# Patent

## Title
Expression of cytosolic malic enzyme in transgenic Yarrowia to increase lipid production

## Abstract

Transgenic Yarrowia species are disclosed herein that comprise a polynucleotide encoding a cytosolic malic enzyme, a lipid content that is at least about 35% by weight of the dry cell weight of the Yarrowia species, and an engineered polyunsaturated fatty acid (PUFA) biosynthetic pathway, wherein overexpression of the cytosolic malic enzyme increases lipid content.

## Background
This application claims the benefit of U.S. Provisional Application No. 61/619,574, filed Apr. 3, 2012, which is incorporated herein by reference in its entirety.


FIELD OF THE INVENTION
This invention is in the field of biotechnology. More specifically, this invention pertains to transgenic Yarrowia species overexpressing cytosolic malic enzyme in order to increase lipid content.
BACKGROUND OF THE INVENTION
Research has been directed to understanding lipid and fatty acid (FA) biosynthetic pathways, and genetic engineering has been used to introduce these biosynthetic pathways into host organisms. For example, 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 omega-6) or alpha-linolenic acid (ALA, 18:3 omega-3) fatty acid production, can be substantially altered to result in high-level production of various long-chain omega-3/omega-6 PUFAs.
Although the literature reports a number of recent examples whereby various portions of the omega-3/omega-6 PUFA biosynthetic pathway responsible for EPA production have been introduced into plants and non-oleaginous yeast, significant efforts have focused on the use of the oleaginous yeast, Yarrowia lipolytica (U.S. Pat. Nos. 7,238,482; 7,932,077; U.S. Pat. Appl. Publ. No. 2009-0093543-A1; U.S. Pat. Appl. Publ. No. 2010-0317072-A1). Oleaginous yeast are defined as those yeast that are naturally capable of oil synthesis and accumulation, wherein oil accumulation is at least 25% of the cellular dry weight, or those yeast genetically engineered such that they become capable of oil synthesis and accumulation, wherein oil accumulation is at least 25% of the cellular dry weight.
Still there remains considerable interest in increasing lipid accumulation in fungi. Expression of malic enzyme in the cytosol in Saccharomyces cerevisiae has been shown to increase NADPH production (2004, dos Santos et al., Metabol. Engineering 6:352-363). Given the role of NADPH as a reducing agent in fatty acid synthesis, malic enzyme has been investigated as a possible factor for altering lipid production. Zhang et al. (2007, Microbiology 153:2013-2025) have found that overexpression of malic enzyme in wild type Mucor circinelloides leads to a 2.5-fold increase in lipid accumulation. Consistent with this finding, studies have shown that malic enzyme expression in M. circinelloides and Mortierella alpina is correlated with lipid accumulation (1999, Wynn et al., Microbiology 145:1911-1917; 2002, Ratledge, Biochem. Soc. Trans. 30:1047-1050). Also, a mutant Aspergillus nidulans isolate lacking malic enzyme activity was shown to accumulate half as much lipid as produced by A. nidulans strains having malic enzyme (1997, Wynn et al., Microbiology 143:253-257).
However, studies in wild type Y. lipolytica suggest that malic enzyme may not play as large a role in lipid production. Beopoulos et al. (2011, Appl. Microbiol. Biotechnol. 90:1193-1206) briefly report that the overexpression of the mitochondrial form of malic enzyme did not affect lipid accumulation in Y. lipolytica. 
Notwithstanding the foregoing disclosures, surprisingly, it has been found that the lipid content of a transgenic Yarrowia species, comprising an engineered polyunsaturated fatty acid biosynthetic pathway and having a lipid content of at least about 35% by weight of the dry cell weight of the Yarrowia species, can be increased by overexpressing cytosolic malic enzyme.
SUMMARY OF THE INVENTION
In one embodiment, the invention concerns a transgenic Yarrowia species that comprises (i) a polynucleotide encoding a cytosolic malic enzyme, (ii) a lipid content that is at least about 35% by weight of the dry cell weight of the Yarrowia species, and (iii) an engineered polyunsaturated fatty acid (PUFA) biosynthetic pathway, wherein overexpression of the cytosolic malic enzyme increases lipid content.
In a second embodiment, the cytosolic malic enzyme encoded by the polynucleotide comprises a dysfunctional mitochondrial targeting sequence. In a third embodiment, the cytosolic malic enzyme lacks a mitochondrial targeting sequence.
In a fourth embodiment, the cytosolic malic enzyme encoded by the polynucleotide comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:5 and has malic enzyme activity.
In a fifth embodiment, the transgenic Yarrowia species has a lipid content that is at least about 50% by weight of the dry cell weight of the Yarrowia species.
In a sixth embodiment, the engineered PUFA biosynthetic pathway comprised by the transgenic Yarrowia species produces at least one PUFA such as linoleic acid, alpha-linolenic acid, gamma-linolenic acid, stearidonic acid, eicosadienoic acid, dihomo-gamma-linolenic acid, arachidonic acid, eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosatetraenoic acid, omega-3 docosapentaenoic acid, omega-6 docosapentaenoic acid, or docosahexaenoic acid. Preferably, the engineered PUFA biosynthetic pathway produces eicosapentaenoic acid.
In a seventh embodiment, the transgenic Yarrowia species is Yarrowia lipolytica. 
In an eighth embodiment, the invention concerns a method for increasing the lipid content of a transgenic Yarrowia species that comprises:
a) culturing the transgenic Yarrowia species of the invention, wherein a microbial oil comprising at least one PUFA is produced, and
b) optionally, recovering the microbial oil of step (a).
With respect to the method, the cytosolic malic enzyme may comprise a dysfunctional mitochondrial targeting sequence or the cytosolic malic enzyme does not comprise a mitochondrial targeting sequence. Furthermore, the cytosolic malic enzyme may comprise an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:5 and has malic enzyme activity.
In still another aspect of the method, the lipid content of the Yarrowia species is at least about 50% by weight of the dry cell weight of the Yarrowia species.
In yet another aspect of the method, the engineered PUFA biosynthetic pathway produces at least one PUFA selected from the group consisting of linoleic acid, alpha-linolenic acid, gamma-linolenic acid, stearidonic acid, eicosadienoic acid, dihomo-gamma-linolenic acid, arachidonic acid, eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosatetraenoic acid, omega-3 docosapentaenoic acid, omega-6 docosapentaenoic acid, and docosahexaenoic acid. Preferably, the at least one PUFA produced is eicosapentaenoic acid.



BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCES
FIG. 1: An alignment of the amino acid sequences of MEs expressed by Saccharomyces cerevisiae (ScME), Schizosaccharomyces pombe (SpME) and Yarrowia lipolytica (YIME) is shown. The underlined amino acids in the depicted YIME sequence represent a predicted mitochondrial targeting sequence (MTS).
FIG. 2: Plasmids for ectopic expression of Y. lipolytica MEs are shown. Construct pME (A) contains a cassette (FBAIN::YIME::PEX20) for overexpression of full length (native) Y. lipolytica ME (YIME), whereas construct pMET2 (B) contains a cassette (FBAIN::YIME-T2::PEX20) for overexpression of truncated (cytosolic) Y. lipolytica ME (YIME-T2). Construct pBlue-YURA3 (C) was used for control purposes.
FIG. 3: An alignment of the amino acid sequences of YIME and YIME-T2 is shown. This version of YIME differs by one residue (second amino acid) compared to the ME natively expressed in Y. lipolytica that is shown in FIG. 1.
FIG. 4: Biosynthetic pathways for producing omega-3 and omega-6 fatty acids in Yarrowia are shown.
FIG. 5: The general scheme for disrupting the ME gene locus in a genome is shown. Briefly, transformants that have been targeted with the ME deletion construct are selected for a Ura+ phenotype, followed by selection with 5-fluoroorotic acid (5-FOA) for a Ura− phenotype. Screening is then performed for Ura− clones in which the ME gene has been recombined out of the genome along with the URA3 gene.
FIG. 6: Shown is plasmid pME-KO for knocking out the ME gene in Y. lipolytica. 







TABLE 1







Summary of Gene and Protein SEQ ID Numbers










Nucleic acid
Protein


Description
SEQ ID NO.
SEQ ID NO.












Malic enzyme, derived from Yarrowia

 1


lipolytica (YIME)

(629 a.a.)


Malic enzyme, derived from Saccharomyces

 2


cerevisiae (ScME)

(669 a.a.)


Malic enzyme, derived from

 3


Schizosaccharomyces pombe (SpME)

(565 a.a.)


Cytosolic Yarrowia lipolytica malic enzyme
4
 5


(YIME-T2), used in construct pMET2

(578 a.a.)


Malic enzyme, derived from Yarrowia
6
 7


lipolytica (YIME), used in construct

(629 a.a.)


pME (comprises L2V mutation)


Primer ME-TN2
8


Primer ME-T2
9


Plasmid construct pMET2
10


Primer ME-F
11


Primer ME-R
12


Plasmid construct pME
13


Plasmid construct pBlue-YURA3
14


Plasmid construct pME-KO
15


Primer YME-5-1
16


Primer YME-5-2
17


Primer YME-3-1
18


Primer YME-3-2
19


Primer YME-5-confirm-1
20


Primer YME-5-confirm-2
21


Primer YME-3-confirm-1
22


Primer YME-3-confirm-2
23


Yarrowia malate dehydrogenase (MDH),
24
25


mitochondrial

(338 a.a.)


Yarrowia malate dehydrogenase (MDH),
26
27


peroxisomal (carboxy terminus ends

(331 a.a.)


with . . . PPAN)


Yarrowia malate dehydrogenase (MDH),
28
29


peroxisomal (carboxy terminus ends

(332 a.a.)


with . . . PPAKI)


Yarrowia malate dehydrogenase (MDH),

30


mitochondrial (Phe2Val)

(338 a.a.)


Primer YMDH1-F
31


Primer YMDH1-R
32












DETAILED DESCRIPTION OF THE INVENTION
All patents, patent applications, and publications cited are incorporated herein by reference in their entirety.
The following definitions are provided.
“Eicosapentaenoic acid” is abbreviated as “EPA”.
“American Type Culture Collection” is abbreviated as “ATCC”.
“Polyunsaturated fatty acid(s)” is abbreviated as “PUFA(s)”.
“Triacylglycerols” are abbreviated as “TAGs”.
“Total fatty acids” are abbreviated as “TFAs”.
“Fatty acid methyl esters” are abbreviated as “FAMEs”.
“Dry cell weight” is abbreviated as “DCW”.
“Weight percent” is abbreviated as “wt %”.
As used herein the term “invention” or “present invention” is intended to refer to all aspects and embodiments of the invention as described in the claims and specification herein and should not be read so as to be limited to any particular embodiment or aspect.
The term “malic enzyme” refers to an (S)-malate:NADP+oxidoreductase (decarboxylating), pyruvic-malic carboxylase, NADP+-specific malic enzyme, or NADP+-malic enzyme. Malic enzyme carries out the irreversible decarboxylation of malate to pyruvate with the formation of NADPH from NADP+. Malic enzyme has the Enzyme Commission entries EC 1.1.1.39 and EC 1.1.1.40. The term “cytosolic malic enzyme” refers to a malic enzyme is targeted to the cytosol (cytoplasm) in the cell. Cytosolic targeting can occur if the malic enzyme lacks a mitochondrial targeting sequence or has a dysfunctional mitochondrial targeting sequence. The terms “malic enzyme” and “ME” are used interchangeably herein.
The term “mitochondrial targeting sequence” refers to an amino acid sequence that directs a protein to localize to the mitochondria. The terms “mitochondrial targeting sequence”, “MTS”, and “mitochondrial signal peptide” are used interchangeably herein. The MTS is generally located at the N-terminus of a protein and comprises one or more amphipathic helixes that have alternating hydrophobic amino acids and positively charged amino acids. The structure of the MTS permits a protein's interaction with mitochondrial surface receptors and subsequent translocation through the inner and outer mitochondrial membrane layers into the mitochondrial matrix, where the MTS is then cleaved. The MTS is generally twenty to eighty amino acids in length. Mitochondrial targeting sequence physiology has been described (e.g., Molecular Biology of the Cell, Alberts et al. 4th Edition, Garland Science: NY (2002).
The term “dysfunctional mitochondrial targeting sequence” refers to an MTS that does not have mitochondrial targeting function. An MTS may be dysfunctional by virtue of containing a deletion, insertion, and/or amino acid changes that alter the structural features of the MTS such that the MTS does not interact with mitochondrial surface receptors or allow mitochondrial membrane translocation. For example, a dysfunctional MTS may be rendered by removing or structurally impairing one or more amphipathic helixes of the MTS.
The term “lipids” refers to any fat-soluble (i.e., lipophilic), naturally-occurring molecule. A general overview of lipids is provided in U.S. Pat. Appl. Publ. No. 2009-0093543-A1 (see Table 2 therein).
The term “oil” refers to a lipid substance that is liquid at 25° C.; oil is hydrophobic and soluble in organic solvents. In oleaginous organisms, oil constitutes a major part of the total lipids “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 lipids are generally similar; thus, an increase or decrease in the concentration of fatty acids in the total lipids will correspond with an increase or decrease in the concentration of fatty acids in the oil, and vice versa.
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 refers to the sum of all cellular fatty acids that can be derivatized 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, e.g., the phosphatidylcholine and the 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”], although 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 lipids is expressed herein as a weight percent of TFAs [“% TFAs”], e.g., milligrams of a given fatty acid per 100 milligrams of TFAs. Unless otherwise specifically stated herein, reference to the percent of a given fatty acid with respect to total lipids or oil is equivalent to the concentration of the fatty acid as % TFAs (e.g., % EPA of total lipids or oil is equivalent to EPA % TFAs).
In some cases, it is useful to express the content of a given fatty acid(s) in a cell as its weight percent of the dry cell weight [“% DCW”]. Thus, for example, a measure of EPA productivity [“EPA % DCW”] would be determined according to the following formula: (EPA % TFAs)*(TFAs % DCW)]/100. The content of a fatty acid(s) such as EPA in a cell as its weight percent of the dry cell weight [“% DCW”] can be approximated, however, as: (EPA % TFAs)*(FAMEs % DCW)]/100.
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 wt % of TFAs. The sum of each individual fatty acid present in the mixture should be 100.
The term “oleaginous” as used in certain embodiments describes those organisms that tend to store their energy source in the form of lipid (Weete, In: Fungal Lipid Biochemistry, 2nd Ed., Plenum, 1980). An oleaginous microorganism can comprise, or can accumulate or produce, about 25% or more of its dry cell weight as oil (i.e., ≧25 TFAs % DCW).
The term “oleaginous yeast” refers to those microorganisms classified as yeasts that make oil. Examples of oleaginous yeast include, for example, the genera Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces. 
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 incorporated herein by reference.
Nomenclature used to describe PUFAs herein is given in Table 2. In the “Shorthand Notation” column, 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 Table 2 summarizes the common names of omega-3 and omega-6 fatty acids and their precursors, the abbreviations that will be used throughout







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 n-6


gamma-Linolenic
GLA
cis-6,9,12-octadecatrienoic
18:3 n-6


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


Dihomo-gamma-
DGLA
cis-8,11,14-eicosatrienoic
20:3 n-6


Linolenic


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




eicosatetraenoic


alpha-Linolenic
ALA
cis-9,12,15-
18:3 n-3




octadecatrienoic


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




octadecatetraenoic


Eicosatrienoic
EtrA
cis-11,14,17-eicosatrienoic
20:3 n-3


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




eicosatetraenoic


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


pentaenoic

eicosapentaenoic


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




docosatetraenoic


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


pentaenoic

docosapentaenoic


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


pentaenoic

docosapentaenoic


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


hexaenoic

docosahexaenoic









The term “PUFA biosynthetic pathway” refers to a metabolic process that converts oleic acid to omega-6 fatty acids such as LA, EDA, GLA, DGLA, ARA, DTA and DPAn-6 and omega-3 fatty acids such as ALA, STA, ETrA, ETA, EPA, DPA and DHA. This process is described in the literature (e.g., U.S. Pat. No. 7,932,077; U.S. Pat. Appl. Publ. No. 2009-0093543-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 these enzymes) associated with the biosynthesis of a PUFA, including: delta-4 desaturase, delta-5 desaturase, delta-6 desaturase, delta-12 desaturase, delta-15 desaturase, delta-17 desaturase, delta-9 desaturase, delta-8 desaturase, delta-9 elongase, C14/16 elongase, C16/18 elongase, C18/20 elongase and/or C20/22 elongase.
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 “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, as described in U.S. Pat. Appl. Pub. No. 2010-0317072-A1, either by manual evaluation or by computer-automated sequence comparison and identification using algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., J. Mol. Biol., 215:403-410 (1993)).
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.
The term “isolated” as used in certain embodiments refers to a polynucleotide or polypeptide molecule that has been completely or partially purified from its native source. In some instances, the isolated polynucleotide or polypeptide molecule is part of a greater composition, buffer system or reagent mix. For example, the isolated polynucleotide or polypeptide molecule can be comprised within a cell or organism in a heterologous manner.
“Gene” refers to a nucleic acid fragment that expresses a specific protein, and which may refer to the coding region alone or may include regulatory sequences upstream and/or downstream to the coding region (e.g., 5′ untranslated regions upstream of the transcription start site of the coding region, 3′ non-coding regions). “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 of the coding sequence's transcription start site, 5′ untranslated regions and 3′ non-coding regions, and which may influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, enhancers, silencers, 5′ untranslated leader sequence, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, stem-loop structures and other elements involved in regulation of gene expression.
“Promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a promoter sequence is 5′ upstream of a coding 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 cell growth and/or development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.
The terms “3′ non-coding sequence”, “transcription terminator” and “terminator” refer to DNA sequences located 3′ 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.
The term “operably linked” in certain embodiments 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. That is, the coding sequence is under the transcriptional control of the promoter. Coding sequences can be operably linked to regulatory sequences in 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 terms “recombinant”, “transgenic”, “transformed”, “engineered” or “modified for exogenous gene expression” are used interchangeably herein.
The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA. Expression also includes translation of mRNA into a polypeptide.
The term “increased” as used in certain embodiments means having a greater quantity, for example a quantity only slightly greater than the original quantity, or for example a quantity in large excess compared to the original quantity, and including all quantities in between. Alternatively, “increased” may refer to a quantity or activity that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% more than the quantity or activity for which the increased quantity or activity is being compared. The terms “increased”, “greater than”, and “improved” are used interchangeably herein. The term “increased” can be used to characterize the expression of a polynucleotide encoding a protein, for example, where “increased expression” can also mean “over-expression”.
“Transformation” refers to the transfer of a nucleic acid molecule into a host organism. The nucleic acid molecule may be a plasmid that replicates autonomously, 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” or “transformed” organisms or as “transformants”.
“Stable transformation” refers to the transfer of a nucleic acid fragment into the genome of a host organism, including both nuclear and organellar genomes, resulting in genetically stable inheritance (i.e., the nucleic acid fragment is “stably integrated”). In contrast, “transient transformation” refers to the transfer of a nucleic acid fragment into the nucleus, or DNA-containing organelle, of a host organism resulting in gene expression without integration or stable inheritance.
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 have autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, and may be 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 comprising 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; 2) a coding sequence (i.e., ORF); and, 3) a terminator that usually contains a polyadenylation site in eukaryotes. 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 term “sequence analysis software” refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. Typical sequence analysis software includes, for example: 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, 111-120. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be 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.
“Sequence identity” or “identity” in the context of nucleic acid or polypeptide sequences in certain embodiments 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.) Humana: NJ (1994); 4) Sequence Analysis in Molecular Bioloqy (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.). Alternately, 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.
Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described by 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).
Various polypeptide amino acid sequences and polynucleotide sequences are disclosed herein as features of certain embodiments of the disclosed invention. Variants of these sequences that are at least about 70-85%, 85-90%, or 90%-95% identical to the sequences disclosed herein may be used in certain embodiments. Alternatively, a variant amino acid sequence or polynucleotide sequence in certain embodiments can have at least 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% identity with a sequence disclosed herein. The variant amino acid sequence or polynucleotide sequence has the same function of the disclosed sequence, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the function of the disclosed sequence.
As is shown in the Examples below, overexpression of malic enzyme, whether mitochondrial or cytosolic, had little or no impact on lipid production in wild type Yarrowia lipolytica. 
Surprisingly and unexpectedly, it has been found that overexpression of cytosolic malic can increase lipid production in a transgenic Yarrowia species comprising:
(i) a polynucleotide encoding a cytosolic malic enzyme;
(ii) a lipid content that is at least about 35% of the dry cell weight of the Yarrowia species; and
(iii) an engineered polyunsaturated fatty acid (PUFA) biosynthetic pathway.
Specifically, the transgenic Yarrowia species of the invention comprises, inter alia, a non-native cytosolic malic enzyme (ME)-encoding polynucleotide. In this sense, the polynucleotide encoding ME may be ectopic or heterologous to the Yarrowia species.
The polynucleotide encoding a cytosolic ME may be a polymer of DNA or RNA, and may be single- or double-stranded. The polynucleotide may contain nucleotides produced by the Yarrowia species containing the polynucleotide, or synthetic, non-natural or altered nucleotides (e.g., nucleotide base analogue). The polynucleotide may be in the form of a linear fragment or as a component of a larger nucleotide construct (e.g., plasmid, vector, linear or circular construct). The polynucleotide or a construct containing the polynucleotide may be chromosomal or episomal. The polynucleotide may alternatively be characterized as a gene, genetic sequence, nucleic acid sequence, DNA sequence, complementary DNA (cDNA) sequence, or RNA sequence.
The polynucleotide may contain an open reading frame (ORF) encoding a cytosolic ME (i.e., cytosolic ME coding sequence), as well as elements that regulate the expression of the cytosolic ME ORF. Alternatively, the polynucleotide may have an amino acid coding sequence having one or more introns that can be removed via gene splicing (e.g., a genomic copy of the cytosolic ME gene). Regulatory elements may include a promoter and/or a 3′ transcriptional termination sequence (i.e., terminator sequence). Other elements may include translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, stem-loop structures and/or other elements involved in regulation of gene expression.
The above regulatory element(s) may be operably linked to the cytosolic ME coding region such that ME expression is functionally modulated by the element. In this sense, the element is active or operational in the Yarrowia species. Also, the polynucleotide may be considered to be expressible or capable of being expressed in the Yarrowia species. The activity of the promoter can be constitutive (for cytosolic ME overexpression) or have specific activity subject to a particular environmental stimulus (i.e., inducible). The regulatory elements may be native to, or heterologous to, the Yarrowia species containing the polynucleotide. A heterologous cytosolic ME gene cassette having one or more non malic enzyme gene regulatory elements and/or non-Yarrowia-derived regulatory elements may be characterized as chimeric. Examples of promoter and terminator sequences that may be used are provided in the below Examples section, and are also disclosed in U.S. Appl. Publ. Nos. 2006/0035351A1 and 2010/0068789A1, which are both incorporated herein by reference.
The expression of the amino acid coding sequence of the polynucleotide encoding the cytosolic ME in the Yarrowia species may be characterized as upregulated, enhanced, increased, elevated, or overexpressed in comparison to the level of expression that may have existed in the Yarrowia species before introducing the polynucleotide thereto (i.e., a control Yarrowia). Since Yarrowia species are believed to not have a native cytosolic ME gene, any level of exogenous expression of cytosolic ME from the polynucleotide can be characterized as upregulated or overexpressed, for example, as compared to the Yarrowia species before it was modified to contain the cytosolic ME-encoding polynucleotide (or as compared to some other suitable control such as a wild type Yarrowia or a transformed Yarrowia containing but not expressing a cytosolic ME-encoding polynucleotide, etc.). Nevertheless, the increased level of cytosolic ME expression in the Yarrowia species modified to contain the polynucleotide may be characterized to be at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 100%, 150%, 200%, 500%, or 1000% above the expression of cytosolic ME in the Yarrowia species before it was modified to contain the cytosolic ME-encoding polynucleotide (or a corresponding Yarrowia control).
The terms “control cell” and “suitable control cell” are used interchangeably and may be referenced with respect to a cell in which a particular modification (e.g., over-expression of a polynucleotide, down-regulation of a polynucleotide) has been made (i.e., an “experimental cell”). A control cell may be any cell that does not have or does not express the particular modification of the experimental cell. Thus, a control cell may be an untransformed wild type cell or may be genetically transformed but does not express the genetic transformation. For example, a control cell may be a direct parent of the experimental cell, which direct parent cell does not have the particular modification that is in the experimental cell. Alternatively, a control cell may be a parent of the experimental cell that is removed by one or more generations. Alternatively still, a control cell may be a sibling of the experimental cell, which sibling does not comprise the particular modification that is present in the experimental cell. A sibling cell that could serve as a control cell could be a cell in which a plasmid for protein over-expression is inserted, but not expressed, in the sibling cell, whereas the plasmid is expressed in the experimental cell. It is well within the skill in the art to determine whether a cell can be a control cell.
The amino acid coding sequence of the polynucleotide encoding a cytosolic ME may be optimized for recognition by the protein translation machinery of the Yarrowia species into which the polynucleotide is placed. For example, the cytosolic ME ORF may be derived from a species other than Yarrowia, but codon-optimized for expression in Yarrowia. Codon optimization in this manner can be performed following the codon usage profile for Yarrowia lipolytica as provided in U.S. Pat. No. 7,125,672.
As an alternative to exogenous polynucleotide expression, the polynucleotide encoding a cytosolic ME may be expressed in Yarrowia from the native ME gene locus itself, but which has been appropriately modified. Since the native ME gene in Yarrowia encodes a mitochondrial ME, this gene would have to be modified using a genetic targeting technique (e.g., sequence knock-out) to remove all or part of the mitochondrial targeting sequence encoded at the 5′-end of the native Yarrowia ME ORF. Other modifications at the native Yarrowia ME gene locus could include the addition of a constitutive promoter, additional regulatory elements for overexpressing the modified gene, and/or modification of the translation start site so that the modified gene will produce ME localized in cytoplasm.
The cytosolic ME encoded by the polynucleotide may be characterized as a polypeptide that comprises the amino acid sequence of a cytosolic ME. The cytosolic ME can also be characterized as a cytosolic (S)-malate:NADP+ oxidoreductase (decarboxylating), pyruvic-malic carboxylase, NADP+-specific malic enzyme, or NADP+-malic enzyme (Enzyme Commission entries EC 1.1.1.39 and EC 1.1.1.40).
Malic enzymes are responsible for various essential physiological functions in living organisms. The end products of the ME reaction (pyruvate, CO2, NAD(P)H; see below) feed into numerous biological pathways such as the TCA cycle and reductive biosynthesis processes. Certain NADP-dependent isoforms of ME are found in bacteria, yeast, fungi, birds and mammals and primarily play a role in biosynthetic reactions such as lipid biosynthesis and desaturation through the provision of NADPH. Several isoforms of NADP-dependent ME exist in fungi through the action of post-translational modifications (either partial proteolytic cleavage, phosphorylation or dephosphorylation) (Saayman et al., 2006, S. Afr. J. Enol. Vitic. 27:113-122).
Malic enzyme activity catalyzes the following reaction:

malate+NADP+→pyruvate+CO2+NADPH,

which can also be expressed as:





This reaction constitutes the oxidative decarboxylation of L-malate to pyruvate and CO2. L-malate may also referred to as (S)-malate. Cytosolic ME activity may be NADP+ (nicotinamide adenine dinucleotide phosphate)-dependent; in this sense, the cytosolic ME encoded by the polynucleotide may also be characterized, for example, as NADP+-dependent ME, NADP+-dependent cytosolic ME, or NADPH-producing ME. Methods for measuring cytosolic ME activity are well known in the art (e.g., Pongratz et al., 2009, Methods. Enzymol. 457:425-450; Geer et al., 1980, Comp. Biochem. Physiol. 65B:25-34; Fukuda et al., 2005, Archaea. 1:293-301).

Catalysis by ME generally proceeds in three steps: dehydrogenation of malate to produce oxaloacetate, decarboxylation of oxaloacetate to produce enolpyruvate, and tautomerisation of enolpyruvate to produce pyruvate. The active site residues of a ME can be roughly divided into four categories: (1) divalent cation-binding residues; (2) substrate-binding residues; (3) NAD(P)+ cofactor binding residues; and (4) catalytic residues. A metal ion serves as a bridge to properly position malate at the active site (Saayman et al., 2006, S. Afr. J. Enol. Vitic. 27:113-122).
In one embodiment of the invention, the cytosolic malic enzyme encoded by the polynucleotide does not comprise a mitochondrial targeting sequence (MTS). The cytosolic ME may be derived from a mitochondrial ME from which the MTS has been removed. Since the cytosolic ME lacks an MTS—therefore it is not a mitochondrial ME—this ME does not locate to mitochondria, but rather locates within the cytosol of the cell. The cytosolic ME can also be characterized as a cytoplasmic ME or as an extramitochondrial ME. Alternatively, the cytosolic ME may be derived from or represent a malic enzyme that localizes to the cytosol in its native form (i.e., no genetic engineering or other modification is necessary to endow the property of cytosolic localization).
The MTS of a mitochondrial ME is located at the N-terminus of the protein. Therefore, removal of the MTS from a mitochondrial ME would involve deleting amino acid residues from or within the N-terminus. In this sense, a cytosolic ME obtained by removing the MTS from a mitochondrial ME can be characterized as amino-truncated or N-terminal-truncated with respect to the mitochondrial ME from which the cytosolic ME is derived.
An MTS can be identified as comprising an alternating pattern of hydrophobic amino acids and positively charged amino acids. Generally, an MTS has one or more helical sequences containing abundant positive charges on one face and hydrophobic residues on the other face (amphipathic helix). Depending on the nature and sequence of the MTS being manipulated, approximately the first 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 amino acids of the N-terminus of a mitochondrial ME can be removed to provide a cytosolic ME, or stretches of about 5, 10, 15, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, or 80 contiguous amino acids within an MTS of a mitochondrial ME can be removed to provide a cytosolic ME. Alternatively, the amino acids of an MTS sub-structure (see Neupert, 1997, Ann. Rev. Biochem. 66:863-917, incorporated herein by reference) may be altered, deleted, or disrupted by insertion.
In another embodiment of the invention, the cytosolic malic enzyme encoded by the polynucleotide comprises a dysfunctional mitochondrial targeting sequence. For example, the cytosolic ME may lack a functional MTS by virtue of (i) lacking all or a portion of the MTS, (ii) containing one or more amino acid changes (e.g., resulting from genetic mutation or alteration) that inhibits MTS function, and/or (iii) providing to, or expressing in, a cell a factor that inhibits malic enzyme MTS function (e.g., small molecule, antibody, antigen-binding antibody fragment, aptamer etc.). An example of a cytosolic ME is a Y. lipolytica mitochondrial ME that lacks a functional MTS (e.g., a Y. lipolytica mitochondrial ME that lacks any or all of the amino acids of an MTS).
An MTS may be identified using an algorithm such as that described by Emanuelsson et al. (2000, J. Mol. Biol. 300:1005-1016). Other algorithms for identifying an MTS in a protein include, for example, MitoProt (Claros et al., 1996, Eur. J. Biochem. 241:779-786), Predotar (Small et al., 2004, Proteomics 4:1581-1590), and pTARGET (Guda et al., 2005, Bioinformatics 21:3963-3969). Alternatively, an MTS may be identified by aligning a query sequence with one or more MTS amino acid sequences that have been characterized in other proteins.
In general, an MTS functions by first binding to a receptor on the outer mitochondrial membrane (transporter of outer membrane, or “Tom”) via interactions through the hydrophobic surface(s) of the MTS (Roise et al., 1988, J. Biol. Chem. 263:4509-4511). Then the MTS, through its positively charged surface(s), transfers to another Tom receptor complex (Brix et al., 1997, J. Biol. Chem. 272:20730-20735) containing a channel. Following translocation through the Tom channel into the mitochondrial inter-membrane space, the basic residues of the MTS mediate interaction with a highly acidic complex (transporter of inner membrane, or “Tim”), which mediates importation of the MTS-containing protein into the mitochondrial matrix (Abe et al., 2000, Cell 100:551-560). Once transport is complete, the MTS is usually cleaved from the protein (Neupert, 1997, Ann. Rev. Biochem. 66:863-917).
The MTS of a malic enzyme may be identified and/or made dysfunctional with respect to any of these molecular interactions. For example, binding assays may be performed to determine if certain ME N-terminal amino acids of a putative MTS bind to the above-described receptor-channel complexes of the mitochondrial outer and inner membranes. Removing and/or altering one or more of those amino acids that mediate ME's binding to these factors may prevent the MTS from targeting ME to mitochondria.
The cytosolic ME encoding-polynucleotide may be derived from a polynucleotide encoding an ME from a Yarrowia species or from a different organism. Malic enzymes are widely distributed in nature and have been reported in the yeasts S. pombe, Rhodotorula glutinis, Z. bailii, S. cerevisiae and C. utilis. The S. cerevisiae, C. utilis and S. pombe MEs are bifunctional and can react with both malate and oxaloacetate. The S. cerevisiae ME can use both NAD+ and NADP+ as an electron acceptor, with NAD+being favoured. The C. utilis ME uses either NAD+ or NADP+ for the decarboxylation of oxaloacetate, but only NADP+ for the decarboxylation of L-malate. Yeast MEs show variability with respect to their substrate affinities and metal requirements. The S. pombe ME has a very high substrate affinity (Km=3.2 mM) as opposed to the ME of S. cerevisiae (Km=50 mM). The C. utilis and S. pombe cytosolic MEs require the divalent cations Mn2+ or Mg2+ for activity, in contrast to the mitochondrial S. cerevisiae ME which prefers Mn2+ (Saayman et al., 2006, S. Afr. J. Enol. Vitic. 27:113-122).
The cytosolic ME can be derived, for example, from any of the polypeptides provided in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 (FIG. 1), or GenBank Accession Nos. NP—012896, ABL67725, XP—504112, XP—001683592.1, AAF54860.1, AAF54859.1, NP—731739.1, NP—524880.2, EHQ58305.1, AEY64427.1, EHP69692.1, EHP69535.1, AEX51047.1, ZP—09413940.1 EHM12738.1, EHM10701.1, AEV69826.1, AEV24956.1, AEV24553.1, ZP—09201798.1, AEV29176.1, AEV33715.1, ACU98038.1, EAZ89719.1, AET68238.1, AET65914.1, EHI48436.1, YP—003543293.1, YP—003134865.1, ADE35429.1, ACL02315.1, ZP—08187924.1, ZP—08182502.1, ZP—08177630.1, EGD19865.1, EGD14411.1, ZP—01730858.1, AAB07709.1, AAA41563.1, XP—001913406.1, ADX74453.1, ACZ23235.1, ACU98385.1, YP—003316069.1, YP—003148857.1, YP—003154265.1, ZP—09629862.1, ACR47743.1, EHP37708.1, AEX51358.1, YP—004063227.1, ADR27874.1, ADN70279.1, ADN63267.1, NP—439983.1, YP—004076148.1, ZP—09517186.1, EHN27331.1, ZP—09329416.1, EHL58360.1, ADT98313.1, ZP—09289264.1, ZP—09285866.1, ZP—09267857.1, BAL27832.1, YP—001655800.1, EHK74844.1, YP—004510107.1, YP—003142912.1, EHJ97543.1, EBA14945.1, ZP—09091420.1, ZP—08429361.1, ABV78885.1, ZP—05120299.1, EGJ31523.1, ADJ15331.1, ADI89962.1, EGJ23089.1, ZP—08663698.1, ZP—05586166.1, ZP—04466958.1, ABM45933.1, ZP—06862982.1, ZP—03833419.1,ZP—03513851.1, ZP—02331708.1, ZP—07448622.1, ZP—02153123.1,ZP—02151206.1, ZP—01916022.1, ZP—01883625.1, ZP—01870789.1, AAC47396.1, BAE47514.1, EHQ60523.1, NP—838014.1, YP—001019404.1, YP—984283.1, AAF37577.1, CAA39690.1, CCC74376.1, BAB76295.1, YP—647281.1, NP—422343.1, NP—244034.1, YP—001239856.1, XP—002283814.1, AEW62565.1, YP—001232212.1, YP—001235403.1, YP—001002607.1, EEU87611.1, NP—002386.1, NP—001155058.1, EFG91746.1, AAF54860.1, NP—524880.2, AEC06242.1, NP—001105383.1, AAA41563.1, EAT42717.1, AAK97531.1, XP—002572611.1, NP—001138325.1, NP—001015690.1, NP—001128692.1, NP—001231187.1, NP—001082582.1, NP—001003627.2, XP—532217.3, XP—848770.1, XP—001499853.2, XP—001499424.2, XP—518610.3, ADK56109.1, EDN59877.1, ABL67725.1, GAA86393.1, XP—001267753.1, ABM30154.1, CAX41101.1, XP—001825515.1, EHA55338.1, XP—001395105.2, XP—001390670.2, XP—003236013.1, EEA25978.1, XP—002548578.1, EGU77660.1, or XP—448858.1. With any of these ME polypeptides, those that are mitochondrial may be appropriately modified (see above) to render cytosolic targeting or localization. A variant of any of these polypeptides may be used, but should have ME enzymatic activity (e.g., see above) and cytosolic localization. Such a variant may comprise an amino acid sequence that is at least about 50%, 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% identical to the amino acid sequence of the reference ME. Preferably, a variant ME comprises an amino acid sequence that is at least about 90% identical to the reference ME. Where one of these reference MEs is mitochondrial, it should be understood that a variant thereof that localizes to the cytosol may contain mutations, deletions, and/or insertions in the MTS that interfere with MTS targeting activity (see above).
The cytosolic ME encoded by the polynucleotide may be prokaryotic or eukaryotic, and may be from bacteria, fungi, yeasts, plants, animals, protozoa, or algae. The Yarrowia species may contain 1, 2, 3, 4, 5, 6, 7, 8, or more polynucleotides encoding the same or a combination of different cytosolic ME polypeptides.
A cytosolic ME polypeptide encoded by the polynucleotide may comprise SEQ ID NO:5 (FIG. 3). SEQ ID NO:5 represents a Yarrowia mitochondrial ME from which the first 53 amino acids have been removed. Alternatively, a variant of this cytosolic ME may comprise an amino acid sequence that is at least about 50%, 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% identical to SEQ ID NO:5. Such a variant should have ME enzymatic activity (e.g., see above) and cytosolic localization. In one embodiment of the invention, the cytosolic malic enzyme comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:5 and has malic enzyme activity.
A cytosolic ME polypeptide encoded by the polynucleotide may comprise of an amino acid sequence that is at least about 50%, 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% identical to SEQ ID NO:7 (FIG. 3); such a variant should localize to the cytosol and have ME enzymatic activity (e.g., see above). Given that SEQ ID NO:7 represents a mitochondrial ME, it should be understood that a variant thereof that localizes to the cytosol has mutations, deletions, and/or insertions (see above) in the MTS that interfere with MTS targeting activity.
An example of a polynucleotide sequence encoding a cytosolic ME is one that comprises SEQ ID NO:4, which encodes SEQ ID NO:5. Alternatively, given the degeneracy of the genetic code, a polynucleotide may comprise a variant of SEQ ID NO:4 that encodes SEQ ID NO:5. For example, such a variant polynucleotide may be at least about 50%, 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% identical to SEQ ID NO:4 and encode SEQ ID NO:5. Another polynucleotide may have a sequence encoding a variant Yarrowia ME polypeptide described above.
The Basic Local Alignment Search Tool (BLAST) algorithm, which is available online at the National Center for Biotechnology Information (NCBI) website, may be used to measure percent identity between or among two or more of the polynucleotide sequences (BLASTN algorithm) or polypeptide sequences (BLASTP algorithm) disclosed herein. Alternatively, percent identity between sequences may be performed using a Clustal algorithm (e.g., ClustalW or ClustalV). For multiple alignments using a Clustal method of alignment, the default values may correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using a Clustal method may be KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids, these parameters may be KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4.
Within a cellular context, a cytosolic ME can alternatively be provided by altering (e.g., amino acid mutation, deletion, or insertion) a protein that interacts with the MTS of a mitochondrial ME to effect mitochondrial transport. Such an alteration may inhibit the MTS-interacting protein from binding the MTS domain, thereby preventing mitochondrial transport of the ME. Thus, the “mitochondrial ME” may become a cytosolic ME by virtue of not being targeted to the mitochondria. Examples of MTS-interacting proteins that play a role in ME mitochondrial targeting that can be altered for the above purpose are disclosed by Neupert (1997, Ann. Rev. Biochem. 66:863-917).
The cytosolic ME encoded by the polynucleotide, including any variant thereof (e.g., homolog, mutant, deletant, etc.), has malic enzyme activity (see above). Where a variant has lower activity compared to its reference ME (e.g., native ME, wild type ME, unaltered ME, endogenous ME, etc.), the activity of the variant ME should have at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the activity of the reference ME.
The amino acid sequence of the cytosolic ME encoded by the polynucleotide may comprise an added protein tag or epitope, such that the ME is expressed as a tagged protein (i.e., fusion protein) that can be more easily detected or isolated. The tag or epitope should not interfere with ME enzymatic activity (e.g., see above) and cytosolic targeting.
The preferred Yarrowia species used to practice the invention is Yarrowia lipolytica. Examples of Y. lipolytica strains that can be used to prepare the transgenic Yarrowia species provided herein are available from the American Type Culture Collection (ATCC, Manassas, Va.): strain designations ATCC #20362, #8862, #8661, #8662, #9773, #15586, #16617, #16618, #18942, #18943, #18944, #18945, #20114, #20177, #20182, #20225, #20226, #20228, #20327, #20255, #20287, #20297, #20315, #20320, #20324, #20336, #20341, #20346, #20348, #20363, #20364, #20372, #20373, #20383, #20390, #20400, #20460, #20461, #20462, #20496, #20510, #20628, #20688, #20774, #20775, #20776, #20777, #20778, #20779, #20780, #20781, #20794, #20795, #20875, #20241, #20422, #20423, #32338, #32339, #32340, #32341, #34342, #32343, #32935, #34017, #34018, #34088, #34922, #34922, #38295, #42281, #44601, #46025, #46026, #46027, #46028, #46067, #46068, #46069, #46070, #46330, #46482, #46483, #46484, #46436, #60594, #62385, #64042, #74234, #76598, #76861, #76862, #76982, #90716, #90811, #90812, #90813, #90814, #90903, #90904, #90905, #96028, #201241, #201242, #201243, #201244, #201245, #201246, #201247, #201249, or #201847.
In addition to the polynucleotide encoding a cytosolic malic enzyme, transgenic Yarrowia species of the invention also comprise an engineered PUFA biosynthetic pathway.
For example, the metabolic process wherein oleic acid is converted to EPA 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 desaturation and elongation enzymes present in the endoplasmic reticulum membrane. However, as described below, multiple alternate pathways exist for EPA production.
Specifically, FIG. 4 depicts the pathways described below. All pathways require the initial conversion of oleic acid to linoleic acid [“LA”], the first of the omega-6 fatty acids, by a delta-12 desaturase. Then, using the “delta-9 elongase/delta-8 desaturase pathway” and LA as substrate, long-chain omega-6 fatty acids are formed as follows: 1) LA is converted to eicosadienoic acid [“EDA”] by a delta-9 elongase; 2) EDA is converted to dihomo-gamma-linolenic acid [“DGLA”] by a delta-8 desaturase; 3) DGLA is converted to arachidonic acid [“ARA”] by a delta-5 desaturase; 4) ARA is converted to docosatetraenoic acid [“DTA”] by a C20/22 elongase; and, and 5) DTA is converted to docosapentaenoic acid [“DPAn-6”] by a delta-4 desaturase.
The delta-9 elongase/delta-8 desaturase pathway can also use alpha-linolenic acid [“ALA”] as substrate to produce long-chain omega-3 fatty acids as follows: 1) LA is converted to ALA, the first of the omega-3 fatty acids, by a delta-15 desaturase; 2) ALA is converted to eicosatrienoic acid [“ETrA”] by a delta-9 elongase; 3) ETrA is converted to eicosatetraenoic acid [“ETA”] by a delta-8 desaturase; 4) ETA is converted to eicosapentaenoic acid [“EPA”] by a delta-5 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 delta-4 desaturase. Optionally, omega-6 fatty acids may be converted to omega-3 fatty acids. For example, ETA and EPA are produced from DGLA and ARA, respectively, by delta-17 desaturase activity. Advantageously for the purposes herein, the delta-9 elongase/delta-8 desaturase pathway enables production of an EPA oil that lacks significant amounts of gamma-linolenic acid [“GLA”].
Alternate pathways for the biosynthesis of omega-3/omega-6 fatty acids utilize a delta-6 desaturase and C18/20 elongase, that is, the “delta-6 desaturase/delta-6 elongase pathway”. More specifically, LA and ALA may be converted to GLA and stearidonic acid [“STA”], respectively, by a delta-6 desaturase; then, a C18/20 elongase converts GLA to DGLA and/or STA to ETA.
Economical commercial production of EPA in a recombinant Yarrowia sp. host cell requires consideration of a variety of variables, including the EPA concentration [“EPA % TFAs”] and total lipid content [“TFAs % DCW”]. Furthermore, it is desirable to reduce the production of intermediate fatty acids and by-product fatty acids in the final oil product in order to maximize production of the desired fatty acid, i.e., EPA.
Intermediate fatty acids are those fatty acids (e.g., oleic acid, LA, ALA, EDA, DGLA, ETA) that can be further converted to EPA by the action of other metabolic pathway enzymes. In contrast, by-product fatty acids (e.g., sciadonic acid, juniperonic acid) refer to any fatty acid produced that is neither EPA nor an intermediate fatty acid that can be further converted to EPA.
U.S. Pat. Appl. Publ. No. 2009-0093543-A1 describes optimized strains of recombinant Yarrowia lipolytica having the ability to produce microbial oils comprising at least about 43.3 EPA % TFAs, with less than about 23.6 LA % TFAs (an EPA:LA ratio of 1.83). The preferred strain was Y4305, whose maximum production was 55.6 EPA % TFAs, with an EPA:LA ratio of 3.03. Generally, the EPA strains of U.S. Pat. Appl. Publ. No. 2009-0093543-A1 comprised the following genes of the omega-3/omega-6 fatty acid biosynthetic pathway:

    
    
        a) at least one gene encoding delta-9 elongase;
        b) at least one gene encoding delta-8 desaturase;
        c) at least one gene encoding delta-5 desaturase;
        d) at least one gene encoding delta-17 desaturase;
        e) at least one gene encoding delta-12 desaturase;
        f) at least one gene encoding C16/18 elongase; and
        g) optionally, at least one gene encoding diacylglycerol cholinephosphotransferase (CPT1).

Examples of preferred genes having the enzymatic functionalities described above are set forth in Table 3 (although these genes are not intended to be limiting).

    
    









TABLE 3







Preferred Desaturases and Elongases for EPA Biosynthesis in Yarrowia lipolytica















Wildtype
Codon-Optimized
Mutant


ORF
Organism
Patent Reference
Abbreviation
Abbreviation
Abbreviation





delta-9
Euglena gracillis
U.S. Pat. No. 7,645,604
“EgD9e”
“EgD9eS”
—


elongase
Eutreptiella sp.
U.S. Pat. No. 7,645,604
“E389D9e”
“E389D9eS”
—



CCMP389



Euglena
U.S. Pat. Appl. Publ. No. 2008-
“EaD9e”*
“EaD9eS”
—



anabaena UTEX
0254522-A1; Intl. App. Publ. No.



373
WO 2008/128241


delta-8
Euglena gracilis
U.S. Pat. No. 7,256,033;
“EgD8”*
“EgD8S”*
“EgD8M”*


desaturase

U.S. Pat. No. 7,709,239



Euglena
U.S. Pat. Appl. Publ. No. 2008-
“EaD8”*
“EaD8S”
—



anabaena UTEX
0254521-A1; Intl. Appl. Publ. No.



373
WO 2008/124194


delta-5
Euglena gracilis
U.S. Pat. No. 7,678,560; U.S. Pat.
“EgD5”
“EgD5S”
“EgD5M”;


desaturase

Appl. Publ. No. 2010-0075386-A1


“EgD5SM”



Peridinium sp.
U.S. Pat. 7,695,950; U.S. Pat.
“RD5”
“RD5S”
—



CCMP626
Appl. Publ. No. 2010-0075386-A1



Euglena
U.S. Pat. Appl. Publ. No. 2008-
“EaD5”*
“EaD5S”*
“EaD5SM”



anabaena UTEX
0274521-A1; U.S. Pat. Appl. Publ.



373
No. 2010-0075386-A1


delta-17
Phytophthora
U.S. Pat. No. 7,465,793
“PrD17”
“PrD17S”
—


desaturase
ramorum



Pythium
U.S. Pat. No. 7,556,949
“PaD17”
“PaD17S”
—



aphanidematum


delta-12
Fusarium
U.S. Pat. No. 7,504,259
“FmD12”*
“FmD12S”
—


desaturase
moniliforme


C16/18
Mortierella alpina
U.S. Pat. No. 7,470,532
“ELO3”

—


elongase


Diacyl-
Yarrowia
Intl. Appl. Publ. No. WO
“YICPT”
—
—


glycerol
lipolytica
2006/052870


choline-


phospho-


transferase





*Notes: EaD9e was identified as “EaD9Elo1” in U.S. Pat. Appl. Publ. No. 2008-0254522-A1; EgD8 was identified as “Eg5” in U.S. Pat. No. 7,256,033; EgD8S was identified as “D8SF” in U.S. Pat. No. 7,256,033; EgD8M was identified as “EgD8S-23” in U.S. Pat. No. 7,709,239; EaD8 was identified as “EaD8Des3” in U.S. Pat. Appl. Publ. No. 2008-0254521-A1; EaD5 was identified as “EaD5Des1” in U.S. Pat. Appl. Publ. No. 2008-0274521-A1; and FmD12 was identified as “Fm2” in U.S. Pat. No. 7,504,259.






As one skilled in the art will appreciate from the foregoing discussion, one or more of the above PUFA biosynthetic pathway enzymes may be derived from Yarrowia and/or from one or more oleaginous organisms. Such other oleaginous organisms may be characterized as an oleaginous microbe, yeast, mold, fungus, oomycete, bacteria, algae, stramenopile, or protist (e.g., euglenoid). Examples of oleaginous yeast, aside from Yarrowia, include species of the genera Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces. Examples of oleaginous fungi include species of the genera Fusarium (e.g., Fusarium lateritium), Mortierella (e.g., Mortierella alpina) and Mucor (e.g., Mucor rouxii and Mucor circinelloides), which are all filamentous fungi. Examples of oleaginous algae include species of the genera Entomophthora, Pythium and Porphyridium. 
In one embodiment of the invention, the engineered PUFA biosynthetic pathway produces at least one PUFA selected from the group consisting of linoleic acid, alpha-linolenic acid, gamma-linolenic acid, stearidonic acid, eicosadienoic acid, dihomo-gamma-linolenic acid, arachidonic acid, eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosatetraenoic acid, omega-3 docosapentaenoic acid, docosahexaenoic acid and omega-6 docosapentaenoic acid. Preferably, the PUFA produced is eicosapentaenoic acid.
In addition to (i) a polynucleotide encoding a cytosolic malic enzyme and (ii) an engineered polyunsaturated fatty acid (PUFA) biosynthetic pathway, the transgenic Yarrowia species of the invention also comprises a lipid content that is at least about 35% by weight of the dry cell weight of said Yarrowia species.
Examples of such high lipid-containing transgenic Yarrowia strains are Z1978, L250, L258, Z5565, Z5567, Z5575, Z5576, Z5620, Z5623, Z5625, Z5581, Z5582, Z5583, Z5584, Z5570, Z5571, Z5572, Z5574, Z5585 and Z5627, all of which are disclosed in U.S. Appl. Publ. No. 2012/0052537 A1, the disclosure of which is incorporated herein by reference. Other examples of high lipid-containing transgenic Yarrowia strains that can be used in practicing the invention are disclosed in U.S. Appl. Publ. No. 2010/0317072 A1 (e.g., strains Y8647, Y9028, Y9029, Y9031, Y9481, Y9502, Y9508 and Y9510), all of which are hereby incorporated by reference. All of these exemplary Yarrowia strains are capable of producing a lipid content greater than about 35% by weight of the dry cell weight of the respective strain.
The transgenic Yarrowia species may have a lipid content (i.e., total lipids or oil) that is by weight at least about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, or 65% of the DCW of the Yarrowia species. In a preferred embodiment of the invention, the lipid content is at least about 50% by weight of the dry cell weight of the Yarrowia species.
The level of total lipids or oil (TFAs % DCW) produced by the transgenic Yarrowia species may increase at least about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% relative to the total lipid/oil content that was in the Yarrowia species prior to insertion of a cytosolic ME-encoding polynucleotide (or relative to another suitable control such as a wild type Yarrowia or a transformed Yarrowia containing but not expressing a cytosolic ME-encoding polynucleotide, etc.).
Constructs or vectors comprising the gene(s) of interest may be introduced into a host cell by any standard technique. These techniques include transformation (e.g., lithium acetate transformation [Methods in Enzymology, 194:186-187 (1991)]), biolistic impact, electroporation, microinjection, or any other method that introduces the gene(s) of interest into the host cell. As an example, U.S. Pat. Nos. 4,880,741 and 5,071,764, and Chen et al. (Appl. Microbiol. Biotechnol., 48(2):232-235 (1997)), describe integration techniques for Yarrowia lipolytica, based on linearized fragments of DNA.
For convenience, a Yarrowia cell that has been manipulated by any method to take up a DNA sequence (e.g., an expression cassette) is referred to herein as “transformed”, “engineered”, “transformant” or “recombinant”. The transformed host will have at least one copy of the expression cassette and may have two or more, depending upon whether the expression cassette is integrated into the genome or is present on an extrachromosomal element having multiple copies. The transformed host cell can be identified by various selection techniques, as described for example in U.S. Pat. Nos. 7,238,482 and 7,259,255.
Preferred selection methods for use herein 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. In alternate embodiments, 5-fluoroorotic acid (5-fluorouracil-6-carboxylic acid monohydrate; “5-FOA”) is used for selection of yeast Ura− mutants (U.S. Pat. Appl. Publ. 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. Appl. Publ. 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. Publ. No. 2009-0093543-A1.
It may be desirable to manipulate a number of different genetic elements that control aspects of transcription, RNA stability, translation, protein stability and protein location, oxygen limitation and secretion from the host cell. More specifically, gene expression in certain embodiments may be controlled by altering the following: the nature of the relevant promoter and terminator sequences; the number of copies of the cloned gene; whether the gene is plasmid-borne or integrated into the genome of the host cell; the final cellular location of the synthesized foreign protein; the efficiency of translation in the host organism; the intrinsic stability of the cloned gene protein 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. Several of these methods of overexpression will be discussed below and are useful during genetic manipulation of recombinant microbial host cells as a means to overexpress genes.
Promoters useful to drive expression of heterologous genes in microbial host cells are numerous and known to those skilled in the art. Expression can be accomplished in an induced or constitutive fashion. Induced expression can be accomplished by inducing the activity of a regulatable promoter operably linked to the gene of interest, while constitutive expression can be achieved by the use of a constitutive promoter operably linked to the gene of interest. Virtually any promoter (i.e., native, synthetic, or chimeric) capable of directing expression of a gene is suitable, although transcriptional and translational regions from the host species are particularly useful.
In general, the terminator can be derived from the 3′ region of the gene from which the promoter was obtained or from a different gene. A large number of terminators 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. The terminator usually is selected more as a matter of convenience rather than because of any particular property. Preferably, the terminator is derived from a yeast gene. The terminator can also be synthetic, as one of skill in the art can utilize available information to design and synthesize a terminator. A terminator may be unnecessary, but it is highly preferred.
Although not intended to be limiting, preferred promoters and terminators for use in a recombinant microbial host cell of the genus Yarrowia are those taught in U.S. Pat. Appl. Publ. No. 2009-0093543-A1, U.S. Pat. Appl. Publ. No. 2010-0068789-A1, U.S. Pat. Pub. No. 2011-0059496-A1, U.S. Provisional Pat. Appl. No. 61/469,933, U.S. Provisional Pat. Appl. No. 61/470,539, U.S. Provisional Pat. Appl. No. 61/471,736, and U.S. Provisional Pat. Appl. No. 61/472,742, the disclosure of each which is hereby incorporated by reference.
Additional copies (i.e., more than one copy) of the PUFA biosynthetic pathway desaturases, elongases, etc. genes may be introduced into the recombinant microbial host cell to thereby increase EPA production and accumulation. Specifically, additional copies of genes may be cloned within a single expression construct; and/or additional copies of the cloned gene(s) may be introduced into the host cell by increasing the plasmid copy number or by multiple integration of the cloned gene into the genome.
It is noted that when preparing an optimized recombinant microbial host cell according to the methodology herein, copies of various desaturases, elongases, DGLA synthases, etc. are often referred to. If, for example, 2 copies of a delta-9 elongase are required, this can refer to: 1) two copies of an identical coding sequence for a particular delta-9 elongase isolated from a single species; or 2) one coding sequence for a delta-9 elongase isolated from a species “A” and one coding sequence for a delta-9 elongase isolated from a species “B”, thus collectively resulting in two delta-9 elongases.
In general, once a DNA cassette (e.g., comprising a chimeric gene comprising a promoter, ORF and terminator) suitable for expression in a recombinant microbial host cell has been obtained, it is either placed in a plasmid vector capable of autonomous replication in the host cell or 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 with the host locus. Although not relied on herein, all or some of the transcriptional and translational regulatory regions can be provided by the endogenous locus where constructs are targeted to an endogenous locus.
The transgenic Yarrowia species in certain embodiments may further comprise a heterologous polynucleotide encoding a malate dehydrogenase (MDH) enzyme. In other words, cytosolic ME can be co-expressed with an MDH. The MDH in certain embodiments is MDH (EC 1.1.1.37), which is an enzyme that reversibly catalyzes the oxidation of malate to oxaloacetate using the reduction of NAD+ to NADH. Since MDH (EC 1.1.1.37) also catalyzes the reverse of this reaction (i.e., oxaloacetate to malate), the expression of MDH can increase the amount of malate substrate available to cytosolic ME. This in turn can help sustain cytosolic ME production of NADPH when converting malate to pyruvate. This intersection of the MDH and cytosolic ME reactions can be illustrated as follows (where MAE is malic enzyme and PYC is pyruvate carboxylase):




Alternatively, the MDH in certain embodiments is a “malate dehydrogenase (NADP+)”, which can also be referred to as “(S)-malate:NADP+ oxidoreductase”. Malate dehydrogenase (NADP+) (EC 1.1.1.82) is an enzyme that catalyzes the chemical reaction: (S)-malate+NADP+→oxaloacetate+NADPH+H+. Malate dehydrogenase (NADP+) belongs to the oxidoreductase family of enzymes, specifically those acting on the CH—OH group of donor with NAD+ or NADP+ as acceptor. The NADPH produced from this reaction represents source of NADPH for fatty acid synthesis.
The MDH (EC 1.1.1.37 or EC 1.1.1.82) in certain embodiments can be prokaryotic or eukaryotic, and may be from bacteria, fungi, yeasts, plants, animals, protozoa, algae, or stramenopiles. The Yarrowia species may contain 1, 2, 3, 4, 5, 6, 7, 8, or more heterologous polynucleotides encoding the same or a combination of different MDH enzymes. The MDH can be a mitochondrial MDH, cytosolic MDH, or peroxisomal MDH, for example. Several MDH enzymes, both EC 1.1.1.37 and EC 1.1.1.82, are known in the art.
In certain embodiments of the invention, the MDH co-expressed with cytosolic ME in the Yarrowia species may be a Yarrowia MDH. Such an MDH can be over-expressed using a heterologous polynucleotide in the Yarrowia species, taking into account native gene expression of the MDH. The Yarrowia MDH can comprise SEQ ID NO:25, for example. SEQ ID NO:25 is a mitochondrial MDH. Alternatively, the Yarrowia MDH can comprise an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:25, and have MDH activity (e.g., oxaloacetate conversion to malate). A polynucleotide encoding any of these MDH amino acid sequences can be used, such as SEQ ID NO:24, for example. In certain embodiments, the MDH comprises SEQ ID NO:30, which differs from SEQ ID NO:25 by one amino acid residue (contains a valine residue instead of a phenylalanine residue at position 2).
The Yarrowia MDH can comprise SEQ ID NO:27 or SEQ ID NO:29 in certain embodiments of the invention. SEQ ID NO:27 and SEQ ID NO:29 are peroxisomal MDH enzymes. Alternatively, the Yarrowia MDH can comprise an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:27 or SEQ ID NO:29, and have MDH activity (e.g., oxaloacetate conversion to malate). A polynucleotide encoding any of these MDH amino acid sequences can be used, such as SEQ ID NO:26 or SEQ ID NO:28, for example.
With respect to engineered recombinant Y. lipolytica host cells, the preferred method of expressing genes in this microbial host is by integration of a linear DNA fragment 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. Preferred loci include those taught in U.S. Pat. Appl. Publ. No. 2009-0093543-A1.
Furthermore, Juretzek et al. (Yeast, 18:97-113 (2001)) note that the stability of an integrated DNA fragment in Y. lipolytica is dependent on the individual transformants, the recipient strain and the targeting platform used. Thus, a skilled artisan will recognize that multiple transformants of a particular recombinant microbial host must be screened in order to obtain a strain displaying the desired expression level and pattern. 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 analysis of protein expression, phenotypic analysis or GC analysis of the PUFA products.
The present invention also concerns a method for increasing the lipid content of a transgenic Yarrowia species that comprises:
a) culturing the transgenic Yarrowia species of the invention wherein a microbial oil comprising at least one PUFA is produced, and
b) optionally, recovering the microbial oil of step (a).
Oil may be recovered or obtained from the transgenic Yarrowia species after about 12, 24, 36, 48, 60, 72, 84, 96, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, or 200 hours of culturing the Yarrowia species to produce a microbial oil comprising at least one PUFA.
The transgenic Yarrowia species of the present disclosure can be grown under conditions that optimize expression of chimeric genes (e.g., encoding desaturases, elongases, etc.) and produce the greatest and the most economical yield of one or more PUFAs. In general, media conditions may be optimized by modifying 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. For example, Yarrowia lipolytica is generally grown in a complex media 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 described in U.S. Pat. No. 7,238,482 and U.S. Pat. Appl. Publ. No. 2011-0059204-A1. Although it is contemplated that the source of carbon utilized in the present invention may encompass a wide variety of carbon-containing sources, preferred carbon sources are sugars (e.g., glucose, invert sucrose, fructose and combinations of thereof), glycerols and/or fatty acids (e.g., those containing between 10-22 carbons).
Nitrogen may be supplied from an inorganic (e.g., (NH4)2SO4) 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 recombinant microbial host cell and the promotion of the enzymatic pathways for EPA 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 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 or corn steep liquors. Other defined or synthetic growth media may also be used. 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 EPA in 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.
Example 10 of U.S. Pat. Appl. Publ. No. 2009-0093543-A1 also provides a detailed description of parameters required for a 2-L fermentation of the recombinant Yarrowia lipolytica strain Y4305 (whose maximum production was 12.1 EPA % DCW [55.6 EPA % TFAs, with a ratio of EPA % TFAs to LA % TFAs of 3.03] over a period of 162 hours). This disclosure includes a description of means to prepare inocula from frozen cultures to generate a seed culture, initially culture the yeast under conditions that promoted rapid growth to a high cell density, and then culture the yeast to promote lipid and PUFA accumulation (via starving for nitrogen and continuously feeding glucose). Process variables including temperature (controlled between 30-32° C.), pH (controlled between 5-7), dissolved oxygen concentration and glucose concentration were monitored and controlled per standard operating conditions to ensure consistent process performance and final PUFA oil quality.
In some aspects of the invention, the primary product is the recombinant microbial biomass. As such, isolation and purification of the PUFA-containing oils from the microbial biomass may not be necessary (i.e., wherein the whole cell biomass is the product). However, certain end uses and/or product forms may require partial and/or complete isolation/purification of the EPA-containing oil from the microbial biomass, to result in partially purified microbial biomass, purified oil, and/or purified EPA. See U.S. Pat. Appl. Publ. No. 2010-0317072-A1 for further details regarding these aspects.
EXAMPLES
Except as specifically delineated herein, the following procedures were used in the Examples. The procedures for culturing Y. lipolytica strains, and for measuring the dry cell weight, lipid content and fatty acid profile thereof, were generally performed as described in U.S. Pat. Appl. Publ. Nos. 2008/0254191 and 2009/0093543, which are incorporated herein by reference. The procedures for transforming Y. lipolytica strains with plasmid expression vectors were generally performed as described in U.S. Pat. Appl. Publ. No. 2009/0093543, which is incorporated herein by reference. Recombinant DNA cloning and manipulation were performed using standard molecular biology procedures.
Example 1
Vector Construction for Native or Truncated (Cytosolic) Y. lipolytica Malic Enzyme Overexpression
The amino acid sequence of Yarrowia lipolytica malic enzyme (ME) was analyzed to determine whether a mitochondrial targeting sequence (MTS) is contained therein. After identifying a putative MTS, vectors for full length (mitochondrial) ME and truncated (cytosolic) ME expression were constructed.
Malic Enzyme MTS Identification
Y. lipolytica ME sequence (YIME, SEQ ID NO:1, GenBank Acc. No. XP—504112) was analyzed using the TargetP 1.1 Server sequence analysis prediction program (Center for Biological Sequence Analysis, Technical University of Denmark, Lyngby, Denmark) as described by Emanuelsson et al. (2000, J. Mol. Biol. 300:10051016) to determine whether this protein may contain any particular subcellular localization sequences such as an MTS. This analysis suggested that Yarrowia ME contains a putative MTS of 26 amino acid residues in length (FIG. 1, underlined in YIME sequence).
It was reported that Saccharomyces cerevisiae malic enzyme (ScME, SEQ ID NO:2, GenBank Acc. No. EDN59877) is mitochondrial, whereas malic enzyme in Schizosaccharomyces pombe (SpME, SEQ ID NO:3, GenBank Acc. No. NP—587760) is cytosolic (Saayman et al., 2006, S. Afr. J. Enol. Vitic. 27(2):113-122). Comparing the sequences of malic enzymes from S. cerevisiae, S. pombe and Y. lipolytica showed that the first 55 and 85 amino acids of YIME and ScME, respectively, have no apparent counterpart in SpME (FIG. 1). This suggested that the MTS may be as long as, or contained within, the first 55 amino acid residues of YIME.
Construction of Vectors for Malic Enzyme Overexpression
A full length Yarrowia ME (YIME) ORF was obtained by polymerase chain reaction (PCR) amplification using 5′-end primer ME-F (SEQ ID NO:11, contains added NcoI site) and 3′-end primer ME-R (SEQ ID NO:12, contains added NotI site). These primers amplified the coding region from the ATG start codon to the stop codon, except that the second codon was changed from TTA (leu) to GTA (val) in order to engineer the added NcoI site. The PCR reaction mixture contained 1 μL of Y. lipolytica genomic DNA, 1 μL each of primers ME-F and ME-R (from 20 μM stocks), 22 μL water, and 25 μL Ex TaqTM premix 2×Taq PCR solution (TaKaRa Bio Inc., Siga, Japan). Amplification was carried out as follows: initial denaturation at 94° C. for 2 min, followed by 30 cycles of denaturation at 94° C. for 30 sec, primer annealing at 55° C. for 30 sec, and elongation at 72° C. for 90 sec. A final elongation period at 72° C. for 7 min was carried out, followed by reaction termination at 4° C. The DNA fragment amplified by the reaction was purified using the MinElute® Gel Extraction kit (QIAGEN, Valencia, Calif.) according to the manufacturer's protocol. The purified DNA was digested with NcoI and NotI, and cloned into an NcoI/NotI-cut plasmid having a pZP2-based vector backbone (see U.S. Pat. Appl. Publ. No. 2010/0159558 to yield the pME (SEQ ID NO:13) expression vector (FIG. 2A).
To construct a cytosolic version of Yarrowia ME, a DNA fragment encoding an N-terminally truncated version of YIME was created by PCR. This truncation removed the first 53 amino acids of YIME; the predicted MTS in this region was consequently removed with this sequence deletion. The YIME ORF lacking N-terminal amino acids 1-53 (“YIME-T2”) was prepared as follows. Using the above PCR conditions, 5′-end primer ME-TN2 (SEQ ID NO:8, contains added NcoI site) and 3′-end primer ME-T2 (SEQ ID NO:9, contains added NotI site) were used to amplify the YIME-T2 ORF plus 203 base pairs of the 3′-untranslated region of the ME gene from Y. lipolytica genomic DNA. The amplified fragment was purified as above, digested with NcoI and NotI, and cloned into NcoI/NotI-cut pME vector to yield pMET2 (FIG. 2B, SEQ ID NO:10).
In both pME and pMET2, the cloned coding sequences (full length or truncated YIME, respectively) were under the transcriptional control of the FBAIN promoter of Y. lipolytica fructose-bisphosphate aldolase gene (“FBAIN”, refer to U.S. Pat. No. 7,202,356). This promoter allows for overexpression of downstream gene sequences in Yarrowia species, for example. Also, both coding sequences are flanked at their 3′-ends by a terminator sequence from the Y. lipolytica PEX20 gene (GenBank Acc. No. AF054613). pMET2 had 203 bp of the 3′ untranslated sequence of the YIME gene between the ME-T2 coding region and the PEX20 terminator. Correctly ligated constructs were confirmed by plasmid minipreparation and digestion analyses accordingly.
The constructed chimeric gene expression cassettes can be characterized in shorthand as FBAIN::YIME::PEX20 and FBAIN::YIME-T2::PEX20. An alignment of the translated YIME and YIME-T2 polypeptides is shown in FIG. 3. The sequences as amplified above can also be considered to represent cDNA sequences given that the coding region of the Y. lipolytica ME gene does not contain introns. The sequences of the YIME and YIME-T2 ORFs are set forth as SEQ ID NOs:6 and 4, respectively.
Example 2
ME Overexpression and its Effect on Lipid Production in Y. lipolytica 
The effect of overexpressing wild type (full length) or cytosolic ME on the lipid content of different Y. lipolytica strains was determined using the constructs described in Example 1.
In the following gene overexpression studies, Y. lipolytica strains were transformed with either the FBAIN::YIME::PEX20 or FBAIN::YIME-T2::PEX20 chimeric gene expression cassette using plasmid pME or pMET2, respectively, that was digested with BssHII and SphI. For transformation control purposes, the strains used in each experiment were transformed with plasmid pBlue-YURA3 (FIG. 2C, SEQ ID NO:14) that was digested with KpnI and Sail. Plasmid pBlue-YURA3 was derived from the cloning vector pBluescript®-SK(−) (Stratagene, La Jolla, Calif.) that was modified to contain the Yarrowia URA3 gene (GenBank Acc. No. AJ306421) in the pBluescript®-SK(−) multiple cloning site. Transformants were selected on plates lacking uracil, as the experimental and control vectors bestow a Ura+ phenotype to the otherwise Ura− cells. Y. lipolytica strain Y2224 was used in these analyses; Y2224 is a Ura− strain of wild type strain ATCC #20362, and has a lipid content representative of wild type Yarrowia. The isolation of strain Y2224 is described in U.S. Pat. Appl. Publ. No. 2008/0254191, which is incorporated herein by reference.
Transformants were grown for 2 days in fermentation medium (FM, per liter: 6.70 g Yeast nitrogen base, 6.00 g KH2PO4, 2.00 g K2HPO4, 1.50 g MgSO4*7H2O, 20 g glucose, 5.00 g Yeast extract [BBL]), followed by 5 days of growth in high glucose medium (HGM, per liter: 80 g glucose, 2.58 g KH2PO4, 5.36 g K2HPO4, pH 7.5 [do not need to adjust]). After this incubation period, the lipid content (TFAs % DCW) and fatty acid profile of transformants were measured by gas chromatography as described in U.S. Pat. Appl. Publ. No. 2008/0254191.
Lipid Production in Strain Y2224 The lipid and fatty acid profiles of Yarrowia strain Y2224 transformed with pBlue-YURA3 (control), YIME, or YIME-T2 sequences are listed in Table 4. Four different (1-4) control transformants and eight (1-8) different transformants for YIME or YIME-T2 overexpression were analyzed. The detected fatty acids included 16:0 (palmitic acid), 16:1 (palmitoleic acid), 18:0 (stearic acid), 18:1 (oleic acid) and 18:2 (linoleic acid); the concentration of each fatty acid is presented as a weight percent of TFAs (i.e., “% TFAs”) in Table 4.







TABLE 4







Lipid content in Y. lipolytica strain Y2224


overexpressing full length or cytosolic ME.









Y2224
TFAs
% TFAs













Transformant
% DCW
16:0
16:1
18:0
18:1
18:2
















pBlue-YURA3-1
15.6
14.8
14.0
5.7
47.6
15.6


pBlue-YURA3-2
14.7
14.9
14.0
5.8
47.4
15.6


pBlue-YURA3-3
14.4
12.8
14.0
5.5
50.6
14.7


pBlue-YURA3-4
14.2
13.4
12.3
5.9
50.4
15.6


Average
14.7
13.98
13.6
5.7
49
15.4


pME-1
15.4
14.6
14.5
5.6
48.1
15.2


pME-2
16.3
14.3
14.2
5.8
49.2
14.3


pME-3
16.9
15.2
13.1
6.0
46.6
16.9


pME-4
15.4
14.9
14.4
5.5
47.4
15.5


pME-5
14.7
13.2
14.0
5.4
50.4
14.4


pME-6
14.7
11.4
13.9
5.0
53.2
14.1


pME-7
15.7
14.3
14.8
5.3
48.6
14.7


pME-8
13.8
14.3
14.8
5.4
48.5
14.6


Average
15.4
14.0
14.2
5.5
49
14.96


pMET2-1
15.1
14.2
14.7
5.2
48.1
14.2


pMET2-2
13.0
11.9
12.3
5.4
49.7
17.6


pMET2-3
17.6
14.0
14.1
5.4
49.0
14.0


pMET2-4
14.5
12.5
14.3
5.1
51.5
13.7


pMET2-5
15.5
13.6
14.2
5.3
49.4
13.9


pMET2-6
15.1
14.0
14.9
5.0
49.0
14.1


pMET2-7
15.0
14.2
14.9
5.0
48.4
14.4


pMET2-8
13.8
15.1
12.1
5.5
48.4
15.9


Average
14.95
13.7
13.9
5.2
49.2
14.7









As shown in Table 4, most transformants for YIME and YIME-T2 overexpression produced a similar amount of total lipids as the ones carrying control plasmid pBlue-YURA3.
These results altogether indicate that neither the overexpression of full length ME or cytosolic ME substantially alters lipid production in a Yarrowia strain having wild type lipid production capacity. Specifically, neither full length or cytosolic ME overexpression significantly elevated lipid production from the wild type baseline level of about 15 TFAs % DCW under the described analytical conditions.
Lipid Production in Strain Z1978U
The pME and pME-T2 overexpression vectors were also used to transform an engineered strain of Y. lipolytica, Z1978U, which is a Ura− strain of Z1978. The Z1978 strain can produce a lipid content greater than about 35 TFAs % DCW, with about 52 EPA % TFAs. Details regarding the development of strains Z1978 and Z1978U are provided in U.S. Appl. Publ. No. 2012/0052537 A1, which is incorporated herein by reference. The lipid and fatty acid profiles of the Z1978U transformants are listed in Table 5. Four different (1-4) control transformants and eight (1-8) different transformants for YIME or YIME-T2 overexpression were analyzed. The fatty acids detected in the total fatty acids included 18:0, 18:1, 18:2, dihomo-gamma-linolenic acid (DGLA) and eicosapentaenoic acid (EPA).







TABLE 5







Lipid content in Y. lipolytica strain Z1978U overexpressing full


length or cytosolic ME











TFAs

EPA


Z1978U
%
% TFAs
%














Transformant
DCW
18:0
18:1
18:2
DGLA
EPA
DCW

















pBlue-YURA3-1
34.1
3.4
6.9
13.5
2.6
51.5
17.6


pBlue-YURA3-2
33.8
3.4
6.9
13.4
2.6
52.2
17.6


pBlue-YURA3-3
33.9
3.3
7.0
13.5
2.6
51.5
17.5


pBlue-YURA3-4
33.9
3.4
7.0
13.6
2.6
51.4
17.4


Average
33.9
3.4
7.0
13.5
2.6
51.7
17.5


pME-1
35.2
3.3
6.9
14.5
2.6
51.7
18.2


pME-2
38.9
3.3
6.9
16.5
2.6
51.8
20.1


pME-3
35.9
3.3
6.9
17.0
2.6
52.2
18.7


pME-4
35.4
3.3
6.9
17.1
2.6
51.6
18.3


pME-5
35.2
3.3
6.9
17.0
2.6
51.8
18.2


pME-6
35.7
3.3
6.9
15.4
2.6
51.7
18.4


pME-7
36.0
3.3
7.0
15.5
2.6
51.6
18.6


pME-8
35.7
3.2
6.9
16.5
2.6
51.8
18.5


Average
36.0
3.3
6.9
16.2
2.6
51.8
18.6


pMET2-1
37.8
2.2
5.7
16.8
3.4
54.4
20.5


pMET2-2
37.9
3.3
7.0
17.2
2.6
51.8
19.6


pMET2-3
39.6
3.3
6.9
17.2
2.6
51.7
20.5


pMET2-4
37.4
3.3
7.0
16.8
2.6
51.5
19.2


pMET2-5
35.7
3.4
7.1
16.8
2.6
51.4
18.3


pMET2-6
37.7
3.1
7.1
16.6
2.7
51.7
19.5


pMET2-7
34.2
3.3
6.9
16.5
2.6
51.9
17.8


pMET2-8
38.4
3.3
7.0
16.6
2.6
51.7
19.9


Average
37.3
3.1
6.8
16.9
2.7
52.0
19.4









As shown in Table 5, most of the pME transformants showed a modest increase in lipid content ( ̃5%) compared to the control transformants. However, most of the pME-T2 transformants produced a significantly higher lipid content (>10%) compared to the control transformants; particular examples are listed in bold in the table. The EPA content (EPA % DCW) in the pME-T2 transformants generally increased with the rise in total lipid content. Expression of cytosolic ME thus allowed increased production of lipids in strain Z1978U. This result is in contrast to the observations made above with strain Y2224 (has lipid levels representative of wild type Yarrowia), which on average did not exhibit a significant enhancement of lipid production with cytosolic ME overexpression.
These results altogether indicate that overexpression of cytosolic ME can significantly increase lipid production in a transgenic Yarrowia strain having a lipid production capacity over about 35 TFAs % DCW. Specifically, cytosolic ME overexpression in this strain increased lipid production by over 10% with respect to the control.
Lipid Production in Strain Z5567U
The above plasmids were similarly used to transform another engineered strain of Yarrowia lipolytica, Z5567U, which is a Ura− strain of Z5567. The Z5567 strain can produce a lipid content greater than about 55.0 TFAs % DCW, with about 27.0 EPA % DCW. Details regarding the development of strains Z5567 and Z5567U are provided in U.S. Appl. Publ. No. 2012/0052537 A1, which is incorporated herein by reference. As shown in Table 6, four different (1-4) control transformants (pBlue-YURA3) and ten (1-10) different transformants for YIME or YIME-T2 overexpression in Z5567U were analyzed for lipid production. The fatty acids detected in the total fatty acids included 18:0, 18:1, 18:2, DGLA and EPA. The control and pME (full length Yarrowia ME) Z5567U transformants produced similar levels of total lipid. However, pMET2 (cytosolic Yarrowia ME) transformants produced significantly more total lipid compared to the control and pME transformants. Particular examples of total lipid levels that were elevated in pMET2 transformants are listed in Table 6 in bold.







TABLE 6







Lipid content in Y. lipolytica strain Z5567U


overexpressing full length or cytosolic ME.











TFAs

EPA


Z5567U
%
% TFAs
%














Transformant
DCW
18:0
18:1
18:2
DGLA
EPA
DCW

















pBlue-YURA3-1
55.5
2.7
6.2
16.9
5.3
49.1
27.2


pBlue-YURA3-2
54.1
2.6
6.2
16.8
5.3
49.0
26.5


pBlue-YURA3-3
54.9
2.7
6.2
16.6
5.3
48.9
26.9


pBlue-YURA3-4
54.7
2.6
6.1
16.5
5.3
49.2
26.9


Average
54.8
2.65
6.2
16.7
5.3
49.05
26.9


pME-1
52.5
1.7
6.0
14.5
5.9
50.8
26.7


pME-2
54.9
2.6
6.0
16.5
5.4
49.8
27.3


pME-3
55.7
2.7
6.3
17.0
5.3
48.8
27.2


pME-4
52.8
2.6
6.6
17.1
5.0
48.2
25.4


pME-5
54.6
2.5
6.8
17.0
5.0
47.7
26.1


pME-6
50.6
2.3
5.8
15.4
5.3
51.2
25.9


pME-7
51.8
2.1
6.0
15.5
5.5
50.4
26.1


pME-8
54.6
2.6
6.0
16.5
5.4
49.2
26.9


pME-9
52.1
2.2
6.6
16.1
5.3
48.7
25.4


pME-10
52.8
2.4
6.4
16.5
5.0
48.0
25.3


Average
53.2
2.4
6.25
16.2
5.3
49.3
26.2


pMET2-1
54.9
2.7
6.2
16.8
5.3
49.3
27.0


pMET2-2
56.0
2.8
6.5
17.2
5.2
47.9
26.8


pMET2-3
59.4
2.7
6.5
17.2
5.2
48.3
28.7


pMET2-4
55.8
2.7
6.3
16.8
5.2
48.6
27.1


pMET2-5
55.8
2.7
6.3
16.8
5.3
49.4
27.6


pMET2-6
57.2
2.7
6.2
16.6
5.3
48.7
27.9


pMET2-7
56.9
2.8
6.2
16.5
5.4
49.0
27.9


pMET2-8
55.5
2.2
5.9
16.6
5.2
50.6
28.1


pMET2-9
58.8
2.7
6.3
16.6
5.4
59.1
28.8


pMET2-10
54.3
2.6
7.1
17.5
4.9
47.3
25.7


Average
56.5
2.7
6.35
16.9
5.2
49.8
27.6









Expression of cytosolic ME also allowed increased production of lipids in strain Z5567U. Therefore, these results further indicate that overexpression of cytosolic ME can significantly increase lipid production in a transgenic Yarrowia strain having a lipid production capacity over about 35 TFAs % DCW. In this particular case, cytosolic ME overexpression increased lipid production in a strain having a lipid production capacity of over 50 TFAs % DCW under the described analytical conditions.
The results obtained using the engineered high lipid strains are in contrast to the observations made above with strain Y2224 (has lipid levels representative of wild type Yarrowia), which on average did not exhibit a significant enhancement of lipid production with cytosolic ME overexpression. Another difference was the effect on lipid content induced by full length ME versus cytosolic ME in Y2224 and Z5567U. In Y2224, the respective effects of overexpressing full length ME and cytosolic ME on lipid content compared to control were relatively similar. However, full length ME overexpression in strain Z5567U generally reduced lipid content compared to control, whereas cytosolic ME induced elevated lipid levels. Yet, in strain Z1978, the full length ME modestly increased lipid content, while cytosolic ME had a significantly more pronounced effect on increasing lipid levels. These data altogether indicate that cytosolic ME enhances the lipid production capacity of high lipid-producing strains of Yarrowia. 
Based on these data, the NADPH reducing equivalents produced by cytosolic ME are likely a factor in lipid biosynthesis in Yarrowia strains that can produce approximately over 35 TFAs % DCW. NADPH produced by cytosolic ME in lower lipid producing Yarrowia strains does not appear to play as significant a role in lipid production. These observations could indicate that NADPH produced by cytosolic ME becomes more necessary for lipid biosynthesis as lipid production capacity increases in a strain (i.e., more lipid synthesis may require more reductive capacity). Interestingly, however, even though Z5567U produces more lipid than Z1978U, the effect of cytosolic ME overexpression on lipid production was more pronounced in Z1978U compared to Z5567U. For example, while cytosolic ME overexpression increased lipid production by over 10% in Z1978U (discussed above), lipid production in Z5567U was increased by over about 3% compared to the control.
In summary, the above results indicate that overexpression of cytosolic ME can significantly increase lipid production in transgenic Yarrowia strains having a lipid production capacity over about 35 TFAs % DCW.
Example 3
Deletion of the Gene Encoding ME and its Impact on Lipid Production in Y. lipolytica 
Lipid production in Yarrowia lacking a native ME gene and in elevated lipid-producing transformants thereof was measured. Elevated lipid production was induced by diacylglycerol acyltransferase-2 (DGAT2) overexpression.
Malic Enzyme Gene Deletion
Plasmid pME-KO (FIG. 6, SEQ ID NO:15) was constructed to delete the ME gene in wild type Yarrowia through homologous recombination. FIG. 5 shows the general scheme of the disruption strategy. The 5′- and 3′-flanking regions of the ME gene were amplified by PCR using the following primer pairs: YME-5-1 (SEQ ID NO:16)/YME-5-2 (SEQ ID NO:17) for the 5′-flanking region, and YME-3-1 (SEQ ID NO:18)/YME-3-2 (SEQ ID NO:19) for the 3′-flanking region.
The PCR amplification was performed using Yarrowia genomic DNA as template. The reaction mixture contained 1 μL of the genomic DNA, 1 μL each primer (from 20 μM stocks), 22 μL water, and 25 μL Ex TaqTM premix 2×Taq PCR solution. Amplification was carried out as follows: initial denaturation at 94° C. for 2 min, followed by 30 cycles of denaturation at 94° C. for 30 sec, annealing at 55° C. for 30 sec, and elongation at 72° C. for 90 sec. A final elongation cycle at 72° C. for 7 min was carried out, followed by reaction termination at 4° C.
The two PCR products were cloned between the BamHI and EcoRI sites of pBlue-YURA3 in a three piece ligation reaction. The PCR product of the 5′-flanking region was digested with EcoRI and XhoI, and the PCR product of the 3′-flanking region was digested with XhoI and BamHI. Plasmid pBlue-YURA3 was digested with BamHI and EcoRI. The three DNA fragments were ligated together such that the two PCR products are joined together at an XhoI site, and the linked PCR products reside in between the BamHI and EcoRI sites of the vector. The resulting plasmid, pME-KO (SEQ ID NO:15) is shown in FIG. 6.
Y. lipolytica strain Y2224 (Ura− representative of wild type Yarrowia, see above) was transformed with pME-KO digested with SphI. Transformants were plated on Ura-minus plates to select for integration of the constructs. Ura+ transformants were screened by colony PCR, using primers YME-5-confirm-1 (SEQ ID NO:20) and YME-5-confirm-2 (SEQ ID NO:21).
The reaction mixture contained 0.5 μL of each primer (20 μM stock), 14 μL water and 15 μL Ex TaqTM premix 2×Taq PCR solution. A small amount of cells was picked from the plate and added to the reaction mixture. PCR conditions were: initial denaturation at 95° C. for 5 min, followed by 35 cycles of denaturation at 94° C. for 20 sec, annealing at 55° C. for 20 sec, and elongation at 72° C. for 60 sec. A final elongation cycle at 72° C. for 7 min was carried out, followed by reaction termination at 4° C.
If the digested plasmid integrated into the Yarrowia genome at the ME locus, a  ̃1-kb fragment would have been generated by PCR. Three out of 36 transformants produced the expected PCR product. These three transformants were patched onto minimal media plates containing 350 μg/mL 5-fluoroorotic acid (5-FOA) (U.S. Pat. Appl. Publ. No. 2009-0093543) to select for cells that would undergo the second round of recombination to become Ura−. Cells that underwent the second round of recombination could either have (i) lost the wild type ME gene along with the deletion construct, or (ii) lost the deletion construct only, in which case the ME gene is left intact (refer to FIG. 5).
Sixteen colonies from the 5-FOA plates were selected and tested by colony PCR using primers YME-3-confirm-1 (SEQ ID NO:22) and YME-3-confirm-2 (SEQ ID NO:23). A  ̃1.3-kb fragment was expected if the ME gene was deleted from the strain. PCR conditions were the same as above with the confirmation PCR for the first round of recombination. Two of the Ura− strains produced a PCR product having the correct size ( ̃1.3 kb), indicating that these transformants contained the deletion. Both transformants were further confirmed for ME gene deletion by PCR using primers YME-5-confirm-2 and YME-3-confirm-1. This primer pair would amplify a 250-bp product if the ME gene was knocked out, or a  ̃1.7-kb product if the ME locus is wild type. Both Ura− strains produced the 250-bp PCR fragment, thereby confirming that they were ME-deleted derivatives of strain Y2224.
Lipid Production in ME-deleted Yarrowia 
Lipid measurements were made in ME-deleted strains to determine the role of ME in Yarrowia lipid production. As described below, ME-deleted Y2224 cells transformed with pBlue-YURA3, pME, or pME-T2 allowed this assessment with respect to native fatty acid synthesis.
A Ura−, ME− strain of Y2224 was transformed with (i) pBlue-YURA3 digested with EcoRI and Sail, (ii) pME digested with BssHII and SphI, or (iii) pME-T2 digested with BssHII and SphI. Transformants (selected on Ura-minus plates) were grown for 2 days in FM, followed by 5 days in HGM. The lipid content and fatty acid profiles of two transformants selected from each of the pBlue-YURA3, pME and pME-T2 transformations are shown below in Table 7; measurements were also made on two separate cultures of wild type Yarrowia strain ATCC #20362. The fatty acids detected in the total fatty acids included 16:0, 16:1, 18:0, 18:1 and 18:2.







TABLE 7







Lipid content in Y2224-ME− strains transformed


with pBlue-YURA3, pME, or pME-T2










TFAs
% TFAs














% DCW
16:0
16:1
18:0
18:1
18:2

















ATCC #20362
17.3
15.1
13.9
5.4
46.6
15.7


ATCC #20362
17.1
15.0
13.7
5.6
46.8
15.6


Y2224-ME− + pBlue-
17.2
15.2
13.7
5.5
46.3
16.0


YURA3-1


Y2224-ME− + pBlue-
16.8
15.1
13.9
5.5
46.8
15.6


YURA3-1


Y2224-ME− + pME-1
15.1
14.5
12.9
5.9
47.9
15.4


Y2224-ME− + pME-2
16.9
17.1
13.0
6.0
42.2
18.1


Y2224-ME− + pMET2-1
17.3
15.1
13.6
5.5
46.4
16.1


Y2224-ME− + pMET2-2
16.2
15.1
13.3
5.6
46.2
16.4









ME deletion did not appear to have any effect on lipid production and fatty acid profile in a wild type Yarrowia context, as TFAs % DCW and % TFAs values were similar between wild type strain ATCC #20362 and Y2224-ME−+pBlue-YURA3 transformants (Table 7). This was a fair comparison given that, aside from the ME deletion, the Y2224-ME− transformants only differed from ATCC #20362 at the URA3 locus and by containing a portion of the pBlue-YURA3 control vector.
The transformation of Y2224-ME− strains with pME or pME-T2 allowed a comparison of the effects of overexpressing cytosolic ME or full length ME on lipid metabolism without any background effect by the native full length ME. Consistent with the results described above in which the lipid content was not significantly altered in strain Y2224 transformed with either pME or pME-T2, there was no apparent effect on lipid metabolism when either full length ME or cytosolic ME was overexpressed in Y2224-ME− (Table 7).
Finally, in comparing the lipid profiles of the Y2224-ME−+pBlue-YURA3 transformants with the lipid profiles of the Y2224-ME−+pME or pME-T2 transformants, there was no significant effect on lipid content with the rescue of ME (cytosolic or full length) expression in Y2224. This is consistent with there being no discernible difference in lipid metabolism between ATCC #20362 and Y2224-ME−+pBlue-YURA3;i.e., if removing ME had no effect on lipid production, then adding ME back should likewise not have had any effect.
The results obtained herein with strain Y2224 altogether indicate that ME, whether full length or cytosolic, does not significantly affect lipid metabolism in Yarrowia that has not been modified for increased lipid production such as with transgenic strains Z1978U and Z5567U.
Example 4
Heterologous Co-expression of Polynucleotides Encoding Cytosolic Malic Enzyme and Malate Dehydrogenase in Y. lipolytica 
This Example describes co-expressing polynucleotides, one encoding cytosolic ME and the other encoding malate dehydrogenase (MDH), in a heterologous manner in Y. lipolytica. This analysis is performed to determine whether MDH expression can augment the increase in lipid production that occurs when expressing cytosolic ME in Y. lipolytica that has a lipid content of at least about 35% of dry cell weight.
Primers YMDH1-F (SEQ ID NO:31) and YMDH1-R (SEQ ID NO:32) are used to PCR-amplify a polynucleotide encoding a Y. lipolytica mitochondrial MDH (SEQ ID NO:30). This polynucleotide is cloned into a vector such that it is contained within an expression cassette having suitable promoter and terminator sequences for expressing the polynucleotide in Yarrowia. 
Transformation procedures are performed to co-express YIME-T2 and Yarrowia mitochondrial MDH in a Y. lipolytica strain that has a lipid content of at least about 35% of dry cell weight. The lipid profiles of transformants are measured as described above.

## Claims

1. A transgenic Yarrowia species comprising:
(i) a polynucleotide encoding a cytosolic malic enzyme;
(ii) a lipid content that is at least about 35% by weight of the dry cell weight of said Yarrowia species; and
(iii) an engineered polyunsaturated fatty acid (PUFA) biosynthetic pathway,
wherein overexpression of the cytosolic malic enzyme increases lipid content.

2. The Yarrowia species of claim 1, wherein said cytosolic malic enzyme comprises a dysfunctional mitochondrial targeting sequence.

3. The Yarrowia species of claim 1, wherein said cytosolic malic enzyme does not comprise a mitochondrial targeting sequence.

4. The Yarrowia species of claim 1, wherein said cytosolic malic enzyme comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:5 and has malic enzyme activity.

5. The Yarrowia species of claim 1, wherein said lipid content is at least about 50% by weight of the dry cell weight of said Yarrowia species.

6. The Yarrowia species of claim 1, wherein said engineered PUFA biosynthetic pathway produces at least one PUFA selected from the group consisting of linoleic acid, alpha-linolenic acid, gamma-linolenic acid, stearidonic acid, eicosadienoic acid, dihomo-gamma-linolenic acid, arachidonic acid, eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosatetraenoic acid, omega-3 docosapentaenoic acid, omega-6 docosapentaenoic acid and docosahexaenoic acid.

7. The Yarrowia species of claim 6, wherein said engineered PUFA biosynthetic pathway produces eicosapentaenoic acid.

8. The Yarrowia species of claim 1, which species is Yarrowia lipolytica.

9. A method for increasing lipid content of a transgenic Yarrowia species that comprises:
a) culturing the transgenic Yarrowia species of claim 1, wherein a microbial oil comprising at least one PUFA is produced, and
b) optionally, recovering the microbial oil of step (a).

10. The method of claim 9, wherein said cytosolic malic enzyme comprises a dysfunctional mitochondrial targeting sequence.

11. The method of claim 9, wherein said cytosolic malic enzyme does not comprise a mitochondrial targeting sequence.

12. The method of claim 9, wherein said cytosolic malic enzyme comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:5 and has malic enzyme activity.

13. The method of claim 9, wherein said lipid content is at least about 50% by weight of the dry cell weight of said Yarrowia species.

14. The method of claim 1, wherein said engineered PUFA biosynthetic pathway produces at least one PUFA selected from the group consisting of linoleic acid, alpha-linolenic acid, gamma-linolenic acid, stearidonic acid, eicosadienoic acid, dihomo-gamma-linolenic acid, arachidonic acid, eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosatetraenoic acid, omega-3 docosapentaenoic acid, omega-6 docosapentaenoic acid, and docosahexaenoic acid.

15. The method of claim 14, wherein said engineered PUFA biosynthetic pathway produces eicosapentaenoic acid.

16. The Yarrowia species of claim 4, wherein said cytosolic malic enzyme comprises SEQ ID NO:5.

17. The Yarrowia species of claim 6, wherein said cytosolic malic enzyme comprises SEQ ID NO:5.

18. The method of claim 12, wherein said cytosolic malic enzyme comprises SEQ ID NO:5.

19. The method of claim 14, wherein said cytosolic malic enzyme comprises SEQ ID NO:5.