least about 99% pure. A polynucleotide is considered to be isolated if, for example, it is cloned into a vector that is not a part of the natural environment.
The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.
These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.
EXAMPLES
Example 1
Generation of DGAT and PAP-Expressing Cyanobacteria
Acinetobacter baylii sp. ADP1, a gram-negative TAG forming prokaryote, contains a well-characterized DGAT (AtfA, also referred to herein as ADP1-DGAT). The ADP1-DGAT nucleotide sequence was synthesized and codon optimized for Selongatus PCC 7942 expression using DNA2.0, received in a plasmid, subcloned using established molecular biology techniques into the IPTG-inducible vector pAM2991trc (this vector contains sequences encoding the tact transcriptional repressor, and the pTrc promoter which is repressed by LacI), and recombined into neutral site 1 (NS1) of S. elongatus PCC 7942. Colonies were selected from BG11-spec/strep plates, restreaked for isolation, and tested by PCR for positive colonies. Inducible transcription of the gene was verified by real-time PCR
Saccharomyces cerevisiae contains three characterized phosphatidate phosphatases, one of which is a soluble, non-integral membrane protein, Pah1p (YMR165C). Pah1 plays a major role in the synthesis of TAGs and phospholipids in S. cerevisiae. The Pah1 nucleotide sequence was synthesized and codon optimized for S. elongatus PCC 7942 expression using DNA2.0, received in a plasmid, subcloned using established molecular biology techniques into the IPTG-inducible vector pAM2991trc, and recombined into neutral site 1 (NS1) of S. elongatus PCC 7942. Colonies were selected from BG11-spec/strep plates, restreaked for isolation, and tested by PCR for positive colonies.
A S. elongatus PCC 7942 strain expressing both the ADP1-DGAT and Pah1 genes described above was generated by transforming an ADP1-DGAT expressing strain (ADP1-DGAT subcloned into IPTG-inducible vector pAM1579trc-kanamycin, which recombined in NS2) with the construct carrying Pah1 from NS1 (described above) and selecting transformants on plates containing kanamycin, streptomycin and spectinomycin. Inducible transcription of these genes was verified by real-time PCR.
Example 2
Generation of DGAT and ACCase-Expressing Cyanobacteria
Synechococcus sp. PCC 7002 contains fours genes encoding the four subunits of bacterial acetyl coenzyme A carboxylase (7002 acc). These genes (accA, accB, accC, and accD) were PCR amplified and two synthetic two-gene operons were constructed using splicing by overlap extenstion PCR techniques. Synthetic operon 1 contains accAD and synthetic operon 2 contains accBC. The two synthetic operons were cloned into vector pTG2087 (pAM2314Ftrc3.) The vector pTG2087 contains regions of homology for recombination into neutral site 1 (NS1) of S. elongatus PCC7942, sequences encoding the lacl transcriptional repressor, and the pTrc promoter which is repressed by Lacl. Synthetic 7002 acc operons 1 and 2 were cloned into pTG2087, in two separate sites, under control of the pTrc promoter to generate plasmid pTG2087-7002acc. Clone candidates were sequenced to confirm that there were no PCR-induced mutations in the coding sequence of any of the 7002 acc genes.
pTG2087-7002acc was transformed into S. elongatus PCC 7942 and recombinants into NS1 were selected by plating on BG11 media containing spectinomycin and streptomycin. Transformants that grew out in the presence of antibiotic were streaked for isolated colonies and single colonies were tested for the presence of the 7002 acc genes in NS1 by PCR. Inducible transcription of the 7002 acc genes was verified by real-time PCR.
Functional expression of the 7002 acc genes was tested by the ability to complement a deletion of the endogenous S. elongatus PCC 7942 accD gene. S. elongatus PCC7942 with synthetic operon 1 (7002 accAD) recombined into NS1 was tested for the ability to complement loss of the native S. elongatus accD gene. Successful complementation indicated that the 7002 acc genes were functionally expressed in S. elongatus PCC7942.
The S. elongatus PCC7942-7002 accADBC strain was transformed with vectors containing one of two DGAT genes (either ADP1-DGAT or ScoDGAT from examples 1 and 7) for recombination into NS2. Transformants were selected by plating on media containing kanamycin. The recombination of ADP1-DGAT or ScoDGAT into NS2 was confirmed by PCR. These strains were tested for triglyceride production using the methods described in examples 3 and 4.
S. cerevisiae contains one characterized gene encoding an acetyl-CoA carboxylase (yAcc1), which was isolated from S. cerevisiae, codon optimized for S. elongatus PCC 7942 expression using DNA2.0, received as a plasmid and subcloned into an IPTG-inducible vector pAM2991 generating vector pTG2035. This vector was recombined into neutral site 1 (NS1) of S. elongatus PCC 7942 containing the ADP1-DGAT gene from example 1 recombined into NS2. Colonies were selected from a BG11-spectinomycin and streptomycin plate, restreaked for isolation, and tested by PCR for positive clones. These strains were tested for triglyceride production using the methods described in examples 3 and 4.
Example 3
Increased Fatty Acid Production in Cyanobacteria
ADP1-DGAT-expressing Cyanobacteria from Example 1 was tested for the ability to produce increased levels of fatty acids. Induction of ADP1-DGAT positive clones was carried out by the addition of 1 mM IPTG when culture reached an OD750=0.2. Samples were taken at 24 hours after induction, and analyzed for lipid content by gas chromatography (GC).
As seen in FIG. 1, GC results showed a 2-fold increase in lipid content for IPTG-induced DGAT compared to un-induced vector control.
Example 4
Triglyceride Production in Cyanobacteria and TLC Analysis of DGATs
Several enzymes with acylCoA: diacylglycerol acyltransferase activity have been described in the literature, and a number of homologs were identified by conducting homology searches of publicly available DNA and protein databases. Several DGAT homologs were synthesized, optimized for expression in Synechoccocus elontatus PCC 7942, and integrated into its genome via homologous recombination as described in Example 1.
A modified version of ADP1-DGAT from Example 1 was cloned into vector pTG2087 (pAM2314Ftrc3), a neutral site 1 expression vector described in Example 2. In this version, the 6 bases immediately following the ATG start codon of the ADP1-DGAT gene from Example 1 were deleted. This strain was named ADP1-DGATn.
Streptomyces coelicolor is a gram-positive TAG forming prokaryote that contains a well-characterized DGAT. The Streptomyces-DGAT (ScoDGAT) nucleotide sequence was synthesized and codon optimized for S. elongatus PCC 7942 expression using DNA2.0. The gene was received in a plasmid, subcloned using established molecular biology techniques into pTG2087 (pAM2314Ftrc3), a neutral site 1 expression vector described in Example 2, and recombined into neutral site 1 (NS1) of S. elongatus PCC 7942. Colonies were selected from BG11-spec/strep plates, restreaked for isolation and tested by PCR for positive colonies. Inducible transcription of this gene was verified by real-time PCR.
Alcanivorax borkumensis is a marine protobacteria gamma TAG forming prokaryote that contains a well-characterized DGAT (atfA1). The Alcanivorax-DGAT (AboDGAT) nucleotide sequence was synthesized and codon optimized for S. elongatus PCC 7942 expression using DNA2.0. The gene was received in a plasmid, subcloned using established molecular biology techniques into pTG2087 (pAM2314Ftrc3), a neutral site 1 expression vector described in Example 2, and recombined into neutral site 1 (NS1) of S. elongatus PCC 7942. Colonies were selected from BG11-spec/strep plates, restreaked for isolation and tested by PCR for positive colonies. Inducible transcription of this gene was verified by real-time PCR.
Induction experiments for ADP1-DGAT, ADP1-DGATn, ScoDGAT and AboDGAT were performed as described in Example 3. Samples were collected at 24 hours post-induction, and total lipid extracts were prepared for TLC analysis as follows. Pellets were resuspended in 100 ul of water, to which 375 ul of a 1:2 mixture of chloroform to methanol was added. Cells were extracted with frequent vortexing for 10 minutes. To this was added 125 ul of chloroform, and the extract was vortexed for another minute. Finally, phase separation was produced by adding 125 ul of 1M NaCl, with another 1 minute of vortexing. To speed separation, the samples were centrifuged in a clinical centrifuge for 10 minutes at an rcf of 1930. The organic phase was removed to a new tube and dried down in a vacuum dryer. The dry lipid extract was resuspended in 40 ul of a 2:1 chloroform:methanol mixture, and either a 6 ul aliquot or the entire volume was applied to TLC plates (200-um thick silica plates). Chromatography was run using a mobile phase comprised of 75% n-hexane, 25% diethylether acidified with 1 ml of glacial acetic acid per 100 ml solvent mixture. Completed runs were dried, and the lipids were imaged with primuline (50 mg/L dissolved in an 80% acetone solution). Images were recorded digitally using a hand-held UV lamp to excite the primuline stained plate.
As shown in FIG. 2, all four DGAT genes resulted in TAG production when expressed in Cyanobacteria. Moreover, increases in fatty acids were observed in ADP1-DGAT, ADP1-DGATn, and AboDGAT expressing strains but not in ScoDGAT. These results demonstrate that heterologous expression of several DGATs in Cyanobacteria results in TAG formation.
Example 5
Triacylglceride and Free Fatty Acid Accumulation in S. elongatus 
The S. elongatus PCC 7942 ADP1-DGAT expressing strain described in Example 1 was grown under induction conditions as described in Example 3, and total lipid extracts prepared as described in Example 4 were subjected to HPLC analysis. 40 microL of total lipid extracts were analyzed on a Shimadzu Prominence UFLC (Ultra Fast Liquid Chromatograph) connected to an ESA Bioscience Corona CAD Plus detector (Charged Aerosol Detector). A Hypersil Gold C8 3 μm 150×4.6 mm column at 0.8 mL/min flow rate was used. A binary gradient system with mobile phase A: methanol/water/acetic acid (750:250:4) and mobile phase B: acetonitrile/methanol/THF/acetic acid (500:375:125:4) was used. The results of a typical run are shown in FIG. 3, in which the y axis indicates the intensity of the peaks for the different lipid species, and the x axis indicates the corresponding retention time. Three major lipid groups, free fatty acids (FFAs), phospholipids, and TAGs are shown, as indentified using representative standards of these lipid species (not shown). As can be seen, the induced strain produced TAGs. In the un-induced strain, these were undetectable. Thus, exogenous expression of DGAT in cyanobacteria results in TAG formation, as shown by TLC.
Example 6
Acyl Chain Composition of TAGs
The ADP1-DGAT and ScoDGAT strains described in Example 1 and 4 were induced for TAG production as described in Example 3. Lipid extracts were prepared, and the non polar lipids were separated on a TLC as per the method described in Example 4. The spots on the TLC plate corresponding to TAGs, as determined by their co-migration with corresponding standards, were extracted from the TLC plates by cutting out a rectangular area encompassing each spot. This material was then subjected to transesterification and GC analysis. As can be seen in FIG. 4, the fatty acid composition of the TAGS produced by these two strains differed in that the TAGs produced by the ADP1-DGAT strain consisted of mixtures of C18 and C16 acyl chains (FIG. 4A), whereas the TAGs from ScoDGAT consisted of mixtures of C16 and C22 acyl chains (FIG. 4B). This highlights the different acyl change specificities of these two DGAT enzymes and supports the introduction of two or more different DGATs into modified Cyanobacteria to generate multiple different TAGs.
Example 7
Triacylglyceride Production in Cyanobacteria
A gene encoding a DGAT was introduced into a different strain, Synechcocystis sp. strain PCC 6803 (hereafter referred to as PCC 6803), to determine if DGAT expression correlated with TAG production outside of S. elongatus PCC 7942. Two mutants were constructed in Synechocystis sp. strain PCC 6803. The first mutant carried a gene encoding ADP1-DGAT under control of the Ptrc promoter, a locus encoding kanamycin resistance (nptA) and the lactose repressor (lacl). As a negative control, a strain was constructed that carried nptA and lacl, but not the ADP1-DGAT gene. Both constructs were built in a neutral site vector devised for use in PCC 6803.
This vector directs recombination into a neutral site in PCC 6803, a region between two convergently transcribed native genes that have been described in the literature as non-essential. The mutagenesis generally followed the protocols of Eaton-Rye (Methods in Molecular Biology, Vol 24, p 309-323), except that transformants were plated on plain BG-11 plates and subjected to increasing kanamycin concentrations by injecting concentrated kanamycin under the agar pad at 12 and 36 hours. Successful incorporation of the ADP1-DGAT gene was demonstrated using colony PCR. The plates used for mutagenesis were comprised of 1×BG-11 (Pasteur formulation), 1.25% Bactoagar, and sodium thiosulfate to 3 g/L.
Transformants confirmed to have the correct insertions were grown to late exponential phase, aliquots of the cultures were centrifuged, washed in BG-11, re-pelleted, and resuspended to 50 ml of BG-11 with kanamycin. Half the cultures were induced with IPTG at a final concentration of 1 mM. Typically, samples were taken at 0, 3 and 6 days of induction. Pelleted samples were stored at −80° C.
Methods similar to those described in Example 4 were used to perform TLC and determine how the expression of ADP1-DGAT affected the TAG content in PCC 6803. As shown in FIG. 5A, strains that did not carry ADP1-DGAT did not exhibit TAGs on TLC, while strains that did carry ADP1-DGAT produced TAGs. These experiments demonstrated that the engineered DGAT-dependent production of TAGs first seen in S. elongatus sp. strain PCC 7942 is not unique to that strain, but instead is a general property of cyanobacteria engineered to contain a diacyl-glycerol acyltransferase activity.
Example 8
Generation of a Salt-Tolerant Synechococcus Elongatus PCC 7942 Strain
S. elongatus sp. PCC 7942 is a freshwater, Cyanobacterium that does not ordinarily grow well in high salts. This example describes the generation of a Cyanobacterium S. elongatus PCC 7942 mutant that grows in salt or brackish water and can produce TAGs. In addition to being able to grow in freshwater media (BG11), this strain can grow in salt concentrations of up to 3% (in BG11 media).
The mutant S. elongatus PCC 7942 strain was selected through several rounds of growth and dilution in high salt (1.5% NaCl) liquid media. Once a salt tolerant strain emerged (after several months of selection), it was tested for its ability to retain salt tolerance after several rounds of growth on BG11 plates made from freshwater. The resulting salt tolerant strain grew to equal density in either BG11 or 1.5% NaCl-BG11 for up to 14 days. The salt tolerant strain grew indistinguishably from wildtype in BG11, but showed a sharp increase in growth compared to wildtype PCC 7942 in media containing NaCl.
An ADP1-DGAT expressing salt tolerant strain of S. elongatus PCC 7942 was generated by transforming the salt strain described above with the ADP1-DGAT construct described in Example 1. This ADP1-DGAT salt tolerant strain showed a growth advantage over the ADP1-DGAT non-salt tolerant strain in media containing up to 3% salt and produced similar amounts of TAGs as the ADP1-DGAT parental non salt tolerant strain (FIG. 5B). This strain could be useful in production settings where it may be advantageous to use brackish water or seawater.

## Claims

1. A modified Cyanobacterium comprising:
an exogenous polynucleotide encoding a diacylglycerol acyltransferase (DGAT) that uses acyl-ACP as a substrate, wherein the modified Cyanobacterium produces a triglyceride, a wax ester or both.

2. The modified Cyanobacterium according to claim 1, wherein the DGAT is a prokaryotic DGAT.

3. The modified Cyanobacterium according to claim 2, wherein said DGAT is an Acinetobacter DGAT, a Streptomyces DGAT, or an Alcanivorax DGAT.

4. The modified Cyanobacterium according to claim 1, wherein said Cyanobacterium is selected from the group consisting of: Synechoccus elongatus PCC 7942, a salt tolerant variant of Synechoccus elongatus PCC 7942, Synechococcus PCC 7002, and Synechocystis PCC 6803.

5. The modified Cyanobacterium according to claim 1, wherein said Cyanobacterium is salt tolerant.

6. A method for producing a triglyceride, a wax ester or both, comprising culturing the Cyanobacterium according to claim 1 to produce a triglyceride, a wax ester or both.

7. The method according to claim 6, wherein the DGAT is a prokaryotic DGAT.

8. The method according to claim 7, wherein said DGAT is an Acinetobacter DGAT, a Streptomyces DGAT, or an Alcanivorax DGAT.

9. The method according to claim 6, wherein said Cyanobacterium is selected from the group consisting of: S. elongatus PCC 7942, a salt tolerant variant of S. elongatus PCC 7942, Synechococcus PCC 7002, and Synechocystis PCC 6803.

10. The method according to claim 6, wherein said Cyanobacterium is salt tolerant.

11. A method for making a modified Cyanobacterium, comprising modifying a Cyanobacterium by introducing into the Cyanobacterium an exogenous polynucleotide encoding a diacylglycerol acyltransferase (DGAT) that uses acyl-ACP as a substrate, wherein said Cyanobacterium produces a triglyceride, a wax ester or both.

12. The method according to claim 11, wherein said DGAT is an Acinetobacter DGAT, a Streptomyces DGAT, or an Alcanivorax DGAT.

13. The method according to claim 11, wherein said Cyanobacterium is salt tolerant.

14. The method according to claim 11, wherein said Cyanobacterium is selected from the group consisting of: S. elongatus PCC 7942, a salt tolerant variant of S. elongatus PCC 7942, Synechococcus PCC 7002, and Synechocystis PCC 6803.