# Patent

## Title
Mutant Δ-5 desaturases mutated in the heme-binding motif (HPGG) and their use in making polyunsaturated fatty acids

## Abstract

The present invention relates to mutant Δ5 desaturases, which have the ability to convert dihomo-γ-linolenic acid [DGLA; 20:3 ω-6] to arachidonic acid [ARA; 20:4 ω-6] and/or eicosatetraenoic acid [ETA; 20:4 ω-3] to eicosapentaenoic acid [EPA; 20:5 ω-3] and which possess at least one mutation within the HPGG motif of the cytochome b5-like domain. Isolated nucleic acid fragments and recombinant constructs comprising such fragments encoding Δ5 desaturases, along with a method of making long chain polyunsaturated fatty acids [“PUFAs”] using these mutant Δ5 desaturases in oleaginous yeast, are disclosed.

## Background
This application is a divisional application of pending application Ser. No. 12/562,161, filed Sep. 18, 2009, which claims the benefit of U.S. Provisional Application No. 61/098,333, filed Sep. 19, 2008, both of which prior applications are herein incorporated by reference in their entirety.


FIELD OF THE INVENTION
This invention is in the field of biotechnology. More specifically, this invention pertains to the creation of nucleic acid fragments encoding mutant Δ5 fatty acid desaturase enzymes (wherein at least one mutation occurs within the HPGG motif of the cytochrome b5-like domain) and the use of these desaturases in making long-chain polyunsaturated fatty acids [“PUFAs”].
BACKGROUND OF THE INVENTION
A variety of different hosts including plants, algae, fungi, stramenopiles and yeast are being investigated as means for commercial polyunsaturated fatty acid [“PUFA”] production. Genetic engineering has demonstrated that the natural abilities of some hosts (even those natively limited to linoleic acid [LA; 18:2 ω-6] and α-linolenic acid [ALA; 18:3 ω-3] fatty acid production) can be substantially altered to result in high-level production of various long-chain ω-3/ω-6 PUFAs. Whether this is the result of natural abilities or recombinant technology, production of arachidonic acid [ARA; 20:4 ω-6], eicosapentaenoic acid [EPA; 20:5 ω-3] and docosahexaenoic acid [DHA; 22:6 ω-3] may all require expression of a Δ5 desaturase.
Most Δ5 desaturase enzymes identified thus far have the primary ability to convert dihomo-γ-linolenic acid [DGLA; 20:3 ω-6] to ARA, with secondary activity in converting eicosatetraenoic acid [ETA; 20:4 ω-3] to EPA. Numerous Δ5 desaturases have been disclosed in both the open literature and the patent literature. General characteristics of Δ5 desaturases, based on desaturase evolution, are well-described by P. Sperling et al. (Prostaglandins Leukot. Essent. Fatty Acids, 68:73-95 (2003). Along with Δ6, Δ8 and Δ4 desaturases, Δ5 desaturases are known as long-chain PUFA “front-end” desaturases (wherein desaturation occurs between a pre-existing double bond and the carboxyl terminus of the fatty acid's acyl group, as opposed to methyl-directed desaturation). These desaturases are characterized by three histidine boxes [H(X)3-4H (SEQ ID NOs:1 and 2), H(X)2-3HH (SEQ ID NOs:3 and 4) and H/Q(X)2-3HH (SEQ ID NOs:5 and 6)] and are members of the cytochrome b5 fusion superfamily, since they possess a fused cytochrome b5 domain at their N-terminus which serves as an electron donor. The cytochrome b5 domain also contains a conserved heme-binding motif (i.e., a histidine-proline-glycine-glycine sequence or “HPGG” [SEQ ID NO:180] sequence), despite divergence of the remaining cytochrome b5 domain sequences. These motif sequences are the subject of U.S. Pat. No. 5,972,664.
A number of studies have suggested that the HPGG motif is implicated in enzyme activity. Sayanova, O. et al. (Plant Physiol., 121:641 (1999)) performed site-directed mutagenesis to replace the histidine residue of the HPGG motif with an alanine residue in the Δ6 desaturase of borage. The mutant enzyme was expressed in Arabidopsis; however, no enzymatic activity could be measured, suggesting that the cytochrome b5 domain of the desaturase was important for function. A similar study was performed in a rat Δ6 desaturase, where an alanine for histidine substitution was engineered within the HPGG motif. The mutated protein also had no activity (Guillou, H., et al., J. Lipid Res., 45:32-40 (2004)). Most recently, Hongsthong, A. et al. (Appl. Microbiol. Biotechnol., 72:1192-1201 (2006)) reported substitution of the histidine residue of the HPGG motif with an alanine residue in the Δ6 desaturase of Spirulina. As with previous reports, the mutation rendered the mutant enzyme unable to produce GLA in E. coli, suggesting that the cytochrome b5 domain was important for activity and that alterations in this motif will result in diminished enzyme activity. Although Δ5 desaturase enzymes are relatively common and well characterized, there remains a need for enzymes that are efficiently expressed at high levels in production host cells capable of making PUFAs.
The problem to be solved therefore is to discover new Δ5 desaturase enzymes having high activity that are well suited for integration into PUFA biosynthetic pathways in commercially useful host cells. Applicants have solved the stated problem through the unexpected discovery that alterations in the HPGG motif of the cytochrome b5 domain of various Δ5 desaturases resulted in up to 38% improvement in enzymatic activity, based on the conversion of DGLA to ARA.
SUMMARY OF THE INVENTION
The present invention relates to new genetic constructs encoding polypeptides having Δ5 desaturase activity, and their use in bacteria, yeast, algae, euglenoids, stramenopiles, oomycetes and fungi for the production of PUFAs.
Accordingly provided herein is a mutant polypeptide having Δ5 desaturase activity comprising an amino acid motif selected from the group consisting of: SEQ ID NO:183 (His-Gly-Gly-Gly or HGGG), SEQ ID NO:184 (His-His-Gly-Gly or HHGG), SEQ ID NO:186 (His-Cys-Gly-Gly or HCGG), SEQ ID NO:187 (His-Trp-Gly-Gly or HWGG) and SEQ ID NO:185 (His-Pro-Gly-Ser or HPGS). Preferred mutant Δ5 desaturase polypeptides are those that demonstrate a dihomo-γ-linolenic acid to arachidonic acid conversion efficiency that is greater than the dihomo-γ-linolenic acid to arachidonic acid conversion efficiency of the parent polypeptide from which the mutant was derived.
In a second embodiment provided herein is an isolated nucleic acid molecule substantially encoding the polypeptide of the invention.
In a third embodiment provided herein is a microbial host cell expressing the polypeptide of the invention.
In a fourth embodiment provided herein is a method for the production of arachidonic acid comprising growing a microbial host cell expressing the polypeptide of claim 1 in the presence of dihomo-γ-linolenic acid, wherein the dihomo-γ-linolenic acid is converted to arachidonic acid.
In a fifth embodiment provided herein is a method of the production of eicosapentaenoic acid comprising growing a microbial host cell expressing the polypeptide of claim 1 in the presence of eicosatetraenoic acid, wherein the eicosatetraenoic acid is converted to eicosapentaenoic acid.



BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS
FIG. 1A and FIG. 1B illustrate the ω-3/ω-6 fatty acid biosynthetic pathway, and should be viewed together when considering the description of this pathway below.
FIG. 2 provides plasmid maps for the following: (A) pDMW369; and, (B) pZUF17.



The invention can be more fully understood from the following detailed description and the accompanying sequence descriptions, which form a part of this application.
The following sequences comply with 37 C.F.R. §1.821-1.825 (“Requirements for patent applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5 (a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.
SEQ ID NOs:7-19, 58, 97-100, 139, 140 and 179-195 are ORFs encoding genes or proteins (or portions thereof), or plasmids, as identified in Table 1.







TABLE 1







Summary Of Nucleic Acid And Protein SEQ ID Numbers










Nucleic acid
Protein


Description
SEQ ID NO.
SEQ ID NO.












His-rich motif: H(X)3H
—
1


His-rich motif: H(X)4H
—
2


His-rich motif: H(X)2HH
—
3


His-rich motif: H(X)3HH
—
4


His-rich motif: (H/Q)(X)2HH
—
5


His-rich motif: (H/Q)(X)3HH
—
6


Euglena gracilis Δ5 desaturase (“EgD5”)
7 (1350 bp) 
8 (449 AA)


Synthetic Δ5 desaturase, derived from Euglena
9 (1350 bp) 
10 (449 AA)


gracilis, codon-optimized for expression in 




Yarrowia lipolytica (“EgD5S”)




Euglena anabaena Δ5 desaturase (“EaD5”)
11 (1362 bp)
12 (454 AA)


Synthetic Δ5 desaturase, derived from Euglena
13 (1362 bp)
14 (454 AA)


anabaena, codon-optimized for expression in




Yarrowia lipolytica (“EaD5S”)




Peridinium sp. CCMP626 Δ5 desaturase
15 (1392 bp)
16 (463 AA)


(“RD5”)




Synthetic Δ5 desaturase, derived from 
17 (1392 bp)
18 (463 AA)


Peridinium sp. CCMP626, codon-optimized for 




expression in Yarrowia lipolytica (“RD5S”)




Plasmid pDMW369
19 (8438 bp)
—


mutant Δ5 desaturase EgD5S-HXGG (i.e.,
—
58 (449 AA)


comprising either a HGGG or a HHGG motif)




mutant Δ5 desaturase EgD5S-HPGS (i.e.,
—
97 (449 AA)


comprising a HPGS motif)




Plasmid pZUFmEaD5S
98 (8357 bp)
—


Plasmid pZUF17
99 (8165 bp)
—


Plasmid pEaD5S
100
—



(3983 bp)



mutant Δ5 desaturase EaD5S-HCGG (i.e.,
—
139


comprising a HCGG motif)

(454 AA)


Plasmid pZURD5S
140
—



(8480 bp)



mutant Δ5 desaturase RD5S-HXGG (i.e., 
—
179


comprising either a HCGG or a HWGG motif)

(463 AA)


HPGG motif
—
180


HXGG motif
—
181


HPGX motif
—
182


HGGG motif
—
183


HHGG motif
—
184


HPGS motif
—
185


HCGG motif
—
186


HWGG motif
—
187


HAGG motif
—
188


HPGA motif
—
189


mutant Δ5 desaturase EgD5S-HGGG
190
—



(1350 bp)



mutant Δ5 desaturase EgD5S-HHGG
191
—



(1350 bp)



mutant Δ5 desaturase EgD5S-HPGS
192
—



(1350 bp)



mutant Δ5 desaturase EaD5S-HCGG
193
—



(1365 bp)



mutant Δ5 desaturase RD5S-HCGG
194
—



(1392 bp)



mutant Δ5 desaturase RD5S-HWGG
194
—



(1392 bp)










SEQ ID NOs:20-57 correspond to oligonucleotide primers utilized to individually mutate the proline residue of the HPGG motif of EgD5S by site-directed mutagenesis.
SEQ ID NOs:59-96 correspond to oligonucleotide primers utilized to individually mutate the second glycine residue of the HPGG motif of EgD5S by site-directed mutagenesis.
SEQ ID NOs:101-138 correspond to oligonucleotide primers utilized to individually mutate the proline residue of the HPGG motif of EaD5S by site-directed mutagenesis.
SEQ ID NOs:141-178 correspond to oligonucleotide primers utilized to individually mutate the proline residue of the HPGG motif of RD5S by site-directed mutagenesis.
DETAILED DESCRIPTION OF THE INVENTION
New mutant Δ5 desaturase enzymes and genes encoding the same that may be used for the manipulation of biochemical pathways for the production of healthful PUFAs are disclosed herein. These mutant Δ5 desaturases possess at least one mutation within the HPGG motif (SEQ ID NO:180) of the cytochome b5 domain.
PUFAs, or derivatives thereof, are used as dietary substitutes, or supplements, particularly infant formulas, for patients undergoing intravenous feeding or for preventing or treating malnutrition. Alternatively, the purified PUFAs (or derivatives thereof) may be incorporated into cooking oils, fats or margarines formulated so that in normal use the recipient would receive the desired amount for dietary supplementation. The PUFAs may also be incorporated into infant formulas, nutritional supplements or other food products and may find use as anti-inflammatory or cholesterol lowering agents. Optionally, the compositions may be used for pharmaceutical use, either human or veterinary.
All patent and non-patent literature cited herein is hereby incorporated by reference.
In this disclosure, a number of terms and abbreviations are used. The following definitions are provided.
“Open reading frame” is abbreviated “ORF”.
“Polymerase chain reaction” is abbreviated “PCR”.
“American Type Culture Collection” is abbreviated “ATCC”.
“Polyunsaturated fatty acid(s)” is abbreviated “PUFA(s)”.
“Triacylglycerols” are abbreviated “TAGs”.
“Total fatty acids” are abbreviated as “TFAs”. The term “invention” or “present invention” as used herein is not meant to be limiting to any one specific embodiment of the invention but applies generally to any and all embodiments of the invention as described in the claims and specification.
The term “fatty acids” refers to long chain aliphatic acids (alkanoic acids) of varying chain lengths, from about C12 to C22, although both longer and shorter chain-length acids are known. The predominant chain lengths are between C16 and C22. The structure of a fatty acid is represented by a simple notation system of “X:Y”, where X is the total number of carbon [“C”] atoms in the particular fatty acid and Y is the number of double bonds. Additional details concerning the differentiation between “saturated fatty acids” versus “unsaturated fatty acids”, “monounsaturated fatty acids” versus “polyunsaturated fatty acids” [“PUFAs”], and “omega-6 fatty acids” [“ω-6” or “n-6”] versus “omega-3 fatty acids” [“ω-3”] or [“n-3”] are provided in U.S. Pat. No. 7,238,482, which is hereby incorporated herein by reference.
Nomenclature used to describe PUFAs herein is shown below in Table 2. In the column titled “Shorthand Notation”, the omega-reference system is used to indicate the number of carbons, the number of double bonds and the position of the double bond closest to the omega carbon, counting from the omega carbon (which is numbered 1 for this purpose). The remainder of the Table summarizes the common names of ω-3 and ω-6 fatty acids and their precursors, the abbreviations that will be used throughout the specification and the chemical name of each compound.







TABLE 2







Nomenclature Of Polyunsaturated Fatty Acids And Precursors













Shorthand


Common Name
Abbreviation
Chemical Name
Notation





Myristic
—
tetradecanoic
14:0


Palmitic
Palmitate
hexadecanoic
16:0


Palmitoleic
—
9-hexadecenoic
16:1


Stearic
—
octadecanoic
18:0


Oleic
—
cis-9-octadecenoic
18:1


Linoleic
LA
cis-9, 12-octadecadienoic
18:2 ω-6


γ-Linolenic
GLA
cis-6, 9, 12-octadecatrienoic
18:3 ω-6


Eicosadienoic
EDA
cis-11, 14-eicosadienoic
20:2 ω-6


Dihomo-γ-
DGLA
cis-8, 11, 14-eicosatrienoic
20:3 ω-6


Linolenic





Arachidonic
ARA
cis-5, 8, 11, 14-
20:4 ω-6




eicosatetraenoic



α-Linolenic
ALA
cis-9, 12, 15-
18:3 ω-3




octadecatrienoic



Stearidonic
STA
cis-6, 9, 12, 15-
18:4 ω-3




octadecatetraenoic



Eicosatrienoic
ETrA
cis-11, 14, 17-eicosatrienoic
20:3 ω-3


Sciadonic
SCI
cis-5, 11, 14-eicosatrienoic
20:3b ω-6


Juniperonic
JUP
cis-5, 11, 14, 17-
20:4b ω-3




eicosatetraenoic



Eicosa-
ETA
cis-8, 11, 14, 17-
20:4 ω-3


tetraenoic

eicosatetraenoic



Eicosa-
EPA
cis-5, 8, 11, 14, 17-
20:5 ω-3


pentaenoic

eicosapentaenoic



Docosa-
DTA
cis-7, 10, 13, 16-
22:4 ω-6


tetraenoic

docosatetraenoic



Docosa-
DPAn-6
cis-4, 7, 10, 13, 16-
22:5 ω-6


pentaenoic

docosapentaenoic



Docosa-
DPA
cis-7, 10, 13, 16, 19-
22:5 ω-3


pentaenoic

docosapentaenoic



Docosa-
DHA
cis-4, 7, 10, 13, 16, 19-
22:6 ω-3


hexaenoic

docosahexaenoic









Although the ω-3/ω-6 PUFAs listed in Table 2 are the most likely to be accumulated in the oil fractions of microbial hosts using the methods described herein, this list should not be construed as limiting or as complete.

The term “oil” refers to a lipid substance that is liquid at 25° C. and usually polyunsaturated. In oleaginous organisms, oil constitutes a major part of the total lipid. “Oil” is composed primarily of triacylglycerols [“TAGs”] but may also contain other neutral lipids, phospholipids and free fatty acids. The fatty acid composition in the oil and the fatty acid composition of the total lipid are generally similar; thus, an increase or decrease in the concentration of PUFAs in the total lipid will correspond with an increase or decrease in the concentration of PUFAs in the oil, and vice versa.
“Neutral lipids” refer to those lipids commonly found in cells in lipid bodies as storage fats and are so called because at cellular pH, the lipids bear no charged groups. Generally, they are completely non-polar with no affinity for water. Neutral lipids generally refer to mono-, di-, and/or triesters of glycerol with fatty acids, also called monoacylglycerol, diacylglycerol or triacylglycerol, respectively, or collectively, acylglycerols. A hydrolysis reaction must occur to release free fatty acids from acylglycerols.
The term “triacylglycerols” [“TAGs”] refers to neutral lipids composed of three fatty acyl residues esterified to a glycerol molecule. TAGs can contain long chain PUFAs and saturated fatty acids, as well as shorter chain saturated and unsaturated fatty acids.
The term “total fatty acids” [“TFAs”] herein refer to the sum of all cellular fatty acids that can be derivitized to fatty acid methyl esters [“FAMEs”] by the base transesterification method (as known in the art) in a given sample, which may be the biomass or oil, for example. Thus, total fatty acids include fatty acids from neutral lipid fractions (including diacylglycerols, monoacylglycerols and TAGs) and from polar lipid fractions (including the phosphatidylcholine and phosphatidylethanolamine fractions) but not free fatty acids.
The term “total lipid content” of cells is a measure of TFAs as a percent of the dry cell weight [“DCW”], athough total lipid content can be approximated as a measure of FAMEs as a percent of the DCW [“FAMEs % DCW”]. Thus, total lipid content [“TFAs % DCW”] is equivalent to, e.g., milligrams of total fatty acids per 100 milligrams of DCW.
The concentration of a fatty acid in the total lipid is expressed herein as a weight percent of TFAs [“% TFAs”], e.g., milligrams of the given fatty acid per 100 milligrams of TFAs. Unless otherwise specifically stated in the disclosure herein, reference to the percent of a given fatty acid with respect to total lipids is equivalent to concentration of the fatty acid as % TFAs, e.g., % EPA of total lipids is equivalent to EPA % TFAs.
The terms “lipid profile” and “lipid composition” are interchangeable and refer to the amount of individual fatty acids contained in a particular lipid fraction, such as in the total lipid or the oil, wherein the amount is expressed as a weight percent of TFAs. The sum of each individual fatty acid present in the mixture should be 100.
The term “PUFA biosynthetic pathway” refers to a metabolic process that converts oleic acid to ω-6 fatty acids such as LA, EDA, GLA, DGLA, ARA, DTA and DPAn-6 and ω-3 fatty acids such as ALA, STA, ETrA, ETA, EPA, DPA and DHA. This process is well described in the literature. See e.g., U.S. Pat. Appl. Pub. No. 2006-0115881-A1. Briefly, this process involves elongation of the carbon chain through the addition of carbon atoms and desaturation of the molecule through the addition of double bonds, via a series of special elongation and desaturation enzymes termed “PUFA biosynthetic pathway enzymes” that are present in the endoplasmic reticulum membrane. More specifically, “PUFA biosynthetic pathway enzymes” refer to any of the following enzymes (and genes which encode said enzymes) associated with the biosynthesis of a PUFA, including: Δ4 desaturase, Δ5 desaturase, Δ6 desaturase, Δ12 desaturase, Δ15 desaturase, Δ17 desaturase, Δ9 desaturase, Δ8 desaturase, Δ9 elongase, C14/16 elongase, C16/18 elongase, C18/20 elongase and/or C20/22 elongase.
The term “desaturase” refers to a polypeptide that can desaturate, i.e., introduce a double bond, in one or more fatty acids to produce a fatty acid or precursor of interest. Despite use of the omega-reference system throughout the specification to refer to specific fatty acids, it is more convenient to indicate the activity of a desaturase by counting from the carboxyl end of the substrate using the delta-system. Of particular interest herein are Δ5 desaturases that desaturate a fatty acid between the fifth and sixth carbon atom numbered from the carboxyl-terminal end of the molecule and that can, for example, catalyze the conversion of DGLA to ARA and/or ETA to EPA. Other fatty acid desaturases include, for example: Δ8 desaturases, Δ6 desaturases, Δ4 desaturases, Δ12 desaturases, Δ15 desaturases, Δ17 desaturases and Δ9 desaturases. In the art, Δ15 and Δ17 desaturases are also occasionally referred to as “omega-3 desaturases”, “w-3 desaturases” and/or “ω-3 desaturases”, based on their ability to convert ω-6 fatty acids into their ω-3 counterparts (e.g., conversion of LA into ALA and ARA into EPA, respectively). It may be desirable to empirically determine the specificity of a particular fatty acid desaturase by transforming a suitable host with the gene for the fatty acid desaturase and determining its effect on the fatty acid profile of the host.
The term “EgD5” refers to a Δ5 desaturase enzyme (SEQ ID NO:8) isolated from Euglena gracilis, encoded by SEQ ID NO:7 herein. Similarly, the term “EgD5S” refers to a synthetic Δ5 desaturase derived from E. gracilis that is codon-optimized for expression in Yarrowia lipolytica (i.e., SEQ ID NOs:9 and 10). Further details concerning EgD5 and EgD5S are described in Intl. App. Pub. No. WO 2007/136671.
The term “EaD5” refers to a Δ5 desaturase enzyme (SEQ ID NO:12) isolated from Euglena anabaena, encoded by SEQ ID NO:11 herein. Similarly, the term “EaD5S” refers to a synthetic Δ5 desaturase derived from E. anabaena that is codon-optimized for expression in Yarrowia lipolytica (i.e., SEQ ID NOs:13 and 14). Further details concerning EaD5 and EaD5S are described in U.S. Pat. Appl. Pub. No. 2008-0274521-A1.
The term “RD5” refers to a Δ5 desaturase enzyme (SEQ ID NO:16) isolated from Peridinium sp. CCMP626, encoded by SEQ ID NO:15 herein. Similarly, the term “RD5S” refers to a synthetic Δ5 desaturase derived from Peridinium sp. CCMP626 that is codon-optimized for expression in Yarrowia lipolytica (i.e., SEQ ID NOs:17 and 18). Further details concerning RD5 and RD5S are described in Intl. App. Pub. No. WO 2007/136646.
The term “conserved domain” or “motif” means a set of amino acids conserved at specific positions along an aligned sequence of evolutionarily related proteins. While amino acids at other positions can vary between homologous proteins, amino acids that are highly conserved at specific positions indicate amino acids that are essential in the structure, the stability, or the activity of a protein. Because they are identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers, or “signatures”, to determine if a protein with a newly determined sequence belongs to a previously identified protein family. Motifs that are universally found in Δ5 desaturase enzymes of animal, plants and fungi include three histidine boxes (i.e., H(X)3-4H [SEQ ID NOs:1 and 2], H(X)2-3HH [SEQ ID NOs:3 and 4] and H/Q(X)2-3HH [SEQ ID NOs:5 and 6]) and a heme-binding motif (i.e., His-Pro-Gly-Gly or HPGG [SEQ ID NO:180]) within the fused cytochrome b5 domain at the N-terminus.
The term “mutant Δ5 desaturase” refers to a Δ5 desaturase as described herein that has at least one mutation within the HPGG motif (SEQ ID NO:180) of the cytochrome b5 domain, wherein said mutation results in an amino acid substitution, either conservative or non-conservative. Although the mutation(s) may include any amino acid substitution, the mutant Δ5 desaturase preferably comprises a mutant motif selected from the group consisting of His-Xaa-Gly-Gly or “HXGG” (SEQ ID NO:181) and His-Pro-Gly-Xaa or “HPGX” (SEQ ID NO:182) and the Δ5 desaturase activity of the mutant Δ5 desaturase is at least about functionally equivalent to the Δ5 desaturase activity of the wildtype Δ5 desaturase. More preferred, the mutant motif is selected from the group consisting of: SEQ ID NO:183 (His-Gly-Gly-Gly or “HGGG”), SEQ ID NO:184 (His-His-Gly-Gly or “HHGG”), SEQ ID NO:186 (His-Cys-Gly-Gly or “HCGG”), SEQ ID NO:187 (His-Trp-Gly-Gly or “HWGG”) and SEQ ID NO:185 (His-Pro-Gly-Ser or “HPGS”). See, e.g., the Δ5 desaturases set forth as SEQ ID NO:58, SEQ ID NO:97, SEQ ID NO:139 and SEQ ID NO:179.
Each “mutant Δ5 desaturase” has a “corresponding wildtype Δ5 desaturase”. Specifically, the mutant Δ5 desaturase and corresponding wildtype Δ5 desaturase share identical amino acid sequences, with the exception that the wildtype will comprise a HPGG motif (SEQ ID NO:180) within the cytochrome b5 domain, while the mutant will comprise at least one mutation within this motif (as described above).
A mutant Δ5 desaturase is “at least about functionally equivalent” to the corresponding wildtype Δ5 desaturase when enzymatic activity and specific selectivity of the mutant Δ5 sequence are comparable to that of the corresponding wildtype Δ5 desaturase. Thus, a functionally equivalent mutant Δ5 desaturase will possess Δ5 desaturase activity that is not substantially reduced with respect to that of the corresponding wildtype Δ5 desaturase when the “conversion efficiency” of each enzyme is compared (i.e., a mutant Δ5 desaturase will have at least about 50-75%, preferably at least about 75-85%, more preferably at least about 85-95%, and most preferably at least about 95% of the enzymatic activity of the wildtype Δ5 desaturase). The Δ5 desaturase activity of the two polypeptides may be substantially identical. Preferably, the mutant Δ5 desaturase will have increased enzymatic activity and specific selectivity when compared to that of the corresponding wildtype Δ5 desaturase, i.e., having at least about 101-105%, more preferably at least about 106-115% and most preferably at least about 116-125% of the enzymatic activity of the wildtype Δ5 desaturase.
The terms “conversion efficiency” and “percent substrate conversion” refer to the efficiency by which a particular enzyme (e.g., a desaturase) can convert substrate to product. The conversion efficiency is measured according to the following formula: ([product]/[substrate+product])*100. Thus, “DGLA to ARA conversion efficiency” refers to the conversion efficiency by which the substrate, DGLA, is converted to the product, ARA.
The term “elongase” refers to a polypeptide that can elongate a fatty acid carbon chain to produce an acid 2 carbons longer than the fatty acid substrate that the elongase acts upon. This process of elongation occurs in a multi-step mechanism in association with fatty acid synthase, as described in U.S. Pat. App. Pub. No. 2005/0132442. Examples of reactions catalyzed by elongase systems are the conversion of GLA to DGLA, STA to ETA and EPA to DPA.
In general, the substrate selectivity of elongases is somewhat broad but segregated by both chain length and the degree and type of unsaturation. For example, a C14/16 elongase will utilize a C14 substrate (e.g., myristic acid), a C16/18 elongase will utilize a C16 substrate (e.g., palmitate), a C18/20 elongase will utilize a C18 substrate (e.g., GLA, STA, LA, ALA) and a C20/22 elongase [also referred to as a Δ5 elongase] will utilize a C20 substrate (e.g., ARA, EPA). For the purposes herein, two distinct types of C18/20 elongases can be defined: a Δ6 elongase will catalyze conversion of GLA and STA to DGLA and ETA, respectively, while a Δ9 elongase is able to catalyze the conversion of LA and ALA to EDA and ETrA, respectively.
It is important to note that some elongases have broad specificity and thus a single enzyme may be capable of catalyzing several elongase reactions e.g., thereby acting as both a C16/18 elongase and a C18/20 elongase. It may be desirable to empirically determine the specificity of a fatty acid elongase by transforming a suitable host with the gene for the fatty acid elongase and determining its effect on the fatty acid profile of the host.
The term “oleaginous” refers to those organisms that tend to store their energy source in the form of oil (Weete, In: Fungal Lipid Biochemistry, 2nd Ed., Plenum, 1980). Generally, the cellular oil content of oleaginous microorganisms follows a sigmoid curve, wherein the concentration of lipid increases until it reaches a maximum at the late logarithmic or early stationary growth phase and then gradually decreases during the late stationary and death phases (Yongmanitchai and Ward, Appl. Environ. Microbiol., 57:419-25 (1991)). It is common for oleaginous microorganisms to accumulate in excess of about 25% of their dry cell weight as oil.
The term “oleaginous yeast” refers to those microorganisms classified as yeasts that can make oil. Examples of oleaginous yeast include, but are no means limited to, the following genera: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces. Alternatively, organisms classified as yeasts that are engineered to make more than 25% of their dry cell weight as oil are also “oleaginous”.
The term “amino acid” will refer to the basic chemical structural unit of a protein or polypeptide. The amino acids are identified by either the one-letter code or the three-letter codes for amino acids, in conformity with the IUPAC-IYUB standards described in Nucleic Acids Research, 13:3021-3030 (1985) and in the Biochemical Journal, 219 (2):345-373 (1984).
The term “conservative amino acid substitution” refers to a substitution of an amino acid residue in a given protein with another amino acid, without altering the chemical or functional nature of that protein. For example, it is well known in the art that alterations in a gene that result in the production of a chemically equivalent amino acid at a given site (but do not affect the structural and functional properties of the encoded, folded protein) are common. For the purposes herein, “conservative amino acid substitutions” are defined as exchanges within one of the following five groups:

    
    
        1. Small aliphatic, nonpolar or slightly polar residues: Ala [A], Ser [S], Thr [T] (Pro [P], Gly [G]);
        2. Polar, negatively charged residues and their amides: Asp [D], Asn [N], Glu [E], Gln [Q];
        3. Polar, positively charged residues: His [H], Arg [R], Lys [K];
        4. Large aliphatic, nonpolar residues: Met [M], Leu [L], Ile [I], Val [V] (Cys [C]); and
        5. Large aromatic residues: Phe [F], Tyr [Y], Trp [W].

Thus, Ala, a slightly hydrophobic amino acid, may be substituted by another less hydrophobic residue (e.g., Gly). Similarly, changes which result in substitution of one negatively charged residue for another (e.g., Asp for Glu) or one positively charged residue for another (e.g., Lys for Arg) can also be expected to produce a functionally equivalent product. As such, conservative amino acid substitutions generally maintain: the structure of the polypeptide backbone in the area of the substitution; the charge or hydrophobicity of the molecule at the target site; or, the bulk of the side chain. Additionally, in many cases, alterations of the N-terminal and C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein.

    
    


The term “non-conservative amino acid substitution” refers to an amino acid substitution that is generally expected to produce the greatest change in protein properties. Thus, for example, a non-conservative amino acid substitution would be one whereby: 1) a hydrophilic residue is substituted for/by a hydrophobic residue (e.g., Ser or Thr for/by Leu, Ile, Val); 2) a Cys or Pro is substituted for/by any other residue; 3) a residue having an electropositive side chain is substituted for/by an electronegative residue (e.g., Lys, Arg or His for/by Asp or Glu); or, 4) a residue having a bulky side chain is substituted for/by one not having a side chain (e.g., Phe for/by Gly). Sometimes, non-conservative amino acid substitutions between two of the five groups will not affect the activity of the encoded protein.
The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acid sequence”, “nucleic acid fragment” and “isolated nucleic acid fragment” are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. Nucleotides (usually found in their 5′-monophosphate form) are referred to by a single letter designation as follows: “A” for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate, “T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” for any nucleotide.
A nucleic acid fragment is “hybridizable” to another nucleic acid fragment, such as a cDNA, genomic DNA, or RNA molecule, when a single-stranded form of the nucleic acid fragment can anneal to the other nucleic acid fragment under the appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989), which is hereby incorporated herein by reference, particularly Chapter 11 and Table 11.1. The conditions of temperature and ionic strength determine the “stringency” of the hybridization. Stringency conditions can be adjusted to screen for moderately similar fragments (such as homologous sequences from distantly related organisms), to highly similar fragments (such as genes that duplicate functional enzymes from closely related organisms). Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C. An additional set of stringent conditions include hybridization at 0.1×SSC, 0.1% SDS, 65° C. and washes with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS, for example.
Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of thermal melting point [“Tm”] for hybrids of nucleic acids having those sequences. The relative stability, corresponding to higher Tm, of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50-9.51). For hybridizations with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8). In one embodiment the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferably a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably at least about 20 nucleotides; and most preferably the length is at least about 30 nucleotides. Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.
A “substantial portion” of an amino acid or nucleotide sequence is that portion comprising enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as Basic Local Alignment Search Tool [“BLAST”] (Altschul, S. F., et al., J. Mol. Biol., 215:403-410 (1993)). In general, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a “substantial portion” of a nucleotide sequence comprises enough of the sequence to specifically identify and/or isolate a nucleic acid fragment comprising the sequence. The disclosure herein teaches the complete amino acid and nucleotide sequence encoding particular microbial proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above, are encompassed in the present disclosure.
The term “complementary” is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, isolated nucleic acid fragments that are complementary to the complete sequences as reported in the accompanying Sequence Listing, as well as those substantially similar nucleic acid sequences, are encompassed in the present disclosure.
The terms “homology” and “homologous” are used interchangeably. They refer to nucleic acid fragments wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype. These terms also refer to modifications of the nucleic acid fragments such as deletion or insertion of one or more nucleotides that do not substantially alter the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment. It is therefore understood, as those skilled in the art will appreciate, that the invention encompasses more than the specific exemplary sequences.
Moreover, the skilled artisan recognizes that homologous nucleic acid sequences are also defined by their ability to hybridize, under moderately stringent conditions, e.g., 0.5×SSC, 0.1% SDS, 60° C., with the sequences exemplified herein, or to any portion of the nucleotide sequences disclosed herein and which are functionally equivalent thereto. Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms.
The term “selectively hybridizes” includes reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids. Selectively hybridizing sequences typically have about at least 80% sequence identity, or 90% sequence identity, up to and including 100% sequence identity (i.e., fully complementary) with each other.
The term “stringent conditions” or “stringent hybridization conditions” includes reference to conditions under which a probe will selectively hybridize to its target sequence. Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, optionally less than 500 nucleotides in length.
Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% sodium dodecyl sulphate [“SDS”] at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. An additional set of stringent conditions include hybridization at 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS, for example.
Specificity is typically the function of post-hybridization washes, the important factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth et al., Anal. Biochem., 138:267-284 (1984): Tm=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1° C. for each 1% of mismatching; thus, Tm, hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the Tm can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the Tm for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3 or 4° C. lower than the Tm; moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9 or 10° C. lower than the Tm; and, low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15 or 20° C. lower than the Tm. Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45° C. (aqueous solution) or 32° C. (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, New York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995). Hybridization and/or wash conditions can be applied for at least 10, 30, 60, 90, 120 or 240 minutes.
“Sequence identity” or “identity” in the context of nucleic acid or polypeptide sequences refers to the nucleic acid bases or amino acid residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
Thus, “percentage of sequence identity” or “percent identity” refers to the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue 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 and multiplying the results by 100 to yield the percentage of sequence identity.
Methods to determine “percent identity” and “percent similarity” are codified in publicly available computer programs. Percent identity and percent similarity can be readily calculated by known methods, including but not limited to those described in: 1) Computational Molecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2) Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY (1993); 3) Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4) Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and, 5) Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991).
Sequence alignments and percent identity or similarity calculations may be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, the MegAlignTM program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences is performed using the “Clustal method of alignment” which encompasses several varieties of the algorithm including the “Clustal V method of alignment” and the “Clustal W method of alignment” (described by Higgins and Sharp, CABIOS, 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in the MegAlignTM (version 8.0.2) program (supra). After alignment of the sequences using either Clustal program, it is possible to obtain a “percent identity” by viewing the “sequence distances” table in the program.
For multiple alignments using the Clustal V method of alignment, the default values correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal V method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4.
Default parameters for multiple alignment using the Clustal W method of alignment correspond to GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergent Seqs(%)=30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB.
The “BLASTN method of alignment” is an algorithm provided by the National Center for Biotechnology Information [“NCBI”] to compare nucleotide sequences using default parameters, while the “BLASTP method of alignment” is an algorithm provided by the NCBI to compare protein sequences using default parameters.
It is well understood by one skilled in the art that many levels of sequence identity are useful in identifying polypeptides, from other species, wherein such polypeptides have the same or similar function or activity. Suitable nucleic acid fragments, i.e., isolated polynucleotides according to the disclosure herein, encode polypeptides that are at least about 70-85% identical, while more preferred nucleic acid fragments encode amino acid sequences that are at least about 85-95% identical to the amino acid sequences reported herein. Although preferred ranges are described above, useful examples of percent identities include any integer percentage from 50% to 100%, such as 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Also, of interest is any full-length or partial complement of this isolated nucleotide fragment.
Suitable nucleic acid fragments not only have the above homologies but typically encode a polypeptide having at least 50 amino acids, preferably at least 100 amino acids, more preferably at least 150 amino acids, still more preferably at least 200 amino acids, and most preferably at least 250 amino acids.
“Codon degeneracy” refers to the nature in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. Accordingly, described herein is any nucleic acid fragment that encodes all or a substantial portion of the amino acid sequence encoding the instant polypeptides as set forth in SEQ ID NO:58, SEQ ID NO:97, SEQ ID NO:139 and SEQ ID NO:179. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.
“Synthetic genes” can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These oligonucleotide building blocks are annealed and then ligated to form gene segments that are then enzymatically assembled to construct the entire gene. Accordingly, the genes can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell, where sequence information is available. For example, the codon usage profile for Yarrowia lipolytica is provided in U.S. Pat. No. 7,125,672.
“Gene” refers to a nucleic acid fragment that expresses a specific protein, and that may refer to the coding region alone or may include regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign” gene refers to a gene that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, native genes introduced into a new location within the native host, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure. A “codon-optimized gene” is a gene having its frequency of codon usage designed to mimic the frequency of preferred codon usage of the host cell.
“Coding sequence” refers to a DNA sequence that codes for a specific amino acid sequence. “Regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include, but are not limited to: promoters, enhancers, silencers, 5′ untranslated leader sequence (e.g., between the transcription start site and translation initiation codon), introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures.
“Promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters that cause a gene to be expressed at almost all stages of development are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences (especially at their 5′ end) have not been completely defined, DNA fragments of some variation may have identical promoter activity.
The terms “3′ non-coding sequences”, “transcription terminator” and “termination sequences” refer to DNA sequences located downstream of a coding sequence. This includes polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor. The 3′ region can influence the transcription, RNA processing or stability, or translation of the associated coding sequence.
“RNA transcript” refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript. A RNA transcript is referred to as the mature RNA when it is a RNA sequence derived from post-transcriptional processing of the primary transcript. “Messenger RNA” or “mRNA” refers to the RNA that is without introns and that can be translated into protein by the cell. “cDNA” refers to a DNA that is complementary to, and synthesized from, a mRNA template using the enzyme reverse transcriptase. The cDNA can be single-stranded or converted into double-stranded form using the Klenow fragment of DNA polymerase I. “Sense” RNA refers to RNA transcript that includes the mRNA and can be translated into protein within a cell or in vitro. “Antisense RNA” refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA, and that blocks the expression of a target gene (U.S. Pat. No. 5,107,065).
The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence, i.e., the coding sequence is under the transcriptional control of the promoter. Coding sequences can be operably linked to regulatory sequences in a sense or antisense orientation.
The term “recombinant” refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.
The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA. Expression may also refer to translation of mRNA into a protein (either precursor or mature).
“Transformation” refers to the transfer of a nucleic acid molecule into a host organism, resulting in genetically stable inheritance. The nucleic acid molecule may be a plasmid that replicates autonomously, for example, or, it may integrate into the genome of the host organism. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic”, “recombinant”, “transformed” or “transformant” organisms.
The terms “plasmid” and “vector” refer to an extra chromosomal element often carrying genes that are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA fragments. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing an expression cassette(s) into a cell.
The term “expression cassette” refers to a fragment of DNA containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host. Generally, an expression cassette will comprise the coding sequence of a selected gene and regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence that are required for expression of the selected gene product. Thus, an expression cassette is typically composed of: 1) a promoter sequence; 2) a coding sequence [“ORF”]; and, 3) a 3′ untranslated region (i.e., a terminator) that, in eukaryotes, usually contains a polyadenylation site. The expression cassette(s) is usually included within a vector, to facilitate cloning and transformation. Different expression cassettes can be transformed into different organisms including bacteria, yeast, plants and mammalian cells, as long as the correct regulatory sequences are used for each host.
The terms “recombinant construct”, “expression construct”, “chimeric construct”, “construct”, and “recombinant DNA construct” are used interchangeably herein. A recombinant construct comprises an artificial combination of nucleic acid fragments, e.g., regulatory and coding sequences that are not found together in nature. For example, a recombinant DNA construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. Such a construct may be used by itself or may be used in conjunction with a vector. If a vector is used, then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art. For example, a plasmid vector can be used.
The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells comprising any of the isolated nucleic acid fragments described herein. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al., EMBO J., 4:2411-2418 (1985); De Almeida et al., Mol. Gen. Genetics, 218:78-86 (1989)), and thus that multiple events must be screened in order to obtain strains displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, Western analysis of protein expression, or phenotypic analysis, among others.
The term “sequence analysis software” refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. “Sequence analysis software” may be commercially available or independently developed. Typical sequence analysis software will include, but is not limited to: 1) the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.); 2) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol., 215:403-410 (1990)); 3) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4) Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and, 5) the FASTA program incorporating the Smith-Waterman algorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Within this description, whenever sequence analysis software is used for analysis, the analytical results are based on the “default values” of the program referenced, unless otherwise specified. As used herein “default values” will mean any set of values or parameters that originally load with the software when first initialized.
Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989); by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience, Hoboken, N.J. (1987).
In general, lipid accumulation in oleaginous microorganisms is triggered in response to the overall carbon to nitrogen ratio present in the growth medium. This process, leading to the de novo synthesis of free palmitate (16:0) in oleaginous microorganisms, is described in detail in U.S. Pat. No. 7,238,482. Palmitate is the precursor of longer-chain saturated and unsaturated fatty acid derivates.
The metabolic process wherein oleic acid is converted to ω-3/ω-6 fatty acids involves elongation of the carbon chain through the addition of carbon atoms and desaturation of the molecule through the addition of double bonds. This requires a series of special elongation and desaturation enzymes present in the endoplasmic reticulum membrane. However, as seen in FIG. 1 and as described below, multiple alternate pathways exist for production of a specific ω-3/ω-6 fatty acid.
Specifically, FIG. 1 depicts the pathways described below. All pathways require the initial conversion of oleic acid to linoleic acid [“LA”], the first of the ω-6 fatty acids, by a Δ12 desaturase. Then, using the “Δ9 elongase/Δ8 desaturase pathway” and LA as substrate, long-chain ω-6 fatty acids are formed as follows: 1) LA is converted to eicosadienoic acid [“EDA”] by a Δ9 elongase; 2) EDA is converted to dihomo-γ-linolenic acid [“DGLA”] by a Δ8 desaturase; 3) DGLA is converted to arachidonic acid [“ARA”] by a Δ5 desaturase; 4) ARA is converted to docosatetraenoic acid [“DTA”] by a C20/22 elongase; and, 5) DTA is converted to docosapentaenoic acid [“DPAn-6”] by a Δ4 desaturase.
The “Δ9 elongase/Δ8 desaturase pathway” can also use a-linolenic acid [“ALA”] as substrate to produce long-chain ω-3 fatty acids as follows: 1) LA is converted to ALA, the first of the ω-3 fatty acids, by a A15 desaturase; 2) ALA is converted to eicosatrienoic acid [“ETrA”] by a Δ9 elongase; 3) ETrA is converted to eicosatetraenoic acid [“ETA”] by a Δ8 desaturase; 4) ETA is converted to eicosapentaenoic acid [“EPA”] by a Δ5 desaturase; 5) EPA is converted to docosapentaenoic acid [“DPA”] by a C20/22 elongase; and, 6) DPA is converted to docosahexaenoic acid [“DHA”] by a Δ4 desaturase. Optionally, ω-6 fatty acids may be converted to ω-3 fatty acids. For example, ETA and EPA are produced from DGLA and ARA, respectively, by Δ17 desaturase activity.
Alternate pathways for the biosynthesis of ω-3/ω-6 fatty acids utilize a Δ6 desaturase and C18/20 elongase, that is, the “Δ6 desaturase/Δ6 elongase pathway”. More specifically, LA and ALA may be converted to GLA and stearidonic acid [“STA”], respectively, by a Δ6 desaturase; then, a C18/20 elongase converts GLA to DGLA and/or STA to ETA. Downstream PUFAs are subsequently formed as described above.
It is contemplated that the particular functionalities required to be introduced into a specific host organism for production of ω-3/ω-6 fatty acids will depend on the host cell (and its native PUFA profile and/or desaturase/elongase profile), the availability of substrate, and the desired end product(s). For example, expression of the Δ9 elongase/Δ8 desaturase pathway may be preferred in some embodiments, as opposed to expression of the Δ6 desaturase/Δ6 elongase pathway, since PUFAs produced via the former pathway are devoid of GLA and/or STA.
One skilled in the art will be able to identify various candidate genes encoding each of the enzymes desired for ω-3/ω-6 fatty acid biosynthesis. Useful desaturase and elongase sequences may be derived from any source, e.g., isolated from a natural source (from bacteria, algae, fungi, plants, animals, etc.), produced via a semi-synthetic route or synthesized de novo. Although the particular source of the desaturase and elongase genes introduced into the host is not critical, considerations for choosing a specific polypeptide having desaturase or elongase activity include: 1) the substrate specificity of the polypeptide; 2) whether the polypeptide or a component thereof is a rate-limiting enzyme; 3) whether the desaturase or elongase is essential for synthesis of a desired PUFA; 4) co-factors required by the polypeptide; and/or, 5) whether the polypeptide was modified after its production (e.g., by a kinase or a prenyltransferase). The expressed polypeptide preferably has parameters compatible with the biochemical environment of its location in the host cell (see U.S. Pat. No. 7,238,482 for additional details).
It will also be useful to consider the conversion efficiency of each particular desaturase and/or elongase. More specifically, since each enzyme rarely functions with 100% efficiency to convert substrate to product, the final lipid profile of unpurified oils produced in a host cell will typically be a mixture of various PUFAs consisting of the desired ω-3/ω-6 fatty acid, as well as various upstream intermediary PUFAs. Thus, each enzyme's conversion efficiency is also a variable to consider, when optimizing biosynthesis of a desired fatty acid.
With each of the considerations above in mind, candidate genes having the appropriate desaturase and elongase activities (e.g., Δ6 desaturases, C18/20 elongases, Δ5 desaturases, Δ17 desaturases, Δ15 desaturases, Δ9 desaturases, Δ12 desaturases, C14/16 elongases, C16/18 elongases, Δ9 elongases, Δ8 desaturases, Δ4 desaturases and C20/22 elongases) can be identified according to publicly available literature (e.g., GenBank), the patent literature, and experimental analysis of organisms having the ability to produce PUFAs. These genes will be suitable for introduction into a specific host organism, to enable or enhance the organism's synthesis of PUFAs.
Once fatty acids are synthesized within an organism (including saturated and unsaturated fatty acids and short-chain and long-chain fatty acids), they may be incorporated into triacylglycerides [“TAGs”]. TAGs, the primary storage unit for fatty acids, are formed by a series of reactions that involve: 1) the esterification of one molecule of acyl-CoA to glycerol-3-phosphate via an acyltransferase to produce lysophosphatidic acid; 2) the esterification of a second molecule of acyl-CoA via an acyltransferase to yield 1,2-diacylglycerol phosphate (commonly identified as phosphatidic acid); 3) removal of a phosphate by phosphatidic acid phosphatase to yield 1,2-diacylglycerol; and, 4) the addition of a third fatty acid by the action of an acyltransferase to form TAG.
Although Δ5 desaturases contain several conserved sequences (i.e., the three histidine boxes [H(X)3-4H (SEQ ID NOs:1 and 2), H(X)2-3HH (SEQ ID NOs:3 and 4) and H/Q(X)2-3HH (SEQ ID NOs:5 and 6)] and the cytochrome b5 domain), only the heme-binding motif (i.e., His-Pro-Gly-Gly or HPGG [SEQ ID NO:180]) lacks variation within the sequence. It was this motif that was first selected as a target for mutagenesis. The literature suggests that the histidine residue within the HPGG motif is important for function (Sayanova, O. et al., Plant Physiol., 121:641 (1999); Guillou, H., et al., J. Lipid Res., 45:32-40 (2004); Hongsthong, A. et al., Appl. Microbiol. Biotechnol., 72:1192-1201 (2006)). Consequently, substitutions for the histidine residue were avoided in favor of substitutions for the proline and glycine residues.
Site-directed mutagenesis was independently performed on the proline and the second glycine within the HPGG motif of several Δ5 desaturases, followed by expression of the resulting mutant polypeptides and determination of their activities with respect to that of the wildtype enzyme. Surprisingly, various mutant Δ5 desaturases were created comprising amino acid mutant motifs including HXGG (SEQ ID NO:181) and HPGX (SEQ ID NO:182), where the Δ5 desaturase activity of the mutant Δ5 desaturase was functionally equivalent to the Δ5 desaturase activity of the corresponding wildtype Δ5 desaturase.
Oligonucleotide-mediated site-directed mutagenesis was utilized to create specific point mutations within the HPGG motif of various target Δ5 desaturases. Numerous site-directed mutagenesis protocols exist (e.g., Ishii, T. M., et al., Methods Enzymol., 293:53-71 (1998); Ling M. M. and B. H. Robinson, Anal. Biochem., 254:157-178 (1997); Braman J. (ed.) In Vitro Mutagenesis Protocols. 2nd Ed., Humania: Totowa, N.J. (2002); Kunkel T. A., et al., Methods Enzymol., 154:367-382 (1987); Sawano A. and Miyawaki, A. Nucleic Acids Res., 28:e78 (2000)); however, the QuikChange® site-directed mutagenesis kit (Stratagene, La Jolla, Calif.) was selected for use based on its facile implementation and high efficiency. The basic procedure utilizes a supercoiled double-stranded DNA vector with an insert of interest and two synthetic oligonucleotide primers containing the desired mutation. The oligonucleotide primers, each complementary to opposite strands of the vector, are extended during temperature cycling by a DNA polymerase. Incorporation of the oligonucleotide primers generates a mutated plasmid containing staggered nicks. Following temperature cycling, the product is treated with Dpn I endonuclease (specific for methylated and hemi-methylated DNA) as a means to digest the parental DNA template and to select for newly synthesized mutant DNA. The nicked vector DNA containing the desired mutations is then transformed and propagated in an Escherichia coli host.
Using the techniques described above, all possible amino acid substitutions were introduced by site-directed mutagenesis into a synthetic Δ5 desaturase, codon-optimized for expression in Yarrowia lipolytica and derived from Euglena gracilis (i.e., EgD5S; SEQ ID NO:10; U.S. Pat. Appl. Pub. No. 2007-0277266-A1), within a plasmid construct comprising a chimeric FBAIN::EgD5S::Pex20 gene. The mutants were transformed into E. coli, sequenced and then transformed into an appropriate strain of Y. lipolytica previously engineered to produce  ̃18% DGLA. This enabled screening for Δ5 desaturase activity based on GC analyses and the production of ARA.
Many mutations were identified that resulted in a completely non-functional mutant Δ5 desaturase (i.e., having no detectable Δ5 desaturase activity) or a mutant Δ5 desaturase having substantially decreased Δ5 desaturase activity with respect to the non-mutant wildtype enzyme. Surprisingly, however, the preliminary screening identified three amino acid residues that could be substituted for the proline within the HPGG motif and that resulted in approximately equivalent or increased Δ5 desaturase activity in the mutant, when compared to the Δ5 desaturase activity in the corresponding wildtype enzyme (i.e., EgD5S). Thus, this preliminary experimentation suggested that the proline residue within the HPGG motif could be substituted with several amino acids without significantly affecting the Δ5 desaturase activity of EgD5S.
Similar experimentation was performed using EgD5S as the template in site-directed mutagenesis reactions, where the second glycine residue of the HPGG motif was mutated. As described above, analyses of the mutant enzymes determined that 2 amino acid residues were sufficient to replace the wildtype amino acid (i.e., glycine) and resulted in a mutant EgD5S enzyme having equivalent or improved Δ5 desaturase activity.
Once the preliminary analyses of amino acid substitutions in the HPGG motif of EgD5S were completed as described above, a quantitative analysis of those mutants that performed at or above the wildtype EgD5S conversion rate was carried out by re-transformation of each mutant EgD5S-containing plasmid into the host strain of Yarrowia lipolytica. GC analysis of the fatty acid methyl esters [“FAMEs”] produced confirmed that Δ5 desaturase activity of three of the initial five mutants performed with increased activity when compared to the corresponding wildtype EgD5S control.
The above experimental protocol was repeated using a synthetic Δ5 desaturase, codon-optimized for expression in Yarrowia lipolytica and derived from Euglena anabaena (i.e., EaD5S; SEQ ID NO:14; U.S. Pat. Appl. Pub. No. 2008-0274521-A1) and a synthetic Δ5 desaturase, codon-optimized for expression in Y. lipolytica and derived from Peridinium sp. CCMP626 (i.e., RD5S; SEQ ID NO:18; U.S. Pat. Appl. Pub. No. 2007-0271632-A1). Results of all site-directed mutagenesis that resulted in an equivalent or increased Δ5 desaturase activity within the mutant as compared to the corresponding wildtype enzyme (i.e., EgD5S, EaD5S or RD5S) are summarized below in Table 3 (see Examples for additional details). Mutants are designated using the following nomenclature, detailing: 1) Wildtype Enzyme; 2) hyphen (-); 3) mutant HPGG motif. Thus, for example, the mutant enzyme created from the synthetic, codon-optimized EgD5S (i.e., SEQ ID NO:10), having a histidine for proline substitution at amino acid 2 (i.e., a P2 to H substitution) of the HPGG motif is identified as EgD5S-HHGG.







TABLE 3







HPGG Motif Mutants Resulting In Increased Δ5 Desaturase


Activity











Mutant Δ5
SEQ ID NO of
Δ5 Desaturase



Desaturase
Mutant Δ5 Desaturase
Activity







EgD5S-HGGG
SEQ ID NO:58
104.6%



EgD5S-HHGG
SEQ ID NO:58
103.6%



EgD5S-HPGS
SEQ ID NO:97
106.9%



EaD5S-HCGG
SEQ ID NO:139
107.9%



RD5S-HCGG
SEQ ID NO:179
 138.6% *



RD5S-HWGG
SEQ ID NO:179
 113.5% *







* % Increase in the Δ5 desaturase activity of the mutant enzyme with respect to the corresponding wildtype non-mutant enzyme is reported based on initial assay results and not quantitative analysis.






The above data does not suggest a consensus with respect to which particular amino acid substitution is sufficient to produce a mutant polypeptide having increased Δ5 desaturase activity. However, contrary to the above mentioned reports in the art, the data is surprising in demonstrating that substitutions for either the proline or glycine residues may result in an enzyme having higher Δ5 desaturase activity than its wildtype parent. Accordingly, it is within the scope of the present invention to provide a polypeptide having Δ5 desaturase activity comprising an amino acid motif selected from the group consisting of: SEQ ID NO:183 (HGGG), SEQ ID NO:184 (HHGG), SEQ ID NO:186 (HCGG), SEQ ID NO:187 (HWGG) and SEQ ID NO:185 (HPGS). Preferably, the polypeptide has the amino acid sequence selected from the group consisting of: SEQ ID NO:58 (EgD5S-HGGG and EgD5S-HHGG), SEQ ID NO:97 (EgD5S-HPGS), SEQ ID NO:139 (EaD5S-HCGG) and SEQ ID NO:179 (RD5S-HCGG and RD5S-HWGG). More preferably, the mutant Δ5 desaturase: 1) comprises a mutant amino acid motif selected from the group consisting of: SEQ ID NO:183 (HGGG), SEQ ID NO:184 (HHGG), SEQ ID NO:186 (HCGG), SEQ ID NO:187 (HWGG) and SEQ ID NO:185 (HPGS); and, 2) the mutant Δ5 desaturase activity is increased relative to the corresponding wildtype Δ5 desaturase having a HPGG (SEQ ID NO:180) amino acid motif.
It will be appreciated by one of skill in the art that useful mutant Δ5 desaturases are not limited to the mutations described above. Instead, the results suggest that similar experimentation could be performed using any Δ5 wildtype desaturase enzyme having a HPGG (SEQ ID NO:180) motif within the cytochrome b5 domain, to thereby engineer a mutant Δ5 desaturase having increased Δ5 desaturase activity wherein the mutation would result in a mutant HXGG motif (SEQ ID NO:181) or a HPGX (SEQ ID NO:182) motif. A mutant enzyme having increased Δ5 desaturase activity can be useful to enable increased production of ω-3/ω-6 fatty acids.
For example, in vitro mutagenesis and selection or error prone PCR (Leung et al., Techniques, 1:11-15 (1989); Zhou et al., Nucleic Acids Res., 19:6052-6052 (1991); Spee et al., Nucleic Acids Res., 21:777-778 (1993); Melnikov et al., Nucleic Acids Res., 27(4):1056-1062 (Feb. 15, 1999)) could also be employed as a means to obtain mutations of naturally occurring Δ5 desaturase genes, such as EgD5S, EaD5S or RD5S, wherein the mutations may include deletions, insertions and point mutations, or combinations thereof. The principal advantage of error-prone PCR is that all mutations introduced by this method will be within the desired desaturase gene, and any change may be easily controlled by changing the PCR conditions. Alternatively, in vivo mutagenesis may be employed using commercially available materials such as the E. coli XL1-Red strain and Epicurian coli XL1-Red mutator strain from Stratagene (La Jolla, Calif.; Greener and Callahan, Strategies, 7:32-34 (1994)). This strain is deficient in three of the primary DNA repair pathways (mutS, mutD and mutT), resulting in a mutation rate 5000-fold higher than that of wildtype. In vivo mutagenesis does not depend on ligation efficiency (as with error-prone PCR); however, a mutation may occur at any region of the vector and the mutation rates are generally much lower.
It is also contemplated that a mutant Δ5 desaturase enzyme with altered or enhanced Δ5 desaturase activity may be constructed using the method of “gene shuffling” (U.S. Pat. No. 5,605,793; U.S. Pat. No. 5,811,238; U.S. Pat. No. 5,830,721; U.S. Pat. No. 5,837,458). The method of gene shuffling is particularly attractive due to its facile implementation and high rate of mutagenesis. The process of gene shuffling involves the restriction of a gene of interest into fragments of specific size in the presence of additional populations of DNA regions of both similarity to (or difference to) the gene of interest. This pool of fragments will denature and then reanneal to create a mutated gene. The mutated gene is then screened for altered activity. Any of these methods may be used to create Δ5 desaturase mutant enzymes having the substituted motifs HXGG (SEQ ID NO:181) and HPGX (SEQ ID NO:182), which may then be screened for improved activity using the methods described herein.
It is expected that introduction of chimeric genes encoding the mutant Δ5 desaturases described herein (i.e., wherein said mutant Δ5 desaturase comprises at least at one mutation in a region encoding an HPGG amino acid motif and wherein said mutant Δ5 desaturase has increased Δ5 desaturase activity with respect to that of the corresponding wildtype Δ5 desaturase), under the control of the appropriate promoters will result in increased production of ARA and/or EPA in the transformed host organism, respectively. As such, disclosed herein are methods for the direct production of PUFAs comprising exposing a fatty acid substrate (i.e., DGLA and/or ETA) to a mutant desaturase enzyme described herein (e.g., SEQ ID NO:58 [EgD5S-HGGG and EgD5S-HHGG], SEQ ID NO:97 [EgD5S-HPGS], SEQ ID NO:139 [EaD5S-HCGG], SEQ ID NO:179 [RD5S-HCGG and RD5S-HWGG]), such that the substrate is converted to the desired fatty acid product (i.e., ARA and/or EPA, respectively).
More specifically, described herein is a method for the production of ARA in a microbial host cell (e.g., bacteria, yeast, algae, euglenoids, stramenopiles, oomycetes and fungi), wherein the microbial host cell comprises:

    
    
        a) a polypeptide having Δ5 desaturase activity comprising an amino acid motif selected from the group consisting of: SEQ ID NO:183 (HGGG), SEQ ID NO:184 (HHGG), SEQ ID NO:186 (HCGG), SEQ ID NO:187 (HWGG) and SEQ ID NO:185 (HPGS); and,
        b) a source of DGLA;

wherein the host cell is grown under conditions such that the mutant Δ5 desaturase is expressed and the DGLA is converted to ARA, and wherein the ARA is optionally recovered.

    
    


In another method described herein, the mutant Δ5 desaturase may be used for the conversion of ETA to EPA. Accordingly set forth is a method for the production of EPA, wherein the host cell comprises:

    
    
        a) a polypeptide having Δ5 desaturase activity comprising an amino acid motif selected from the group consisting of: SEQ ID NO:183 (HGGG), SEQ ID NO:184 (HHGG), SEQ ID NO:186 (HCGG), SEQ ID NO:187 (HWGG) and SEQ ID NO:185 (HPGS); and,
        b) a source of ETA;

wherein the host cell is grown under conditions such that the mutant Δ5 desaturase is expressed and the ETA is converted to EPA, and wherein the EPA is optionally recovered.

    
    


Alternatively, each mutant Δ5 desaturase gene and its corresponding enzyme product described herein can be used indirectly for the production of various ω-6 and ω-3 PUFAs (see FIG. 1; U.S. Pat. No. 7,238,482; Intl. App. Pub. No. WO 2007/136671 and Intl. App. Pub. No. WO 2007/136646). Indirect production of ω-3/ω-6 PUFAs occurs wherein the fatty acid substrate is converted indirectly into the desired fatty acid product, via means of an intermediate step(s) or pathway intermediate(s). Thus, it is contemplated that the mutant Δ5 desaturases described herein may be expressed in conjunction with additional genes encoding enzymes of the PUFA biosynthetic pathway (e.g., Δ6 desaturases, C18/20 elongases, Δ17 desaturases, Δ8 desaturases, Δ15 desaturases, Δ9 desaturases, Δ12 desaturases, C14/16 elongases, C16/18 elongases, Δ9 elongases, Δ5 desaturases, Δ4 desaturases, C20/22 elongases) to result in higher levels of production of longer-chain ω-3/ω-6 fatty acids, such as e.g., ARA, EPA, DTA, DPAn-6, DPA and/or DHA.
Preferably, the Δ5 desaturases described herein will minimally be expressed in conjunction with a Δ9 elongase and a Δ8 desaturase. The Δ5 desaturases could also be minimally expressed in conjunction with a Δ6 desaturase and a Δ6 elongase. However, the particular genes included within a particular expression cassette will depend on the host cell (and its PUFA profile and/or desaturase/elongase profile), the availability of substrate and the desired end product(s).
It is necessary to create and introduce a recombinant construct comprising an ORF encoding a mutant Δ5 desaturase (i.e., wherein said mutant comprises an amino acid motif selected from the group consisting of: SEQ ID NO:183 (HGGG), SEQ ID NO:184 (HHGG), SEQ ID NO:186 (HCGG), SEQ ID NO:187 (HWGG) and SEQ ID NO:185 (HPGS)) into a suitable host cell. One of skill in the art is aware of standard resource materials that describe: 1) specific conditions and procedures for construction, manipulation and isolation of macromolecules, such as DNA molecules, plasmids, etc.; 2) generation of recombinant DNA fragments and recombinant expression constructs; and, 3) screening and isolating of clones. See, Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989) (hereinafter “Maniatis”); by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience, Hoboken, N.J. (1987).
In general, the choice of sequences included in the construct depends on the desired expression products, the nature of the host cell and the proposed means of separating transformed cells versus non-transformed cells. The skilled artisan is aware of the genetic elements that must be present on the plasmid vector to successfully transform, select and propagate host cells containing the chimeric gene. Typically, however, the vector or cassette contains sequences directing transcription and translation of the relevant gene(s), a selectable marker and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5′ of the gene that controls transcriptional initiation, i.e., a promoter, the gene coding sequence, and a region 3′ of the DNA fragment that controls transcriptional termination, i.e., a terminator. It is most preferred when both control regions are derived from genes from the transformed host cell, although they need not be derived from the genes native to the production host.
Transcriptional initiation control regions (also initiation control regions or promoters) useful for driving expression of the instant Δ5 desaturase ORFs in the desired microbial host cell are well known. These control regions may comprise a promoter, enhancer, silencer, intron sequences, 3′ UTR and/or 5′ UTR regions, and protein and/or RNA stabilizing elements. Such elements may vary in their strength and specificity. Virtually any promoter, i.e., native, synthetic, or chimeric, capable of directing expression of these genes in the selected host cell is suitable, although transcriptional and translational regions from the host species are particularly useful. Expression in a host cell can be accomplished in an induced or constitutive fashion. Induced expression occurs by inducing the activity of a regulatable promoter operably linked to the gene of interest, while constitutive expression occurs by the use of a constitutive promoter.
When the host cell is yeast, transcriptional and translational regions functional in yeast cells are provided, particularly from the host species. See, e.g., U.S. Pat. Appl. Pub. No. 2006-0115881-A1, corresponding to Intl. App. Pub. No. WO 2006/052870 for preferred transcriptional initiation regulatory regions for use in Yarrowia lipolytica. Any one of a number of regulatory sequences can be used, depending upon whether constitutive or induced transcription is desired, the efficiency of the promoter in expressing the ORF of interest, the ease of construction and the like.
Nucleotide sequences surrounding the translational initiation codon ‘ATG’ have been found to affect expression in yeast cells. If the desired polypeptide is poorly expressed in yeast, the nucleotide sequences of exogenous genes can be modified to include an efficient yeast translation initiation sequence to obtain optimal gene expression. For expression in yeast, this can be done by site-directed mutagenesis of an inefficiently expressed gene by fusing it in-frame to an endogenous yeast gene, preferably a highly expressed gene. Alternatively, one can determine the consensus translation initiation sequence in the host and engineer this sequence into heterologous genes for their optimal expression in the host of interest.
3′ non-coding sequences encoding transcription termination regions may be provided in a recombinant construct and may be from the 3′ region of the gene from which the initiation region was obtained or from a different gene. A large number of termination regions are known and function satisfactorily in a variety of hosts, when utilized both in the same and different genera and species from which they were derived. Termination regions may also be derived from various genes native to the preferred hosts. The termination region usually is selected more as a matter of convenience rather than because of any particular property. The 3′-region can also be synthetic, as one of skill in the art can utilize available information to design and synthesize a 3′-region sequence that functions as a transcription terminator. A termination site may be unnecessary, but is highly preferred.
Merely inserting a gene into a cloning vector does not ensure its expression at the desired rate, concentration, amount, etc. In response to the need for a high expression rate, many specialized expression vectors have been created by adjusting certain properties that govern transcription, RNA stability, translation, protein stability and location, oxygen limitation and secretion from the microbial host cell. These properties include: the nature of the relevant transcriptional promoter and terminator sequences; the number of copies of the cloned gene (wherein additional copies may be cloned within a single expression construct and/or additional copies may be introduced into the host cell by increasing the plasmid copy number or by multiple integration of the cloned gene into the genome); whether the gene is plasmid-borne or integrated into the host cell genome; the final cellular location of the synthesized foreign protein; the efficiency of translation and correct folding of the protein in the host organism; the intrinsic stability of the mRNA and protein of the cloned gene within the host cell; and, the codon usage within the cloned gene, such that its frequency approaches the frequency of preferred codon usage of the host cell. Each of these may be used in the methods and host cells described herein, to further optimize expression of the mutant Δ5 desaturases.
After a recombinant construct is created comprising at least one chimeric gene comprising a promoter, a Δ5 desaturase ORF and a terminator, it is placed in a plasmid vector capable of autonomous replication in a host cell, or it is directly integrated into the genome of the host cell. Integration of expression cassettes can occur randomly within the host genome or can be targeted through the use of constructs containing regions of homology with the host genome sufficient to target recombination within the host locus. Where constructs are targeted to an endogenous locus, all or some of the transcriptional and translational regulatory regions can be provided by the endogenous locus.
Where two or more genes are expressed from separate replicating vectors, it is desirable that each vector has a different means of selection and should lack homology to the other construct(s) to maintain stable expression and prevent reassortment of elements among constructs. Judicious choice of regulatory regions, selection means and method of propagation of the introduced construct(s) can be experimentally determined so that all introduced genes are expressed at the necessary levels to provide for synthesis of the desired products.
Constructs comprising the gene(s) of interest may be introduced into a microbial host cell by any standard technique. These techniques include transformation, e.g., lithium acetate transformation (Methods in Enzymology, 194:186-187 (1991)), bolistic impact, electroporation, microinjection, or any other method that introduces the gene(s) of interest into the host cell.
For convenience, a host cell that has been manipulated by any method to take up a DNA sequence, for example, in an expression cassette, is referred to herein as “transformed”, “transformant” or “recombinant”. The transformed host will have at least one copy of the expression construct and may have two or more, depending upon whether the expression cassette is integrated into the genome, amplified, or is present on an extrachromosomal element having multiple copy numbers. The transformed host cell can be identified by selection for a marker contained on the introduced construct. Alternatively, a separate marker construct may be co-transformed with the desired construct, as many transformation techniques introduce many DNA molecules into host cells.
Typically, transformed hosts are selected for their ability to grow on selective media, which may incorporate an antibiotic or lack a factor necessary for growth of the untransformed host, such as a nutrient or growth factor. An introduced marker gene may confer antibiotic resistance, or encode an essential growth factor or enzyme, thereby permitting growth on selective media when expressed in the transformed host. Selection of a transformed host can also occur when the expressed marker protein can be detected, either directly or indirectly. Additional selection techniques are described in U.S. Pat. No. 7,238,482, U.S. Pat. No. 7,259,255 and Intl. App. Pub. No. WO 2006/052870.
Following transformation, substrates suitable for the instant mutant Δ5 desaturases (and, optionally other PUFA enzymes that are co-expressed within the host cell) may be produced by the host either naturally or transgenically, or they may be provided exogenously.
A variety of eukaryotic organisms are suitable as host, to thereby yield a transformant comprising mutant Δ5 desaturases as described herein, including bacteria, yeast, algae, stramenopiles, oomycetes, euglenoids and/or fungi. This is contemplated because transcription, translation and the protein biosynthetic apparatus is highly conserved. Thus, suitable hosts may include those that grow on a variety of feedstocks, including simple or complex carbohydrates, fatty acids, organic acids, oils, glycerols and alcohols, and/or hydrocarbons over a wide range of temperature and pH values.
Preferred microbial hosts are oleaginous organisms. These oleaginous organisms are naturally capable of oil synthesis and accumulation, wherein the total oil content can comprise greater than about 25% of the dry cell weight, more preferably greater than about 30% of the dry cell weight, and most preferably greater than about 40% of the dry cell weight. Various bacteria, algae, euglenoids, moss, fungi, yeast and stramenopiles are naturally classified as oleaginous. In alternate embodiments, a non-oleaginous organism can be genetically modified to become oleaginous, e.g., yeast such as Saccharomyces cerevisiae. 
In more preferred embodiments, the microbial host cells are oleaginous yeast. Genera typically identified as oleaginous yeast include, but are not limited to: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces. More specifically, illustrative oil-synthesizing yeasts include: Rhodosporidium toruloides, Lipomyces starkeyii, L. lipoferus, Candida revkaufi, C. pulcherrima, C. tropicalis, C. utilis, Trichosporon pullans, T. cutaneum, Rhodotorula glutinus, R. graminis, and Yarrowia lipolytica (formerly classified as Candida lipolytica). Alternately, oil biosynthesis may be genetically engineered such that the microbial host cell (e.g., a yeast) can produce more than 25% oil of the cellular dry weight, and thereby be considered oleaginous.
Most preferred is the oleaginous yeast Yarrowia lipolytica. In a further embodiment, most preferred are the Y. lipolytica strains designated as ATCC #20362, ATCC #8862, ATCC #18944, ATCC #76982 and/or LGAM S(7)1 (Papanikolaou S., and Aggelis G., Bioresour. Technol., 82(1):43-9 (2002)).
Specific teachings applicable for transformation of oleaginous yeasts (i.e., Yarrowia lipolytica) include U.S. Pat. No. 4,880,741 and U.S. Pat. No. 5,071,764 and Chen, D. C. et al. (Appl. Microbiol. Biotechnol., 48(2):232-235 (1997)). Specific teachings applicable for engineering ARA, EPA and DHA production in Y. lipolytica are provided in U.S. patent application Ser. No. 11/264,784 (Intl. App. Pub. No. WO 2006/055322), U.S. Pat. Appl. No. 11/265,761 (Intl. App. Pub. No. WO 2006/052870) and U.S. patent application Ser. No. 11/264,737 (Intl. App. Pub. No. WO 2006/052871), respectively.
The preferred method of expressing genes in this yeast is by integration of linear DNA into the genome of the host. Integration into multiple locations within the genome can be particularly useful when high level expression of genes is desired, such as into the Ura3 locus (GenBank Accession No. AJ306421), the Leu2 gene locus (GenBank Accession No. AF260230), the Lys5 gene locus (GenBank Accession No. M34929), the Aco2 gene locus (GenBank Accession No. AJ001300), the Pox3 gene locus (Pox3: GenBank Accession No. XP—503244; or, Aco3: GenBank Accession No. AJ001301), the Δ12 desaturase gene locus (U.S. Pat. No. 7,214,491), the Lip1 gene locus (GenBank Accession No. Z50020), the Lip2 gene locus (GenBank Accession No. AJ012632), the SCP2 gene locus (GenBank Accession No. AJ431362), Pex3 gene locus (GenBank Accession No. CAG78565), Pex16 gene locus (GenBank Accession No. CAG79622), and/or the Pex10 gene locus (GenBank Accession No. CAG81606).
Preferred selection methods for use in Yarrowia lipolytica are resistance to kanamycin, hygromycin and the amino glycoside G418, as well as ability to grow on media lacking uracil, leucine, lysine, tryptophan or histidine. 5-fluoroorotic acid (5-fluorouracil-6-carboxylic acid monohydrate; “5-FOA”) may also be especially useful for the selection of yeast Ura− mutants (U.S. Pat. Appl. Pub. No. 2009-0093543-A1), or a native acetohydroxyacid synthase (or acetolactate synthase; E.C. 4.1.3.18) that confers sulfonyl urea herbicide resistance (Intl. App. Pub. No. WO 2006/052870) is utilized for selection of transformants. A unique method of “recycling” a pair of preferred selection markers for their use in multiple sequential transformations, by use of site-specific recombinase systems, is also taught in U.S. Pat. Appl. Pub. No. 2009-0093543-A1.
Based on the above, disclosed herein is a method of producing either ARA or EPA, respectively, comprising:

    
    
        (a) providing an oleaginous yeast (e.g., Yarrowia lipolytica) comprising:
        
            (i) a first recombinant nucleotide molecule encoding a mutant Δ5 desaturase polypeptide, operably linked to at least one regulatory sequence; and,
            (ii) a source of desaturase substrate consisting of DGLA and/or ETA, respectively; and,
        
        
        (b) growing the yeast of step (a) in the presence of a suitable fermentable carbon source wherein the gene encoding the mutant Δ5 desaturase polypeptide is expressed and DGLA is converted to ARA and/or ETA is converted to EPA, respectively; and,
        (c) optionally recovering the ARA and/or EPA, respectively, of step (b).

Substrate feeding may be required. In preferred embodiments, the mutant Δ5 desaturase polypeptide is selected from the group consisting of SEQ ID NO:58, SEQ ID NO:97, SEQ ID NO:139 and SEQ ID NO:179. Thus, for example, the nucleotide sequence of the gene encoding the mutant Δ5 desaturase polypeptide may be, for example, selected from the group consisting of SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194 and SEQ ID NO:195.

    
    


Since naturally produced PUFAs in oleaginous yeast are limited to 18:2 fatty acids (i.e., LA), and less commonly, 18:3 fatty acids (i.e., ALA), the oleaginous yeast may be genetically engineered to express multiple enzymes necessary for long-chain PUFA biosynthesis (thereby enabling production of e.g., DPAn-6, DPA and DHA), in addition to the mutant Δ5 desaturases described herein.
Specifically, an oleaginous yeast is contemplated herein, wherein said yeast comprises:
a) a first recombinant DNA construct comprising an isolated polynucleotide encoding a mutant Δ5 desaturase polypeptide, operably linked to at least one regulatory sequence; and,
b) at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of: desaturase, Δ6 desaturase, Δ9 desaturase, Δ12 desaturase, Δ15 desaturase, Δ17 desaturase, Δ8 desaturase, Δ9 elongase, C14/16 elongase, C16/18 elongase, C18/20 elongase and C20/22 elongase.
Other suitable microbial hosts include oleaginous bacteria, algae, euglenoids, stramenopiles, oomycetes and fungi. Within this broad group of microbial hosts, of particular interest are microorganisms that synthesize ω-3/ω-6 fatty acids, or those that can be genetically engineered for this purpose (e.g., other yeast such as Saccharomyces cerevisiae). Thus, for example, transformation of Mortierella alpina (which is commercially used for production of ARA) with any of the present Δ5 desaturase genes under the control of inducible or regulated promoters could yield a transformant organism capable of synthesizing increased quantities of ARA. The method of transformation of M. alpina is described by Mackenzie et al. (Appl. Environ. Microbiol., 66:4655 (2000)). Similarly, methods for transformation of Thraustochytriales microorganisms (e.g., Thraustochytrium, Schizochytrium) are disclosed in U.S. Pat. No. 7,001,772.
Irrespective of the host selected for expression of the mutant Δ5 desaturases described herein, multiple transformants must be screened in order to obtain a strain displaying the desired expression level and pattern. For example, Juretzek et al. (Yeast, 18:97-113 (2001)) note that the stability of an integrated DNA fragment in Yarrowia lipolytica is dependent on the individual transformants, the recipient strain and the targeting platform used. Such screening may be accomplished by Southern analysis of DNA blots (Southern, J. Mol. Biol., 98:503 (1975)), Northern analysis of mRNA expression (Kroczek, J. Chromatogr. Biomed. Appl., 618(1-2):133-145 (1993)), Western and/or Elisa analyses of protein expression, phenotypic analysis or GC analysis of the PUFA products.
Knowledge of the sequences of the present mutant Δ5 desaturases will be useful for manipulating ω-3 and/or ω-6 fatty acid biosynthesis in various host cells. Methods for manipulating biochemical pathways are well known to those skilled in the art; and, it is expected that numerous manipulations will be possible to maximize ω-3 and/or ω-6 fatty acid biosynthesis in oleaginous yeasts, and particularly, in Yarrowia lipolytica. This manipulation may require metabolic engineering directly within the PUFA biosynthetic pathway or additional manipulation of pathways that contribute carbon to the PUFA biosynthetic pathway. Methods useful for up-regulating desirable biochemical pathways and down-regulating undesirable biochemical pathways are well known to those skilled in the art.
For example, biochemical pathways competing with the ω-3 and/or ω-6 fatty acid biosynthetic pathways for energy or carbon, or native PUFA biosynthetic pathway enzymes that interfere with production of a particular PUFA end-product, may be eliminated by gene disruption or down-regulated by other means, e.g., antisense mRNA.
Detailed discussion of manipulations within the PUFA biosynthetic pathway as a means to increase ARA, EPA or DHA and associated techniques thereof are presented in Intl. App. Pub. No. WO 2006/055322 [U.S. Pat. Appl. Pub. No. 2006-0094092-A1], Intl. App. Pub. No. WO 2006/052870 [U.S. Pat. Appl. Pub. No. 2006-0115881-A1] and Intl. App. Pub. No. WO 2006/052871 [U.S. Pat. Appl. Pub. No. 2006-0110806-A1], respectively, as are desirable manipulations in the TAG biosynthetic pathway and the TAG degradation pathway (and associated techniques thereof).
It may be useful to modulate the expression of the fatty acid biosynthetic pathway by any one of the strategies described above. For example, provided herein are methods whereby genes encoding key enzymes in the Δ9 elongase/Δ8 desaturase biosynthetic pathway and Δ6 desaturase/Δ6 elongase biosynthetic pathway are introduced into oleaginous yeasts for the production of ω-3 and/or ω-6 fatty acids. It will be particularly useful to express the present mutant Δ5 desaturase genes in oleaginous yeasts that do not naturally possess ω-3 and/or ω-6 fatty acid biosynthetic pathways and coordinate the expression of these genes, to maximize production of preferred PUFA products using various means for metabolic engineering of the host organism.
The transformed microbial host cell is grown under conditions that optimize expression of chimeric genes (e.g., desaturase, elongase) and produce the greatest and most economical yield of desired PUFAs. In general, media conditions that may be optimized include the type and amount of carbon source, the type and amount of nitrogen source, the carbon-to-nitrogen ratio, the amount of different mineral ions, the oxygen level, growth temperature, pH, length of the biomass production phase, length of the oil accumulation phase and the time and method of cell harvest. Microorganisms of interest, such as oleaginous yeast (e.g., Yarrowia lipolytica) are generally grown in a complex medium such as yeast extract-peptone-dextrose broth [“YPD”] or a defined minimal media that lacks a component necessary for growth and thereby forces selection of the desired expression cassettes (e.g., Yeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.)).
Fermentation media for the methods and host cells described herein must contain a suitable carbon source such as are taught in U.S. Pat. No. 7,238,482. Although it is contemplated that the source of carbon utilized in the methods herein may encompass a wide variety of carbon-containing sources, preferred carbon sources are sugars (e.g., glucose), glycerols, and/or fatty acids.
Nitrogen may be supplied from an inorganic (e.g., (NH4)2SC4) or organic (e.g., urea or glutamate) source. In addition to appropriate carbon and nitrogen sources, the fermentation media must also contain suitable minerals, salts, cofactors, buffers, vitamins and other components known to those skilled in the art suitable for the growth of the oleaginous host and promotion of the enzymatic pathways necessary for PUFA production. Particular attention is given to several metal ions, such as Fe+2, Cu+2, Mn+2, Co+2, Zn+2 and Mg+2, that promote synthesis of lipids and PUFAs (Nakahara, T. et al., Ind. Appl. Single Cell Oils, D. J. Kyle and R. Colin, eds. pp 61-97 (1992)).
Preferred growth media for the methods and host cells described herein are common commercially prepared media, such as Yeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.). Other defined or synthetic growth media may also be used and the appropriate medium for growth of the transformant host cells will be known by one skilled in the art of microbiology or fermentation science. A suitable pH range for the fermentation is typically between about pH 4.0 to pH 8.0, wherein pH 5.5 to pH 7.5 is preferred as the range for the initial growth conditions. The fermentation may be conducted under aerobic or anaerobic conditions, wherein microaerobic conditions are preferred.
Typically, accumulation of high levels of PUFAs in oleaginous yeast cells requires a two-stage process, since the metabolic state must be “balanced” between growth and synthesis/storage of fats. Thus, most preferably, a two-stage fermentation process is necessary for the production of PUFAs in oleaginous yeast (e.g., Yarrowia lipolytica). This approach is described in U.S. Pat. No. 7,238,482, as are various suitable fermentation process designs (i.e., batch, fed-batch and continuous) and considerations during growth.
PUFAs may be found in the host microorganisms as free fatty acids or in esterified forms such as acylglycerols, phospholipids, sulfolipids or glycolipids, and may be extracted from the host cells through a variety of means well-known in the art. One review of extraction techniques, quality analysis and acceptability standards for yeast lipids is that of Z. Jacobs (Critical Reviews in Biotechnology, 12(516):463-491 (1992)). A brief review of downstream processing is also available by A. Singh and O. Ward (Adv. Appl. Microbiol., 45:271-312 (1997)).
In general, means for the purification of PUFAs may include extraction (e.g., U.S. Pat. No. 6,797,303 and U.S. Pat. No. 5,648,564) with organic solvents, sonication, supercritical fluid extraction (e.g., using carbon dioxide), saponification and physical means such as presses, or combinations thereof. See U.S. Pat. No. 7,238,482 for additional details.
There are a plethora of food and feed products incorporating ω-3 and/or ω-6 fatty acids, particularly e.g., ALA, GLA, ARA, EPA, DPA and DHA. It is contemplated that the microbial biomass comprising long-chain PUFAs, partially purified microbial biomass comprising PUFAs, purified microbial oil comprising PUFAs, and/or purified PUFAs will function in food and feed products to impart the health benefits of current formulations. More specifically, oils containing ω-3 and/or ω-6 fatty acids will be suitable for use in a variety of food and feed products including, but not limited to: food analogs, meat products, cereal products, baked foods, snack foods and dairy products (see U.S. Pat. Appl. Pub. No. 2006-0094092 for details).
The present compositions may be used in formulations to impart health benefit in medical foods including medical nutritionals, dietary supplements, infant formula and pharmaceuticals. One of skill in the art of food processing and food formulation will understand how the amount and composition of the present oils may be added to the food or feed product. Such an amount will be referred to herein as an “effective” amount and will depend on the food or feed product, the diet that the product is intended to supplement or the medical condition that the medical food or medical nutritional is intended to correct or treat.
EXAMPLES
The present invention is further described in the following Examples, which illustrate reductions to practice of the invention but do not completely define all of its possible variations.
General Methods
Standard recombinant DNA and molecular cloning techniques used in the Examples are well known in the art and are described by: 1) Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989) (Maniatis); 2) T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1984); and, 3) Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience, Hoboken, N.J. (1987).
Materials and Methods suitable for the maintenance and growth of microbial cultures are well known in the art. Techniques suitable for use in the following examples may be found as set out in Manual of Methods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, Eds), American Society for Microbiology: Washington, D.C. (1994)); or by Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, 2nd ed., Sinauer Associates: Sunderland, Mass. (1989). All reagents, restriction enzymes and materials used for the growth and maintenance of microbial cells were obtained from Aldrich Chemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.), unless otherwise specified. E. coli strains were typically grown at 37° C. on Luria Bertani [“LB”] plates.
General molecular cloning was performed according to standard methods (Sambrook et al., supra). DNA sequence was generated on an ABI Automatic sequencer using dye terminator technology (U.S. Pat. No. 5,366,860; EP 272,007) using a combination of vector and insert-specific primers. Sequence editing was performed in Sequencher (Gene Codes Corporation, Ann Arbor, Mich.). All sequences represent coverage at least two times in both directions. Comparisons of genetic sequences were accomplished using DNASTAR software (DNASTAR Inc., Madison, Wis.).
The meaning of abbreviations is as follows: “sec” means second(s), “min” means minute(s), “h” or “hr” means hour(s), “d” means day(s), “μL” means microliter(s), “mL” means milliliter(s), “L” means liter(s), “μM” means micromolar, “mM” means millimolar, “M” means molar, “mmol” means millimole(s), “μmole” mean micromole(s), “g” means gram(s), “μg” means microgram(s), “ng” means nanogram(s), “U” means unit(s), “bp” means base pair(s) and “kB” means kilobase(s).
Nomenclature for Expression Cassettes:
The structure of an expression cassette will be represented by a simple notation system of “X::Y::Z”, wherein X describes the promoter fragment, Y describes the gene fragment, and Z describes the terminator fragment, which are all operably linked to one another.
Transformation and Cultivation of Yarrowia lipolytica:
Yarrowia lipolytica strains with ATCC Accession Nos. #20362, #76982 and #90812 were purchased from the American Type Culture Collection (Rockville, Md.). Y. lipolytica strains were typically grown at 28-30° C. in several media, according to the recipes shown below. Agar plates were prepared as required by addition of 20 g/L agar to each liquid media, according to standard methodology.

    YPD agar medium (per liter): 10 g of yeast extract [Difco], 20 g of Bacto peptone [Difco]; and 20 g of glucose.
    Basic Minimal Media (MM) (per liter): 20 g glucose; 1.7 g yeast nitrogen base without amino acids; 1.0 g proline; and pH 6.1 (not adjusted).
    Minimal Media+Leucine (MM+leucine or MMLeu) (per liter): Prepare MM media as above and add 0.1 g leucine.
    High Glucose Media (HGM) (per liter): 80 glucose, 2.58 g KH2PO4 and 5.36 g K2HPO4, pH 7.5 (do not need to adjust).


Transformation of Y. lipolytica was performed as described in U.S. Pat. Appl. Pub. No. 2009-0093543-A1, hereby incorporated herein by reference.
Fatty Acid Analysis of Yarrowia lipolytica:
For fatty acid analysis, cells were collected by centrifugation and lipids were extracted as described in Bligh, E. G. & Dyer, W. J. (Can. J. Biochem. Physiol., 37:911-917 (1959)). Fatty acid methyl esters [“FAMES”] were prepared by transesterification of the lipid extract with sodium methoxide (Roughan, G. and Nishida I., Arch Biochem Biophys., 276(1):38-46 (1990)) and subsequently analyzed with a Hewlett-Packard 6890 GC fitted with a 30 m×0.25 mm (i.d.) HP-INNOWAX (Hewlett-Packard) column. The oven temperature was from 170° C. (25 min hold) to 185° C. at 3.5° C./min.
For direct base transesterification, Yarrowia culture (3 mL) was harvested, washed once in distilled water, and dried under vacuum in a Speed-Vac for 5-10 min. Sodium methoxide (100 μL of 1%) was added to the sample, and then the sample was vortexed and rocked for 20 min. After adding 3 drops of 1 M NaCl and 400 μL hexane, the sample was vortexed and spun. The upper layer was removed and analyzed by GC as described above.
Construction of Yarrowia lipolytica Strain Y4036U
Y. lipolytica strain Y4036U (Leu-, Ura-), described in Intl. App. Pub. No. WO 2008/073367, was used as the host in Examples 2-4, 6-7 and 9, infra.
The development of strain Y4036U required the construction of strain Y2224 (a FOA resistant mutant from an autonomous mutation of the Ura3 gene of wildtype Yarrowia strain ATCC #20362), strain Y4001 (producing 17% EDA with a Leu-phenotype), strain Y4001U1 (producing 17% EDA with a Leu- and Ura-phenotype) and strain Y4036 (producing 18% DGLA with a Leu-phenotype).
The final genotype of strain Y4036U with respect to wildtype Yarrowia lipolytica ATCC #20362 was as follows: GPD::FmD12::Pex20, YAT1::FmD12::Oct, YAT1::ME3S::Pex16, GPAT::EgD9e::Lip2, EXP1::EgD9eS::Lip, FBAINm::EgD9eS::Lip2, FBAINm::EgD8M::Pex20 (wherein FmD12 is a Fusarium moniliforme Δ12 desaturase gene [Intl. App. Pub. No. WO 2005/047485]; MESS is a codon-optimized C16/18 elongase gene, derived from Mortierella alpina [Intl. App. Pub. No. WO 2007/046817]; EgD9e is a Euglena gracilis Δ9 elongase gene [Intl. App. Pub. No. WO 2007/061742]; EgD9eS is a codon-optimized Δ9 elongase gene, derived from Euglena gracilis [Intl. App. Pub. No. WO 2007/061742]; and, EgD8M is a synthetic mutant Δ8 desaturase [Intl. App. Pub. No. WO 2008/073271], derived from Euglena gracilis [U.S. Pat. No. 7,256,033]).
Example 1
Construct pDMW369, Comprising EgD5S
The present Example describes plasmid pDMW369, comprising a chimeric FBAIN::EgD5S::Pex20 gene (plasmid construction is described in Intl. App. Pub. No. WO 2007/136671). Plasmid pDMW369 (FIG. 2A; SEQ ID NO:19) contained the following components:







TABLE 7







Components Of Plasmid pDMW369 (SEQ ID NO:19)








RE Sites And



Nucleotides



Within SEQ ID



NO:19
Description Of Fragment And Chimeric Gene Components





EcoR I/BsiW I
FBAIN::EgD5S::Pex20, comprising:


(6063-318)
FBAIN: Yarrowia lipolytica FBAIN promoter (U.S. Pat. 



No. 7,202,356)



EgD5S: codon-optimized Δ5 desaturase (SEQ ID NO:9),



derived from Euglena gracilis



Pex20: Pex20 terminator sequence of Yarrowia Pex20 gene



(GenBank Accession No. AF054613)


1354-474
ColE1 plasmid origin of replication


2284-1424
ampicillin-resistance gene (AmpR) for selection in E. coli


3183-4476
Yarrowia autonomous replication sequence (ARS18; 



GenBank Accession No. A17608)


6020-4533
Yarrowia Ura 3 gene (GenBank Accession No. AJ306421)









Example 2
Identification of HXGG Mutations that Result in Improved Δ5 Desaturase Activity In EgD5S
Single amino acid mutations were carried out using pDMW369 (Example 1) as the template and 19 pairs of oligonucleotides (SEQ ID NOs:20-57; Table 8) as primers to individually mutate the proline residue of the HPGG motif (SEQ ID NO:180) of EgD5S (SEQ ID NO:10) by site-directed mutagenesis (QuickChange Kit, Stratagene, Calif.), thereby generating all amino acid substitutions possible (i.e., His-Xaa-Gly-Gly [HXGG] mutants, wherein Xaa can be any amino acid). Plasmids comprising each mutation were transformed into E. coli XL2Blue cells (Stratagene). Four colonies from each of the 19 transformations were picked and grown individually in liquid media at 37° C. overnight. Plasmids (i.e., 76 total) were isolated from these cultures and sequenced individually to confirm the mutations.
The wild type pDMW369 plasmid and the isolated mutant plasmids were transformed into strain Y4036U individually, as described in the General Methods. The transformants were selected on MMLeu plates. After 2 days growth at 30° C., two transformants from each transformation reaction were streaked out onto new MMLeu plates and incubated for an additional 2 days at 30° C. The colonies were then used to inoculate 3 mL of MMLeu in a 24 well Qiagen block. The blocks were incubated in a 30° C. incubator shaking at 200 rpm. After the cultures were incubated for 2 days, the blocks were centrifuged, the supernatant was removed and 3 mL of HGM was added. The blocks were placed back in a 30° C. incubator shaking at 200 rpm for an additional 5 days. The cells were collected by centrifugation, lipids were extracted, and FAMEs were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
The Δ5 desaturase activity attributed to each mutation within the HPGG motif (SEQ ID NO:180) is summarized below in Table 8. EgD5S mutants are designated according to the sequence of the mutant HXGG motif (i.e., the HPGG motif (SEQ ID NO:180) in mutant EgD5S-HAGG had a P2 to A substitution, thereby yielding a His-Ala-Gly-Gly [HAGG] motif (SEQ ID NO:180), while mutant EgD5S-HRGG possessed a P2 to R substitution, etc.). The conversion efficiency was measured according to the following formula: ([product]/[substrate+product])*100. Results are compared to that of the wildtype EgD5S (SEQ ID NO:10) within plasmid pDMW369, wherein GC analysis determined 8.8% DGLA and 4.5% ARA of total lipids were produced by the transformants (i.e., average conversion efficiency was 33.8%).







TABLE 8







Δ5 Desaturase Activity In EgD5S And HXGG Motif Mutants













Percent




Average
Activity 



Primers Used
Conversion
With 


Y4036U
For Mutant
Efficiency of 
Respect 


Transformant *
Motif Construction
DGLA to ARA (%)
to EgD5S













EgD5S
—
33.8
100


EgD5S-HAGG
SEQ ID NOs:20 and 21
31.4
92.9


EgD5S-HRGG
SEQ ID NOs:22 and 23
29.7
87.9


EgD5S-HNGG
SEQ ID NOs:24 and 25
30.6
88.8


EgD5S-HDGG
SEQ ID NOs:26 and 27
ND**
—


EgD5S-HCGG
SEQ ID NOs:28 and 29
ND**
—


EgD5S-HQGG
SEQ ID NOs:30 and 31
31.2
92.3


EgD5S-HEGG
SEQ ID NOs:32 and 33
ND**
—


EgD5S-HGGG
SEQ ID NOs:34 and 35
33.6
99.4


EgD5S-HHGG
SEQ ID NOs:36 and 37
32.8
97.0


EgD5S-HIGG
SEQ ID NOs:38 and 39
28.0
82.8


EgD5S-HLGG
SEQ ID NOs:40 and 41
27.4
81.1


EgD5S-HKGG
SEQ ID NOs:42 and 43
32.4
95.9


EgD5S-HMGG
SEQ ID NOs:44 and 45
30.1
89.1


EgD5S-HFGG
SEQ ID NOs:46 and 47
ND**
—


EgD5S-HSGG
SEQ ID NOs:48 and 49
28.4
84.0


EgD5S-HTGG
SEQ ID NOs:50 and 51
29.7
87.9


EgD5S-HWGG
SEQ ID NOs:52 and 53
ND**
—


EgD5S-HYGG
SEQ ID NOs:54 and 55
34.6
102


EgD5S-HVGG
SEQ ID NOs:56 and 57
31.2
92.3





* Each EgD5S gene (mutant or wildtype) was expressed within pDMW369.


**ND: Did not get mutant in this experiment.






Based on the above, it is clear that the proline residue within the HPGG motif (SEQ ID NO:180) can be substituted with several amino acids without substantially affecting the Δ5 desaturase activity of EgD5S. Preferred proline substitutions, wherein Δ5 desaturase activity was equaled or improved with respect to EgD5S, were present in EgD5S-HGGG (33.6% conversion) and EgD5S-HYGG (34.6% conversion). EgD5S-HHGG (32.8% conversion) functioned with 97% of the Δ5 desaturase activity of EgD5S.
Example 3
Identification of HPGX Mutations that Result in Improved Δ5 Desaturase Activity In EgD5S
Single amino acid mutations were carried out using pDMW369 (Example 1) as the template and 19 pairs of oligonucleotides (SEQ ID NOs:59 to 96; Table 9) as primers to individually mutate the second glycine residue of the HPGG motif (SEQ ID NO:180) of EgD5S (SEQ ID NO:10) by site-directed mutagenesis (QuickChange Kit, Stratagene, Calif.), thereby generating all amino acid substitutions possible (i.e., His-Pro-Gly-Xaa [HPGX] mutants). Following mutagenesis, plasmids were transformed into Y4036U, transformants were selected and grown in MMLeu and HGM, and FAMEs were prepared and analyzed by GC, as described in Example 2.
The Δ5 desaturase activity attributed to each mutation within the HPGG motif (SEQ ID NO:180) is summarized below in Table 9. EgD5S mutants are designated according to the sequence of the mutant HPGX motif (i.e., the HPGG motif (SEQ ID NO:180) in mutant EgD5S-HPGA had a G4 to A substitution, thereby yielding a His-Pro-Gly-Ala [HPGA] motif (SEQ ID NO:189), while mutant EgD5S-HPGR possessed a G4 to R substitution, etc.). Conversion efficiency was measured according to the formula described in Example 2. Results are compared to that of the wildtype EgD5S (SEQ ID NO:10) within plasmid pDMW369, wherein GC analysis determined 8.8% DGLA and 4.5% ARA of total lipids were produced by the transformants (i.e., average conversion efficiency was 33.8%).







TABLE 9







Δ5 Desaturase Activity In EgD5S And HPGX Motif Mutants












Average
Percent




Conversion
Activity




Efficiency of
With


Y4036U
Primers Used For Mutant
DGLA to ARA
Respect to


Transformant*
Motif Construction
(%)
EgD5S













EgD5S
—
33.8
100


EgD5S-HPGA
SEQ ID NOs:59 and 60
31.3
92.6


EgD5S-HPGR
SEQ ID NOs:61 and 62
26.9
79.6


EgD5S-HPGN
SEQ ID NOs:63 and 64
31.5
93.2


EgD5S-HPGD
SEQ ID NOs:65 and 66
ND**
—


EgD5S-HPGC
SEQ ID NOs:67 and 68
ND**
—


EgD5S-HPGQ
SEQ ID NOs:69 and 70
ND**
—


EgD5S-HPGE
SEQ ID NOs:71 and 72
ND**
—


EgD5S-HPGH
SEQ ID NOs:73 and 74
ND**
—


EgD5S-HPGI
SEQ ID NOs:75 and 76
ND**
—


EgD5S-HPGL
SEQ ID NOs:77 and 78
ND**
—


EgD5S-HPGK
SEQ ID NOs:79 and 80
32.0
94.7


EgD5S-HPGM
SEQ ID NOs:81 and 82
ND**
—


EgD5S-HPGF
SEQ ID NOs:83 and 84
ND**
—


EgD5S-HPGP
SEQ ID NOs:85 and 86
ND**
—


EgD5S-HPGS
SEQ ID NOs:87 and 88
37.3
110.4


EgD5S-HPGT
SEQ ID NOs:89 and 90
35.5
105.0


EgD5S-HPGW
SEQ ID NOs:91 and 92
ND**
—


EgD5S-HPGY
SEQ ID NOs:93 and 94
ND**
—


EgD5S-HPGV
SEQ ID NOs:95 and 96
ND**
—





*Each EgD5S gene (mutant or wildtype) was expressed within pDMW369.


**ND: Did not get mutant in this experiment.






The results demonstrated that the second glycine residue within the HPGG motif can be substituted with several amino acids without substantially affecting the Δ5 desaturase activity of EgD5S. Preferred glycine substitutions, wherein Δ5 desaturase activity was equaled or improved with respect to EgD5S, were present in EgD5S-HPGS (37.3% conversion) and EgD5S-HPGT (35.5% conversion).
Example 4
Quantitative Analysis of EqD5 Mutants that Performed at or Above Wildtype EgD5S Level
Once the preliminary analyses of the amino acid substitutions were complete (Examples 2 and 3), a quantitative analysis of those mutations that performed approximately equivalently or above the wildtype EgD5S conversion rate was carried out (i.e., EgD5S-HGGG, EgD5S-HHGG, EgD5S-HYGG, EgD5S-HPGS and EgD5S-HPGT). The plasmids containing the above mutations were designated as pDMW369-HGGG, pDMW369-HHGG, pDMW369-HYGG, pDMW369-HPGS and pDMW369-HPGT, respectively. These plasmids, along with pDMW369, were re-transformed into Y4036U (General Methods) and plated on MMLeu. The plates were incubated at 30° C. for about 4 days. Twelve transformants from each plate were restreaked onto fresh MMLeu plates and incubated again at 30° C. The transformants were inoculated into 3 mL of MMLeu in a 24 well block format. The blocks were incubated at 30° C. at 200 rpm for 2 days. After 2 days' growth the blocks were centrifuged, the supernatant decanted and the pellets resuspended in HGM. The blocks were incubated at 30° C. for an additional 5 days. The cells were collected by centrifugation, lipids were extracted, and FAMEs were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
The average DGLA to ARA conversion rate of 12 samples are summarized below in Table 10:







TABLE 10







Δ5 Desaturase Activity In EgD5S HXGX Motif Mutants










Average Conversion
Percent Activity



Efficiency of DGLA to
With Respect to


Y4036U Transformant*
ARA (%)
EgD5S












EgD5S
30.4
100


EgD5S-HGGG
31.8
104.6


EgD5S-HHGG
31.5
103.6


EgD5S-HYGG
26.0
85.5


EgD5S-HPGS
32.5
106.9


EgD5S-HPGT
30.1
99.0





*Each EgD5S gene (mutant or wildtype) was expressed within pDMW369.






This experiment confirmed that the Δ5 desaturase activities of EgD5S-HGGG and EgD5S-HHGG (SEQ ID NO:58) and EgD5S-HPGS (SEQ ID NO:97) mutants were increased relative to the wildtype EgD5S control. A suitable nucleotide sequence encoding EgD5S-HGGG is set forth as SEQ ID NO:190, a suitable sequence encoding EgD5S-HHGG is set forth as SEQ ID NO:191 and a suitable nucleotide sequence encoding EgD5S-HPGS is set forth as SEQ ID NO:192.
Example 5
Generation of Construct pZUFmEaD5S, Comprising EaD5S
The present Example describes the construction of plasmid pZUFmEaD5S comprising a chimeric FBAINm::EaD5S::Pex20 gene. Plasmid pZUFmEaD5S (SEQ ID NO:98) was constructed by replacing the Nco I/Not I fragment of pZUF17 (FIG. 2B; SEQ ID NO:99) with the Nco I/Not I EaD5S fragment from pEaD5S (SEQ ID NO:100) [wherein plasmid pEaD5S (SEQ ID NO:100) was created when the EaD5S gene (SEQ ID NO:13) was cloned into pUC57 (GenBank Accession No. Y14837)]. The product of this ligation was pZUFmEaD5S, which thereby contained the following components:







TABLE 11







Components Of Plasmid pZuFmEaD5S (SEQ ID NO:98)








RE Sites And



Nucleotides



Within SEQ ID



NO:98
Description Of Fragment And Chimeric Gene Components





Swa I/BsiW I 
FBAIN::EaD5S::Pex20, comprising:


(7435-1686)
FBAIN: Yarrowia lipolytica FBAIN promoter (U.S. PAT.



NO. 7,202,356)



EaD5S: codon-optimized Δ5 desaturase (SEQ ID NO:13),



derived from Euglena anabaena



Pex20: Pex20 terminator sequence of Yarrowia Pex20 gene



(GenBank Accession No. AF054613)


2722-1842
ColE1 plasmid origin of replication


3652-2792
ampicillin-resistance gene (AmpR) for selection in E. coli


4554-5855
Yarrowia autonomous replication sequence (ARS18; 



GenBank Accession No. A17608)


7399-5898
Yarrowia Ura 3 gene (GenBank Accession No. AJ306421)









Example 6
Identification of HXGG Mutations that Result in Improved Δ5 Desaturase Activity In EaD5S
Single amino acid mutations were carried out using pZUFmEaD5S (Example 5) as the template and 19 pairs of oligonucleotides (SEQ ID NOs:101 to 138; Table 12) as primers to individually mutate the proline residue of the HPGG motif (SEQ ID NO:180) of EaD5S (SEQ ID NO:14) by site-directed mutagenesis (QuickChange Kit, Stratagene, Calif.), thereby generating all amino acid substitutions possible (i.e., His-Xaa-Gly-Gly [HXGG] mutants). Plasmids from each mutation were transformed into E. coli XL2Blue cells. Four colonies from each of the 19 transformations were picked and grown individually in liquid media at 37° C. overnight. Plasmids (i.e., 76 total) were isolated from these cultures and sequenced individually to confirm the mutations.
The wild type pZUFmEaD5S plasmid and the isolated mutant plasmids were transformed into strain Y4036U individually, as described in the General Methods. The transformants were selected on MMLeu plates and then grown in liquid MMLeu and HGM media, as described in Example 2 (except that the speed of the incubator was increased from 200 to 250 rpm). The cells were collected by centrifugation, lipids were extracted, and FAMEs were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
The Δ5 desaturase activities attributed to each mutation within the HPGG motif (SEQ ID NO:180) are summarized below in Table 12. EaD5S mutants are designated according to the sequence of the mutant HXGG motif (i.e., the HPGG motif (SEQ ID NO:180) in mutant EaD5S-HAGG had a P2 to A substitution, thereby yielding a His-Ala-Gly-Gly [HAGG] motif (SEQ ID NO:188), while mutant EaD5S-HRGG possessed a P2 to R substitution, etc.). The conversion efficiency was measured according to the following formula: ([product]/[substrate+product])*100. Results are compared to that of the wildtype EaD5S (SEQ ID NO:14) within plasmid pZUFmEaD5S, wherein GC analysis determined the average DGLA to ARA conversion efficiency of 2 transformants was 25%.







TABLE 12







Δ5 Desaturase Activity In EaD5S And HXGG Motif Mutants












Average





Conversion
Percent




Efficiency of
Activity With


Y4036U
Primers Used For Mutant
DGLA to
Respect to


Transformant*
Motif Construction
ARA (%)
EaD5S













EaD5S
—
25.0
100


EaD5S-HAGG
SEQ ID NOs:101 and 102
26.4
105.6


EaD5S-HRGG
SEQ ID NOs:103 and 104
24.9
99.0


EaD5S-HNGG
SEQ ID NOs:105 and 106
23.2
92.8


EaD5S-HDGG
SEQ ID NOs:107 and 108
8.3
33.2


EaD5S-HCGG
SEQ ID NOs:109 and 110
26.2
104.8


EaD5S-HQGG
SEQ ID NOs:111 and 112
20.7
82.8


EaD5S-HEGG
SEQ ID NOs:113 and 114
8.8
35.2


EaD5S-HGGG
SEQ ID NOs:115 and 116
18.9
75.6


EaD5S-HHGG
SEQ ID NOs:117 and 118
20.4
81.6


EaD5S-HIGG
SEQ ID NOs:119 and 120
ND**
—


EaD5S-HLGG
SEQ ID NOs:121 and 122
21.1
84.4


EaD5S-HKGG
SEQ ID NOs:123 and 124
25.2
100.8


EaD5S-HMGG
SEQ ID NOs:125 and 126
23.6
94.4


EaD5S-HFGG
SEQ ID NOs:127 and 128
21.2
84.8


EaD5S-HSGG
SEQ ID NOs:129 and 130
23.0
95.6


EaD5S-HTGG
SEQ ID NOs:131 and 132
25.8
103.2


EaD5S-HWGG
SEQ ID NOs:133 and 134
14.0
56.0


EaD5S-HYGG
SEQ ID NOs:135 and 136
19.9
79.6


EaD5S-HVGG
SEQ ID NOs:137 and 138
ND**
—





*Each EaD5S gene (mutant or wildtype) was expressed within pZuFmEaD5S.


**ND: Did not get mutant in this experiment.






Based on the above, it is clear that the proline residue within the HPGG motif can be substituted with several amino acids without substantially affecting the Δ5 desaturase activity of EaD5S. Preferred proline substitutions, wherein Δ5 desaturase activity was improved with respect to EaD5S, were present in EaD5S-HAGG (26.3% conversion), EaD5S-HCGG (26.2% conversion), EaD5S-HKGG (25.2% conversion) and EaD5S-HTGG (25.8% conversion).
Quantitative Analysis of EaD5 Mutants that Performed at or Above Wildtype EaD5S Level
A more quantitative analysis of those mutations that performed with approximately equivalent or improved activity with respect to the wildtype EaD5S conversion rate was carried out (i.e., EaD5S-HAGG, EaD5S-HRGG, EaD5S-HNGG, EaD5S-HCGG, EaD5S-HHGG, EaD5S-HLGG, EaD5S-HKGG, EaD5S-HMGG, EaD5S-HFGG, EaD5S-HSGG and EaD5S-HTGG). The plasmids containing the above mutations were designated as pZuFmEaD5S-HAGG, pZuFmEaD5S-HRGG, pZuFmEaD5S-HNGG, pZuFmEaD5S-HCGG, pZuFmEaD5S-HHGG pZuFmEaD5S-HLGG, pZuFmEaD5S-HKGG, pZuFmEaD5S-HMGG, pZuFmEaD5S-HFGG, pZuFmEaD5S-HSGG, and pZuFmEaD5S-HTGG, respectively. These plasmids, along with pZuFmEaD5S, were re-transformed into Y4036U (General Methods) and plated on MMLeu. The plates were incubated at 30° C. for about 4 days. Six transformants from each plate were re-streaked onto fresh MMLeu plates and incubated again at 30° C. The transformants were inoculated into 3 mL of MMLeu in a 24 well block format. The blocks were incubated at 30° C. at 200 rpm for 2 days. After 2 days' growth the blocks were centrifuged, the supernatants were decanted and the pellets were re-suspended in HGM. The blocks were incubated at 30° C. for an additional 5 days. The cells were collected by centrifugation, lipids were extracted, and FAMEs were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
The average DGLA to ARA conversion rate of 6 samples are summarized below in Table 13:







TABLE 13







Δ5 Desaturase Activity In EaD5S HXGG Motif Mutants










Average Conversion
Percent Activity


Y4036U
Efficiency of DGLA to
With Respect to


Transformant*
ARA (%)
EaD5S












EaD5S
24.0
100


EaD5S-HAGG
23.8
99.2


EaD5S-HRGG
23.0
95.8


EaD5S-HNGG
20.7
86.2


EaD5S-HCGG
25.9
107.9


EaD5S-HHGG
20.4
85.0


EaD5S-HLGG
16.7
69.6


EaD5S-HKGG
20.7
86.3


EaD5S-HMGG
23.4
97.5


EaD5S-HFGG
21.2
88.3


EaD5S-HSGG
23.8
99.2


EaD5S-HTGG
21.4
89.2





*Each EaD5S gene (mutant or wildtype) was expressed within pZuFmEaD5S.






This experiment confirmed that the Δ5 desaturase activity of mutant EaD5S-HCGG (SEQ ID NO:139) was increased relative to the wildtype EaD5S control. A suitable nucleotide sequence encoding EaD5S-HCGG is set forth as SEQ ID NO:193.
Example 7
Generation of Construct pZUFmRD5S, Comprising RD5S
The present Example describes plasmid pZURD5S, comprising a chimeric FBAIN::RD5S::Pex20 gene (plasmid construction is described in Intl. App. Pub. No. WO 2007/136646). Plasmid pZURD5S (SEQ ID NO:140) is identical in construction to pDMW369 (Example 1; SEQ ID NO:19), with the exception that RD5S (SEQ ID NO:17) was substituted in place of EgD5S (SEQ ID NO:9).
Example 8
Identification of HXGG Mutations that Result in Improved Δ5 Desaturase Activity in RD5S
Single amino acid mutations were carried out by using pZURD5S (Example 7) as the template and 19 pairs of oligonucleotides (SEQ ID NOs:141 to 178; Table 14) as primers to individually mutate the proline residue of the HPGG motif (SEQ ID NO:180) of RD5S (SEQ ID NO:17) by site-directed mutagenesis (QuickChange Kit, Stratagene, Calif.), thereby generating all amino acid substitutions possible (i.e., His-Xaa-Gly-Gly [HXGG] mutants). Plasmids from each mutation were transformed into E. coli XL2Blue cells. Four colonies from each of the 19 transformations were picked and grown individually in liquid media at 37° C. overnight. Plasmids (i.e., 76 total) were isolated from these cultures and sequenced individually to confirm the mutations.
The wild type pZURD5S plasmid and the isolated mutant plasmids were transformed into strain Y4036U individually, as described in the General Methods. The transformants were selected on MMLeu plates and then grown in liquid MMLeu and HGM media, as described in Example 2 (except that the speed of the incubator was increased from 200 to 250 rpm). The cells were collected by centrifugation, lipids were extracted, and FAMEs were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
The Δ5 desaturase activities attributed to each mutation within the HPGG motif are summarized below in Table 14. RD5S mutants are designated according to the sequence of the mutant HXGG motif (i.e., the HPGG motif (SEQ ID NO:180) in mutant RD5S-HAGG had a P2 to A substitution, thereby yielding a His-Ala-Gly-Gly [HAGG] motif (SEQ ID NO:188), while mutant RD5S-HRGG possessed a P2 to R substitution, etc.). The conversion efficiency was measured according to the following formula: ([product]/[substrate+product])*100. Results are compared to that of the wildtype RD5S (SEQ ID NO:18) within plasmid pZURD5S, wherein GC analysis determined the average DGLA to ARA conversion efficiency of 2 transformants was 25.1%.







TABLE 14







Δ5 Desaturase Activity In RD5S And HXGG Motif Mutants












Average 




Primers Used
Conversion
Percent 


Y4036U
For Mutant
Efficiency of 
Activity With 


Trans-
Motif 
DGLA
Respect


formant*
Construction
to ARA (%)
to RD5S













RD5S
—
25.1
100


RD5S-HAGG
SEQ ID NOs:141 and 142
23.2
92.4


RD5S-HRGG
SEQ ID NOs:143 and 144
ND**
—


RD5S-HNGG
SEQ ID NOs:145 and 146
ND**
—


RD5S-HDGG
SEQ ID NOs:147 and 148
13.1
52.2


RD5S-HCGG
SEQ ID NOs:149 and 150
34.8
138.6


RD5S-HQGG
SEQ ID NOs:151 and 152
20.2
80.5


RD5S-HEGG
SEQ ID NOs:153 and 154
18.6
74.1


RD5S-HGGG
SEQ ID NOs:155 and 156
18.7
74.1


RD5S-HHGG
SEQ ID NOs:157 and 158
ND**
—


RD5S-HIGG
SEQ ID NOs:159 and 160
ND**
—


RD5S-HLGG
SEQ ID NOs:161 and 162
ND**
—


RD5S-HKGG
SEQ ID NOs:163 and 164
22.2
88.4


RD5S-HMGG
SEQ ID NOs:165 and 166
21.2
84.1


RD5S-HFGG
SEQ ID NOs:167 and 168
ND**
—


RD5S-HSGG
SEQ ID NOs:169 and 170
ND**
—


RD5S-HTGG
SEQ ID NOs:171 and 172
22.6
90.0


RD5S-HWGG
SEQ ID NOs:173 and 174
28.5
113.5


RD5S-HYGG
SEQ ID NOs:175 and 176
ND**
—


RD5S-HVGG
SEQ ID NOs:177 and 178
20.6
82.0





*Each RD5S gene (mutant or wildtype) was expressed within pZURD5S.


**ND: Did not get mutant in this experiment.






Based on the above, it is clear that the proline residue within the HPGG motif (SEQ ID NO:180) can be substituted with several amino acids without substantially affecting the Δ5 desaturase activity of RD5S. Preferred proline substitutions, wherein Δ5 desaturase activity was improved with respect to RD5S, were present in RD5S-HCGG (34.8% conversion) and RD5S-HWGG (28.5% conversion).
A quantitative analysis of those mutations that performed at or above the wildtype RD5S conversion rate (i.e., RD5S-HCGG and RD5S-HWGG (SEQ ID NO:179)) will be carried out, as described previously for EgD5S and EaD5S mutants. A suitable nucleotide sequence encoding RD5S-HCGG is set forth as SEQ ID NO:194 and a suitable nucleotide sequence encoding RD5S-HWGG is set forth as SEQ ID NO:195.

## Claims

1. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a Euglena gracilis delta-5 desaturase mutant polypeptide, wherein the mutant polypeptide comprises an amino acid sequence that has at least 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO:8, wherein the amino acid sequence of the mutant polypeptide has Ser at the position corresponding to amino acid 36 of SEQ ID NO:8, and wherein said mutant polypeptide has delta-5 desaturase enzymatic activity and has a dihomo-gamma-linolenic acid to arachidonic acid conversion efficiency that is greater than the dihomo-gamma-linolenic acid to arachidonic acid conversion efficiency of a wildtype Euglena gracilis delta-5 desaturase polypeptide having the heme-binding motif as set forth as SEQ ID NO:180 (HPGG).

2. A microbial host cell transformed with the isolated nucleic acid molecule of claim 1.

3. The microbial host cell of claim 2 selected from the group consisting of: bacteria, yeasts, algae, euglenoids, stramenopiles, oomycetes and fungi.

4. The microbial host cell of claim 3 wherein the microbial host cell is an oleaginous yeast.

5. The microbial host cell of claim 4 wherein the oleaginous yeast is selected from the group consisting of: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.

6. A method for producing arachidonic acid comprising growing the microbial host cell according to claim 2 in the presence of dihomo-gamma-linolenic acid, wherein the dihomo-gamma-linolenic acid is converted to arachidonic acid.

7. A method for producing eicosapentaenoic acid comprising growing the microbial host cell according to claim 2 in the presence of eicosatetraenoic acid, wherein the eicosatetraenoic acid is converted to eicosapentaenoic acid.

8. The microbial host cell of claim 2 wherein the microbial host cell is an oleaginous bacterium, yeast, algae, euglenoid, stramenopile, oomycete or fungus and produces a polyunsaturated fatty acid selected from the group consisting of omega-6 fatty acids and omega-3 fatty acids.

9. The isolated nucleic acid of claim 1, wherein said mutant polypeptide comprises the amino acid sequence of SEQ ID NO:97.

10. The isolated nucleic acid of claim 9, wherein said nucleotide sequence is SEQ ID NO:192.