WO2014182657A1 - Increasing homologous recombination during cell transformation - Google Patents

Abstract

Disclosed is a method of increasing the efficiency of targeted integration during genetic transformation protocols comprising the steps of synchronizing cells prior to transforming them. The inventive methods increase the prevalence of homologous recombination during cell transformation, thus allowing the isolation of desired recombinant cells without screening a large number of transformants or using mutant cells.

Description

Increasing Homologous Recombination

Buying Cell Transformation

5 RELATED APPLICATIONS

This application claims the benefit of priority to United States Provisional Patent Application serial number 61/819,746, filed May 6, 2013, which is hereby incorporated by reference.

BACKGROUND

H⁾ Integration of a UNA fragment in a host genome requires action of a double-strand break (DSB) repair mechanism. Two major DSB repair pathways have been identified: Non Homologous End Joining (NHEJ) and Homologous Recombination (HR). NH.EJ results in random integration of a nucleotide fragment. NHEJ is referred to as "nonhomologous" because the break ends are directly ligated without the need for a homologous

1 S template; in contrast, HR requires a homologous sequence to guide repair.

NHEJ typically utilizes short homologous D A sequences called microhomologies to guide repair. These inicrohomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately. Imprecise repair leading to loss of nucleotides can also 0 occur, but is much more common when the overhangs are not compatible. Inappropriate NHEJ cart lead to translocations and telomere fusion, which are hallmarks of tumor cells.

NHEJ is observed, for example, when cycling (asynchronous) Y mmia Upolytica cells are transformed with integrating constructs. As a result, the introduced UNA integrates into the genome randomly, and so the number of transformants that must be screened to 5 obtain targeted integrations can be prohibiti vely large.

On the other hand, HR is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. HR is conserved across all three domains of life, as well as viruses, suggesting that it is a nearly uni versal biological mechanism. In diploid organisms, double-strand breaks can be repaired 0 via HR using the second copy of the affected genomic locus as a template. HR is also observed during horizontal gene transfer to exchange genetic material between different strains and species of bacteria and viruses, in addition, the HR pathway is utilized by investigators to direct specific changes to a chromosomal (gene targeting) or extra- chromosomal (often in recombination cloning) locus. When a nucleotide fragment introduced into the cells is flanked by sequences homologous to a genomic or extra- chromosomal locus or a second fragment flanked by similar sequences, HR results in targeted integration of the nucleotide fragment at that locus or the joining of^" the fragments by recombination.

Differences in gene targeting efficiency between even closely related organisms can be explained by a more active NHEJ system, a less efficient HR system, or both. For example, in organisms with an inefficient HR system targeting of genes at a desired locus is difficult or impossible.

Whether NHEJ or HR is used to repair double-strand breaks also depends on the particular phase of the ceil cycle. HR repairs ^'DNA before a cell eaters mitosis (M phase); it occurs during and shortly after DNA replication (i.e., in the S and G? phases of the cell cycle), when sister chromatids are more easily available. Compared to homologous chromosomes, which are similar to other chromosomes but often have different alleles, sister chromatids are an ideal template for HR because they are identical copies of a given chromosome. In contrast to HR, NHEJ is predominant in the Gt phase of the cell cycle, when the cell is growing but not yet ready to divide, it occurs less frequently after the Oj phase, but maintains at least some activity throughout the cell cycle. See Figure 1.

Currently, when introducing genetic material into a microorganism, cycling cells are transformed and a large number of transfonaants are screened to identify those with targeted insertions. Alternatively, mutant cells that favor HR ma be used; however, such mutations can compromise the ability of die mutant cells to repair double stranded breaks in their chromosomes, which affects telomere protection and leads to genomic instability. Accordingly, there exists a need for a method of increasing the prevalence of HR during cell transformation to obtain the desired recombinant ceils without screening a large number of trans forsiiauts or using mutant ceils .

SUMMARY OF THE INVENTION

In certain embodiments, the invention relates to a method, comprising the steps of: providing a plurality of cells;

7 arresting the cell cycle of the plurality of cells, thereby forming a first mixture comprising a plurality of arrested cells; and

subjecting the plurality of arrested cells to transformation conditions, thereby forming a plurality of genetically engineered cells comprising a first fraction of genetically engineered cells and a second fraction of genetically engineered cells, wherein the first fraction of genetically engineered cells comprises the desired transf miation and the second fraction of genetically engineered cells does not.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein arresting the eel! cycle of the plurality of ceils comprises (i) elutriation, (ti) utilizing ceil cycle mutants, (iii) exposing the plurality of ceils to a chemical, or (iv) limiting the nutrition of the ceils.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the fraction of the plurality of genetically engineered cells comprising the desired transformation (i.e., the first fraction) is larger than the fraction of genetically engineered cells comprising the desired transformation if the plurality of cells had been subjected to transformation conditions without first being arrested.

in certain embodiments, the invention relates to a method, comprising the steps of; providing a plurality of cells;

contacting the plurality of cells with a ribonucleotide reductase inhibitor at a first temperature for a first period of time, thereby forming a first mixture comprising a plurality of arrested ceils; and

subjecting the plurality of arrested cells to transformation, conditions, thereby formin a plurality of genetically engineered cells comprising a first fraction of genetically engineered cells and a second fraction of genetically engineered cells, wherein the first fraction of genetically engineered cells comprises the desired transformation and the second fraction of genetically engineered cells does not.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the fraction of the plurality of genetically engineered cells comprising the desired transformation (i.e., the first fraction) is larger than the fraction of genetically engineered cells comprising the desired transformation if the pluralit of cells had been subjected to transformation conditions without first being contacted with the ribonucleotide reductase inhibitor at the first temperature for the first period of time.

In certain embodiments, the invention, relates to a genetically engineered cell made by any one of the aforementioned methods,

BRIEF ^'DESCRIPTION OF THE FIGURES

Figure 1 depicts a schematic showing the cell cycle, BR usually -repairs DNA before the ceil eaters mitosis (M phase). HR is dominant daring and shortly after DMA replication, during the S and Ga phases of the cell cycle.

Figure 2 depicts results from PCR amplification of NAT and YAIJ0D2 !3S4g interna! sequences, ^'Tie results suggest that 4 of 10 transformants have replaced YAIJ0D2l3S4g with NAT when cells were arrested in S phase prior to transformation. In contrast, in the absence of cell cycle arrest, no transformants showed both lack of YAL1QD2 i S4g and presence of NA F, suggesting only mndom integration of the NAT gene in the genome.

Figure 3 depicts results from PCR analysis of genomic DNA isolated from the transformants identified in Figure 2 and the wild type control strain, reconfirming the presence of NAT and absence of YALI()D21384g sequences in three of the four transformants (#7 genomic DNA preparation failed). The size of a PCR product amplified with primers external to the YAI 0D213S4g locus shows that the YAIJ0D213S4g gene has been replaced with the smaller NA T gene.

Figure 4 depicts various yeast cells useful in the methods of the invention.

Figure 5 depicts the results of PC analysis of 48 hygro ycin resistant transformants isolated from differing transformation conditions (top: no hydroxyurea; bottom: with 50 mM hydroxyurea) with the external forward primer NP1033 and the hph marker-specific reverse primer NP656. Correct integration of hph at the TGL3 locus yields a 990-bp deletion-specific product, while random integration leads to no product amplification. DETAILED DESCRIPTION OF THE INVENTION

Overview

In certain embodiments, the invention relates to a method of increasing the efficiency of targeted integration during genetic transformation protocols, comprising the steps of synchronizing ceils in S phase prior to transforming them.. Transforming cells that are in S phase with DNA carrying sequences homologous to genomic DNA increases the likelihood that the introduced DNA will integrate at the homologous locus (via MR), rather than randomly in the genome (via NHEJ). This targeting of DNA integration allows for accurate deletion or alteration of genomic information with high efficiency and without permanently altering the capacity of the organism to repair its own genome. This method should also be applicable to increasing the efficiency of homologous recombination in extrachromosomal DNA (e.g., linear DNA, plasmids, YACs), and could be relevant in organisms with an unfavorable balance of HR to NHEJ.

Definitions

The term "activate" or "activation" as used herein with reference to a biologically active molecule, such as an enzyme, indicates any modification in the genome and/or proteome of a microorganism that increases the biological activity of the biologically active molecule in the microorganism. Exemplary activations include but, are not limited, to modifications that result in the conversion of the molecule from a biologically inactive form to a biologically active form and from a biologically active form to a biologically more active form, and modifications that result in the expression of the biologically active molecule in a microorganism wherein the biologically active molecule was previously not expressed. For example, activation of a biologically active molecule can be performed by expressing a native or heterologous polynucleotide encoding for the biologically active molecule in the microorganism, by expressing a native or heterologous polynucleotide encoding for an enzyme involved in the pathway for the synthesis of the biological active moiecuie in the microorganism, by expressing a native or heterologous molecule that enhances the expression of the biologically acti ve molecule in the microorganism.

The term "enzyme" as used herein refers to an substance that catalyzes or promotes one or more chemical or biochemical reactions, which usually includes enzymes totally or partially composed of a polypeptide, but can include enzymes composed of a different molecule including polynucleotides. Art "exogenous gene" or "heterologou gene" is a nucleic acid thai codes for the expression of an RNA and/or protein that has been introduced into a cell (e.g., by transfornrntion/transfecfion), and is also referred to as a "transgene," A cell comprising an exogenous gene may be referred to as a recombinant ceil, into which additional exogenous gene(s) may be introduced. The exogenous gene may be from a different species (and so heterologous), or from the same species (and so homologous), relative to the cell being transformed. Thus, an exogenous gene can include a homologous gene that occupies a different location in the genome of the cell or is under different control relative to the endogenous copy of the gene. An exogenous gene may be present in more than one copy in the ceil. An exogenous gene may he maintained in a cell as an insertion into the genome (nuclear or plastic!) or as an episoma.l molecule.

A gene or DNA sequence is "heterologous" to a microorganism if it is not part of the genome of that microorganism as it normally exists (i.e., it is not naturally part of the genome of the wild-type version microorganism).

The terms "host", "host cells" and "recombinant host cells" are used interchangeably herein and refer not only to the particular subject eel! but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not in fact, be iden tical to the parent cell but are still included within the scope of the term as used herein.

"Inducible promoter" is a promoter that mediates transcription of an operably linked gene in response to a particular stimulus.

"In operable linkage" describes a functional linkage between two nucleic acid sequences, such as control sequence (typically a promoter) and the linked sequence (typically a sequence that encodes a. protein, also called a coding sequence). A promoter is in operable linkage with an exogenous gene if it can mediate transcription of the gene.

As used herein, the term "microorganism'' includes prokaryotic and eukaryotic microbial species from the Domains Bacteria and Euk rytm, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista. The terms "microbial cells" and "microbes" are used interchangeably with the term microorganism.

The term "native" or "endogenous" as used herein with reference to molecules, and in particular enzymes and polynucleotides, indicates molecules that are expressed in the organism in which they originated or are Found in nature, independently on the level of expression that can be lower, equal or higher than the level of expression of the molecule in the native microorganism.

The terms "piasniid," "vector," "construct," and "cassette" 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 molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a ceil, "Transformation cassette" refers to a specific vector containing a foreign gene and having elements, in addition to the foreign gene, that facilitate transformation of a particular host cell. "Expression cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.

The term "polynucleotide" is used herein interchangeably with the term "nucleic acid" and refers to an organic polymer composed of two or more monomers including nucleotides, nucleosides or analogs thereof, including but not limited to single-stranded or double-stranded, sense or antisense deoxyribonucleic acid (DNA) of any length and, where appropriate, single stranded or double stranded, sense or antisense ribonucleic acid (RNA) of any length, including siRNA. The term "nucleotide" refers to any of several compounds that consist of a ribose or deoxyribose sugar joined to a purine or a. pyrimidine base and to a phosphate group, and that are the basic structural units of nucleic acids. The term "nucleoside" refers to a compound (as guanostne or adenosine) that consists of a purine or pyrimidine base combined with deoxyribose or ribose and is found especially in nucleic acids. The term "nucleotide analog" or "nucleoside analog" refers, respectively, to a nucleotide or nucleoside in which one or more individual atoms have been replaced with a different atom or with a different functional group. Accordingly, the term polynucleotide includes nucleic acids of any length, DNA, RNA, analogs and fragments thereof. A polynucleotide of three or more nucleotides is also called nucleotidic oligomer or oligonucleotide. The term "portion" refers to peptides, oligopeptides, polypeptides, protein domains, and proteins. A nucleotide sequence encoding a "portion of a protein" includes both nucleotide sequences that can be transcribed and/or translated and nucleotide sequences thai must undergo one or more recombination events to be transcribed and/or translated- For example, a nucleic acid may comprise a nucleotide sequence encoding one or more amino acids of a selectable marker protein. This nucleic acid can be engineered to recombine with one or more different nucleotide sequences that encode the remaining portion of the protein. Such nucleic acids are useful for generating knockout mutations because only recombination with the target sequence is likely to reconstitute the full-length selectable marker gene whereas random-integration events are unlikely to result in a nucleotide sequence that can produce a functional marker protein. A "biologically-active portion" of polypeptide is any amino acid sequence found in the polypeptide's amino acid sequence that is less than the full amino acid sequence but can perform the same function as the full- length polypeptide.

"Promoter" is a nucleic acid control sequence that directs transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

The ter "protein" or "polypeptide" as used herein indicates an organic polymer composed of two or more amino acidic monomers and/or analogs thereof. As used herein, the term "amino acid" or "amino acidic monomer" refers to any natural and/or synthetic amino acids including glycine and both D or L optical isomers. The term "amino acid analog" refers to an amino acid in which one or more individual atoms have been replaced, either with a different atom, or with a different functional group.

Accordingly, the term polypeptide includes amino acidic polymer of any length including full length proteins, and peptides as well as analogs and fragments thereof A polypeptide of three or more amino acids is also called a protein oligomer or oligopeptide.

A cell, nucleic acid, protein, or vector is "recombinant" if it has been modified by the introduction of an exogenous nucleic acid or the alteration of a native nucleic acid. Thus, e.g., recombinant ceils can express genes that are not found within the native (non- reeombinani) form of the cell, or express native genes differently than those genes are expressed by a non-reeornbiiiant ceil. Recombinant cells can, without limitation, include recombinant nucleic acids that encode for a gene product or for suppression elements such as imitations, knockouts, antisense, interfering RNA (RNAi) or dsRNA that reduce the levels of active gene product in a ceil. A "recombinant nucleic acid" is a nucleic acid 5 originally formed in vitro, in general, by the manipulation of nucleic acid, e.g., using polymerases, Hgases, exonucleases, and endonucleases, or otherwise is in a form not normally found in nature. Recombinant nucleic acids may be produced, for example, to place two or more nucleic acids in operable linkage. Thus, an isolated nucleic acid or an expression vector formed in vitro by ligat ig DNA molecules that are not normally joined

H⁾ in nature, are both considered recombinant for the purposes of this invention. Once a recombinant nucleic acid is made and introduced into a host cell or organism, it may replicate using the in vivo cellular machinery of the host cell; however, such nucleic acids,, once produced recombinant!)', although subsequently replicated intraecllolariy, are still considered recombinant for purposes of this invention. Similarly, a "recombinant protein" is

I S a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid.

"Transformation" refers to the transfer of a nucleic acid fragment into a host organism or the genome of a host organism:, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as

20 "recombinant", "transgenic" or "transformed" organisms. Thus, isolated polynucleotides of the present invention can be incorporated into recombinant constructs, typically DNA constructs, capable of introduction into and replication in a host cell. Such a construct can be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell. Typically,

25 expression vectors include, for example, one or more cloned genes under the transcriptional control of 5' and 3' regulatory sequences and a selectable marker. Such vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentaily-regulated, or location-specific expression), a transcription initiation start site, a ribosorae binding site, a transcription 0 termination site, and/or a polyadenyla ion signal. Microbe Engineering

A. Oyeryiew

In certain embodiments of the invention, a microorganism is genetically modified to improve or provide e novo growth characteristics on a variet of feedstock materials.

Genes and gene products may be introduced into microbial host cells. Suitable host cells for expression of the genes and nucleic acid molecules are microbial hosts within the fungal or bacterial families and which grow over a wide range of temperature, pH values, and solvent, tolerances.

E. coli is well suited for use as the host microorganism in the fermentative processes of the invention.

Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are known to those skilled in the art. Any of these could be used to construct chimeric genes to produce any one of the gene products of the instant sequences. These chimeric genes could then be introduced into appropriate microorganisms via transformation techniques to provide high- level expression of the enzymes.

For example, a gene encoding an enzyme can be cloned into a suitable plasraid, and the aforementioned starting parent strain (i.e., as a production host) can be transformed with the resulting plasmid. This approach can increase the copy number of each of the genes encoding the enzymes and, as a result, the activities of these enzymes can be increased. The plasmid i not particularly limited so long as it can autonomously replicate in the microorganism.

Vectors or cassettes useful for the transformation of suitable host cells are known in the art. Typically the vector or cassette contains sequences directing transcription and translation of the relevant, gene, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5' of the gene harboring transcriptional initiation controls, and a region 3' of the DNA fragment which controls transcriptional termination. One or both controls of the regions may be derived from genes homologous to the transformed host cell although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a production host. Promoters, cD As, and 3'UTRs, as well as other elements of the vectors, can be generated through cloning techniques using fragments isolated from native sources (see, for example, Molecular Cloning: A Laboratory Manual, Sambrook et al. (3d edition, 2001, Cold. Spring Harbor Press; and U.S. Pat. No. 4,683,202 (incorporated by reference}). Alternatively, elements can be generated synthetically using known methods (see for example Gene. 1995 Oct. 16; 164(l):49-53).

B. Homologous Recombination

Homologous recombination (HR) is the ability of complementary DNA sequences to align and exchange regions of homology. Transgenic DNA ("donor") containing sequences homologous to the genomic sequences being targeted ("template") is introduced into the organism, and then undergoes recombination into the genome at the site of the corresponding genomic homologous sequences.

The ability to cany out homologous recombination in a host organism has many practical implications for what, can be carried out at the -molecular genetic level and is useful in the generation of an oleaginous microbe that can produced tailored oils. By its very nature, homologous recombination is a precise gene targeting event, hence most transgenic lines generated with the same targeting sequence will be essentially identical in terms of phenotype, necessitating the screening of far fewer transformation events. Homologous recombination aiso targets gene insertion events into the host chromosome, potentially resulting in excellent genetic stability, even in the absence of genetic selection. Because different chromosomal loci will likely impact gene expression, even from heterologous promoters/UTRs, homologous recombination can be a method of querying ioci in an unfamiliar genome environment and assessing the impact of these environments on gene expression,

A particularly useful genetic engineering approach using homologous recombination is to co-opt specific host regulatory elements, such as promoters/UTRs, to drive heterologous gene expression in a highly specific fashion.

Because homologous recombination is a precise gene targeting event, it can be used to precisely modify any nucieotide(s) within a gene or region of interest, so long as sufficient flanking regions have been identified. Therefore, homologous recombination can be used as a means to modify regulatory sequences impacting gene expression of R A and/or proteins. It can also be used to modify protein coding regions in an effort to modify enzyme activities,, such as substrate specificity, affinities and Km, thus affecting the desired change in metabolism of the host cell. Homologous recombination provide a powerful means to manipulate the host genome resulting in gene targeting, gene conversion, gene deletion, gene duplication, gene inversion and exchanging gene expression regulatory elements, such as promoters, enhancers and 3'lJTRs,

Homologous recombination cart be achieved by using targeting constructs containing pieces of endogenous sequences to "target" the gene or region of interest within the endogenous host cell genome. Such targeting sequences can either be located 5' of the gene or region of interest, 3' of the gene/region of interest, or even flank the gene/region of interest. Such targeting constructs can be transformed into the host eel! either as a supercoiied plasmid DNA with additional vector backbone, a PC product with no vector backbone, or as a linearized molecule. In some cases, it may be advantageous first to expose the homologous sequences within the transgenic DNA (donor DNA) by cutting the transgenic DNA with a restriction enzyme; this step can increase the recombination efficiency and decrease the occurrence of imdesired events. Other methods of increasing recombination efficiency include using PCR to generate transforming transgenic DNA containing linear ends ^'homologous to the genomic sequences being targeted.

Two or more homologous recombination events can be used to help screen cells that were correctly targeted. For example, a first nucleic acid may be designed to target a particular .nucleotide sequence and encode a portion of a selectable marker protein, A second nuclic acid may be designed to target an adjacent nucleotide sequence and encode the remaining portion of the selectable marker protein. Thus, only cells that successfully undergo homologous recombination with both nucleic acids are likely to express the full- length selectable marker protein.

Vectors for transformation of microorganisms in accordance with the present invention can be prepared by techniques known to those skilled in the art in view of the disclosure herein. A vector typically contains one or more genes, in which each gene codes for the expression of a desired product (the gene product), and is operabiy linked to one or more control sequences that regulate gene expression or target the gene product to a particular location in the recombinant cell. This subsection is itself further divided into subsections. Subsection 1 describes control sequences typically contained on vectors, as well as novel control sequences provided by the present invention. Subsection 2 describes genes typically contained in vectors, as well as novel codon optimization methods and genes prepared using them.

./. Control Sequences

Control sequences are nucleic acids that regulate the expression of a coding sequence or direct a gene product to a particular location within or outside a cell. Control sequences that regit! ate expression include, for example, promoters that regulate transcription of a coding sequence and terminators that terminate transcription of a coding sequence. Another control sequence is a 3' untranslated sequence located at the end of a coding sequence that encodes a poiyadenylation signal Control sequences that direct gene products to particular locations include those that encode signal peptides, which direct the protein to which they are attached to a particular location within or outside the cell.

Thus, an exemplary vector design lor expression of an exogenous gene in a microbe contains coding sequence for desired gene product (for example, a selectable marker, or an enzyme) in operable linkage with a promoter active in mieroaigae. Alternatively, if the vector does not contain a promoter in operable linkage with the coding sequence of interest, the coding sequence can be transformed into the cells, such that it becomes operably linked to an endogenous promoter at the point of vector integration.

The promoter used to express an exogenous gene can be the promoter naturally linked to that gene or can be a heterologous promoter,

A promoter can generally be characterized as either constitutive or inducible. Constitutive promoters are generally active or function to drive expression at all times (or at certain times in the ceil life cycle) at the same level . inducible promoters, on the other hand, are active (or rendered inacti ve) or are significantly up- or down-regulated only in response to a stimulus. Both types of promoters find application in the methods of the invention, inducible promoters useful in the invention include those that mediate transcription of an operably linked gene in response to a stimulus, such as an exogenously provided small molecule, temperature (beat or cold), or lack of nitrogen in culture media. Suitable promoters can activate transcription of an essentially silent gene or upregulate, preferably substantially, transcription of an operably linked gene that is transcribed at a low level. inclusion of a termination region control sequence is optional and, if employed, the choice is primarily one of convenience, as the termination region i relatively interchangeable. The termination region may he native to the transcriptional initiation region (the promoter), may be native to the DNA sequence of interest, or may be obtainable from another source, See_f for example, Chen and Orozco, Nucleic Acids Res. (1988) 16:841 1 ,

2. Genes and Codon Optimization

Typically, a gene includes a promoter, a coding sequence, and one or more termination control sequences. When assembled by recombinant DMA technology, a. gene may he termed an expression cassette and may be flanked by restriction sites for convenient insertion into a vector that is used to introduce the recombinant gene into a host cell. The expression cassette can be flanked by DNA sequences from the genome or other nucleic acid target to facilitate stable integration of the expression cassette into the genome by homologous recombination. Alternatively, the vector and its expression cassette may remain unietegrated (e.g., an episome), in which ease, the ^'vector typically includes an origin of replication, which is capable of providing for replication of the heterologoits vector DNA.

A gene commonly present on a vector is a gene that codes for a protein, the expression of which allows the recombinant cell containing the protein to be differentiated from cells that do not express the protein. Such a gene, and its corresponding gene product, is called a selectable marker or selection marker. Any of a wide variety of selectable markers can be employed in a transgenc construct useful for transforming an organism.

For optimal expression of a recombinant protein, it is beneficial to employ coding sequences that produce mR A with codons optimally used by the host cell to be transformed. Thus, proper expression of tensgenes cart require that the eodon usage of the transgene matches the specific codon bias of the organism in which the transgenc is being expressed. ^"The precise mechanisms underlying this effect are many, but include the proper balancing of available aminoaeyiated t A pools with proteins being synthesized in the cell, coupled with more efficient translation of the transgenic messenger R A (mRNA) when this need is met. When codon usage in the transgene is not optimized, available tRNA pool are not sufficient to allow for efficient translation of the heterologous mRNA,

1.4 resulting in nbosomal stalling and termination, and possible instability of the transgenic mRNA.

D. Expression of T wo or More Exogenous Genes

A genetically engineered microorganism may comprise arid express more than one exogenous gene. One or more genes can be expressed using an inducible promoter, which allows the relative timing of expression of the genes to be controlled. Expression of the two or more exogenous genes may be under control of the same inducible promoter or under control of different inducible promoters. In the latter situation, expression of a first exogenous gene can be induced for a first period of time (during which expression of a second exogenous gene may or may not be induced), and expression of a second or further exogenous gene can be induced tor a second period of time (during which expression of a first exogenous gene may or may not be induced). Provided herein are vectors and methods for engineering microbes, e.g., to grow on non-traditional growth media.

E. Trans formati on

Ceils can be transformed by any suitable technique, including, e.g., biolistics, electtoporation, glass bead transformation, and silicon carbide whisker transformation.. Any convenient technique for introducing a transgene into a microorganism can be employed in the present invention. Transformation can be achieved by, for example, the method of D.M. Morrison (Method in Bnzymoiogy 68, 326 ( 1979)), the method of increasing the permeability of recipient cells to D A with calcium chloride (Mandel, M, and Riga, A., j. Mol. Biol,, 53, 159 (1970)), or the like.

Examples of expression of transgenes in oleaginous yeast (e.g. , Yarro ia lipoidica) can be found in the literature (see, for example, Bordes et al„ J Microbiol Methods, Jun. 27 (2007)). Examples of expression of exogenous genes in bacteria, such as £ eo/7, are well known; see, for example. Molecular Cloning: A Laboratory Manual, Sambrook et al. (3d edition, 2001 , Cold Spring Harbor Press).

Vectors for transformation of microorganisms in accordance with the present invention can be prepared by techniques familiar to those skilled in the art. In one embodiment, an exemplary vector design for expression of a gene in a microorganism contains a gene encoding an enzyme in operable linkage with a promoter active in the microorganism. Alternatively, if the vector does not contain a promoter in operable linkage with the gene of interest, the gene can be transformed into the cells, such that if becomes operably linked to an endogenous promoter at the point of vector integration. The vector can also contain a second gene that encodes a protein. Optionally, one or both gene(s) is/are followed by a 3' untranslated sequence containing a polyadenylation signal. Expression cassettes encoding the two genes can be physically linked in the vector or on separate vectors. Co-transformation of microbes can also be used, in which distinct vector molecules are simultaneously used to transform ceils (see, for example, Protist 2004 December; 155(4):38f-93). The transformed cells can be optionally selected based upon the ability to grow in the presence of die antibiotic or other selectable marker under conditions in which cells lacking the resistance cassette would not grow.

Exemplary Methods of the invention

Iti certain embodiments, the invention relates to a method, comprising the steps of: providing a pluralit of cells;

arresting the cell cycle of the plurality of cells, thereby forming a first mixture comprising a plurality of arrested cells; and

subjecting the plurality of arrested ceils to transformation conditions, thereby forming a plurality of genetically engineered ceils comprising a first fraction of genetically engineered cells and a second fraction of genetically engineered cells, wherein the first fraction of genetically engineered cells comprises the desired transformation and the second fraction of genetically engineered cells does not.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein arresting the cell cycle of the plurality of ceils comprises eiutriation. In certain embodiments, the cell cycles are arrested without chemicals or mutations, i certain embodiments, eiutriation comprises separating the cells according to their size. In certain embodiments, the cells are separated by centrifugation. m certain embodiments a desired fraction of cells is removed. In certain embodiments, the desired fraction of cells are in S~ phase. In certain embodiments, the desired fraction of cells is substantially uniform. In certain embodiments, the desired fraction of ceils is then returned to rich media so that it undergoes a synchronous eel! cycle {because all starting ceils will begin growing from the same phase). In certain embodiments, the invention further comprises monitoring the growth of the desired fraction of cells, in certain embodiments, the growth of the cells is monitored until they reach S-phase. in certain embodiments, the monitoring comprises microscopy or FACS ana^'lysis/ceil-cycle profiling. In certain embodiments, the cell sorting function of FACS may be used to sort out a population of cells.

in certain embodiments, the invention relates to any oae of the aforementioned methods, wherein arresting the cell cycle of the plurality of ceils comprises utilizing cell cycle mutants. In certain embodiments, the ceil cycle mutant is temperature sensitive. In certain embodiments, the ceil cycle mutant reversibly substantially blocks cells in specific cell cycle stages. In certain embodiments, the cell cycle mutant arrests the cells in S-phase upon exposure to a trigger. In certain embodiments, the ceil cycle mutant arrests the cells elsewhere in the cell cycle. In certain embodiments, the ceil cycle mutants are arrested, and then released synchronously. In certain embodiments,, the ceil cycle mutants are cdcl5 mutants (which arrest in late M phase (after anaphase, before mitotic exit)), cdc20 mutants (which arrest prior to anaphase), or cdc^'7 mutants (which arrest cells at the Gl/S transition). in certain embodiments, the invention relates to any one of the aforementioned methods, wherein arresting the cell cycle of the plurality of cells comprises exposing the plurality of cells to a chemical, in certain embodiments, the chemical comprises noeodazole or benomyl. Noeodazole and benomyl interfere with microtubules and trigger the spindle assembly checkpoint arresting cells in G2/M. in certain embodiments, the chemical comprises hydroxyurea. Hydroxyurea is a ribonucleotide reductase inhibitor that results in low nucleotide pools and triggers the replication checkpoint, arresting cells in S-phase. in certain embodiments, the chemical comprises thymidine, anrinopterin, or eytosine arabi.oos.tde. In certain embodiments, the chemical comprises alpha-factor, a yeast pheroroone. Alpha-factor signals yeast cells of the "a" mating type (as opposed to "alpha" mating type) to prepare for mating, thus leading to Gl. arrest.

in certain embodiments, the invention relates to any one of the aforementioned methods, wherein arresting the ceil cycle of the plurality of cells comprises limiting the nutrition of the cells. In certain embodiments, after a period of limited nutrition, nutrition is reestablished and the cells are released synchronously. In certain embodiments, the cells are then monitored by microscopy or F ACS until they reach the desired phase of the cell cycle.

In certain embodiments, the invention relates to a method, comprising the steps of; providing a plurality of cells; contacting the plurality of ceiis with a ribonucleotide reductase inhibitor at a first temperature for a first period of time, thereby forming a first mixture comprising a plurality of arrested ceils; and

subjecting the plurality of arrested cells to transformation conditions, thereby forming a plurality of genetically engineered ceils comprising a first fraction of genetically engineered cells and a second fraction of genetically engineered cells, wherein the first fraction of genetically engineered ceils comprises the desired transformation and die second fraction of genetically engineered ceils does not.

In certain embodiments, the invention: relates to any one of the aforementioned methods, wherein the fraction of the plurality of genetically engineered cells compri sing the desired transformation (i.e., the first, fraction) is larger than the traction of genetically engineered cells comprising the desired transformation if the plurality of cells had been subjected to transformation conditions without first being contacted with the ribonucleotide reductase inhibitor at the first temperature for the first period of time.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the method is a method of increasing gene targeting efficiency, as compared to a method involving only subjecting the plurality of cells to transformation conditions. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the gene targeting efficiency is from about 1% to about 99%. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the gene targeting efficiency is from about 30% to about 99%. I» certain embodiments, the invention relates to any one of the aforementioned methods, wherein the gene targeting efficiency is about .1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 1.5%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. in certain embodiments, the invention relates to any one of the aforementioned methods, wherein the gene targeting efficiency is independent of the size of the homologous flanks.

In certain embodiments, the invention relates to any one of the aforementioned methods, further comprising the step of contacting the plurality of cells with a liquid culture at a second concentration at a second temperature for a second period of time before contacting the plurality of ceils with the ribonucleotide reductase inhibitor. in certaiii embodiments, the invention relates to any one of the aforementioned methods, wherein the second concentration corresponds to an Dm from about 0.2 to about 0.8. in certain embodiments, the invention relates to any one of the aforementioned methods, wherein the second concentration corresponds to an O of about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, or about 0.8. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the second concentration corresponds to an ODm of about 0.5,

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the iiquid cuiture comprises yeast extract. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the liquid culture comprises peptone. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the liquid culture comprises water. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the Iiquid culture comprises glucose. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the liquid culture consists essentially of yeast extract, peptone, water, and glucose.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the liquid culture is yeast extract -peptone dextrose (Y PD or YEPD).

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the liquid culture and plurality of cells are shaken at the second temperature for the second period of time.

in certain embodiments, the invention relates to any one of the aforementioned methods, wherein the second temperature is from about 15 °C to about 45 "C, In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the second temperature is about 20 °C_« about 25 C, about 30 "C, about 35 or about 40 °C In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the second temperature is about 30 " .

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the second period of time is from about 15 min to about 45 mm. In certain embodiments, the invention relates to an one of the aforementioned methods, wherein the second period of time is about 20 min, about 25 min, about 30 min, about 35 rain, or about 40 mm. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the second period of time is about 30 niin.

In certain embodiments, the invention relates to any one of the aforementioned methods, further comprising the steps of culturing the plurality of cells at a third temperature for a third period of time; and collecting a plurality of cultured cells before contacting the plurality of cultured ceSis with the liquid culture.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the plurality of cells are cultured on a medium. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the medium comprises yeast extract, In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the medium comprises -peptone. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the medium comprises water. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the medium comprises glucose, in certain embodiments, the invention relates to any one of the aforementioned methods, wherein the medium comprises yeast extract, peptone, water, and glucose, in certain embodiments, the invention relates to any one of the aforementioned methods, wherein the medium comprises yeast extract, peptone, water, glucose, and agar. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the medium is yeast extract peptone dextrose (YP or YEPD).

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the third temperature is from about 15 °C to about 45 "C, In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the third temperature is about 20 °C, about 25 "C, about 30 °C, about 35 "C, or about 40 "C. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the third temperature is about 30 °C.

in certain embodiments, the invention relates to any one of the aforementioned methods, wherein the third period of time is aboitt 6 h, about 8 h, aboitt 10 h, aboitt 12 h, or about 14 h,

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the ribonucleotide reductase inhibitor ( RT) is hydroxyurea (HU), In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the concentration of the R.RJ in the first mixture is from about 20 mM to about 80 mM, In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the concentration of the i in the first mixture is about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 raM, about 75 mM, or about 80 mM. In certain embodiments, the invention relates to arty one of the aforementioned methods, wherei n the concentration of the RRi in the first mixture is about 50 mM.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the first period of time is from about 1 h to about 3 h. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the first period of time is about 1 h, about 1.5 h, about 2 h, about 2,5 h, or about 3 h . In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the first period of time is about 2 h.

in certain embodiments, the invention relates to any one of the aforementioned methods, wherein the first mixture is shaken for the first period of time.

In certain embodiments, the invention relate to any one of the aforementioned methods, wherein the first temperature is from about 15 °C to about 45 ^yC. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the first temperature is about 20 ^C, about 25 °C, about 30 °C, about 35 ^"(^', or about 40 °C, in certain embodiments, the invention relates to any one of the aforementioned methods, wherein the first temperature is about 30 °

In certain embodiments, the invention relate to any one of the aforementioned methods, wherein the arrested ceils arc in the S-phase of their cell cycle, in certain embodiments, the invention relates to any one of the aforementioned methods, wherein the arrested cells are in a budded state. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein a plurality of the arrested celts are in the S-phase of their celt cycle, in certain embodiments, the invention relates to any one of the aforementioned methods, wherein a plurality of the arrested ceils are in a budded state.

In certain embodiments, the invention relates to any one of the aforementioned methods, further comprising the step of: collecting the plurality of arrested cells. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the plurality of arrested cells are collected by centrifugaiion. In certain embodiments, the invention relates to any one of the aforementioned methods, further comprising the step of washing the plurality of collected arrested cells. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the collected arrested cells are washed with water. In certain embodiments, the invention relates to any one of the aforementioned methods, further comprising the step of; contacting with water the plurality of collected arrested cells.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein transforming the cells comprises utilizing cells that are naturally capable of taking up DNA under !aborabory conditions (natural competence).

In certain embodiments, the invention relates to arty one of the aforementioned methods, wherein transforming the cells comprises exposing the arrested cells to a chemical. In certain embodiments,, the chemical comprises a divalent cation. In certain embodiments, the chemical comprises calcium. In certain embodiments, the chemical comprises lithium acetate/PEG singie stranded D A.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein transforming the cells comprises electroporation. In this method, the arrested cells are briefly shocked with an electric field of about 10-20 kV cm.

in certain embodiments, the invention relates to any one of the aforementioned methods, wherein transforming the cells comprises bombardment (for example, using a gene gun), in certain embodiments, particles of gold or tungsten are coated with DM A and then shot into the arrested cells.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein transforming the ceils comprises viral infection. In certain embodiments, the desired genetic material is packaged into a suitable virus and allowed to infect the arrested cells.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein transforming the cells comprises protoplast fusion. In certain embodiments, a chemical is added to facilitate fusion of two or more desired types of cells. In certain embodiments, the chemical is PEG. in certain embodiments, the invention relates to any one of the aforementioned methods, wherein transforming the ceils comprises suspending the cells in a transformation mix and adding a polynucleotide, thereby forming a second mixture. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the polynucleotide is linear DNA. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the transformation mix comprises PEG. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein transformation mix comprises PEG 4000. in certain embodiments, the invention relates to any one of the aforementioned methods, wherein transformation mix comprises a lithium sail. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein transformation mix comprises lithium acetate. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein transformation mix comprises dimiothreitol (DTT). in certain embodiments, the invention relates to any one of the aforementioned methods, wherein transformation mix comprises salmon sperm DNA. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein transformation mix comprises PEG 4000, lithium acetate, dithiothreitol (DTT), and salmon sperm DNA.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein transforming the cells further comprises subjecting the second mixture to heat shock, in certain embodiments, the invention relates to any one of the aforementioned methods, wherein the heat shock comprises heating the second mixture at a fourth temperature for a fourth period of time.

in certain embodiments, the invention relates to any one of the aforementioned methods, wherein the fourth temperature is from about 20 °C to about 60 "'C. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the fourth temperature is about 20 °C, about 25 *C, about 30 "C about 35 °C, about 40 °C, about 45 °C, about 50 *C, about 55 °C, or about 60 °C. in certain embodiments, the invention relates to any one of the aforementioned methods, wherein the fourth temperature is about 39 °C.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the fourth period of time is about 20 rain to about 2 h. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the fourth period of time is about 20 rain, about 40 min, about i h, about 80 min, about 100 min, or about 2 h. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the fourth period of time is about 1 h.

In certain embodiments, the invention relates to any one of the aforementioned methods, further comprising the step of selecting the first fraction of genetically engineered cells.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the cells, before being arrested and transformed, are native or wild-type cells. In certain embodiments, the invention, relates to any one of the aforementioned methods, wherein the ceils, before being arrested and transformed, have not been genetically altered to increase HR or decrease NHEJ.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the cells, before being arrested and transformed, are genetically engineered cells.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the ceils are prokaryotic.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the ceils are eukaryotic. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the cells are higher cukaryotes. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the cells are eukaryotic; and the cells, in their native state, tends to repair double stranded DNA breaks by NHEJ.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the ceils are yeast cells.

in certain embodiments, the invention relates to any one of the aforementioned methods, wherein the genera of the cell is selected from the group consisting

Candida, Cryptococcus, Debaryomyces, Hatwe uht, Klo cker , Khtyveromyces, iJpomyces\ Myroihedum, Phqffk^' Pichia, Pse domon s, Rhodosporidiwn, Sacekammyees, Schtosaec aromyces, Sc w nmomyc x, R odotortda, Trichosporon, and. Yarrowia,

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the cells are selected from the group consisting of Yatrowia Hpolytica, Saccharomyce cerevisiae, Saccharomyces buideri, Saccharomyces b rmtti, Saccharomyces exiguus, Saccharomyces imtrum, Saccharomyces diestaticus, Khfyveromyces lactis, Kiuymmmyc s arxhmus, Kluyveromy s fragilis, Candida albicans, Pichia pastoria, Fichia stipitis, Hamen la polymorpha, Fhaffia rhodozyma, Candida utilis, Arxul adeninivorans, Deb ryomyces ansentt, Candida glahrafa, Debaryomyces polymorphs _> Schizosaccharomyces pombe, Schwatmiomyces occid&ilalis, Rhodosporidhm tondoid s, Cryptococats curvatm, Lipomyces starkeyi, Rhodotonda glutinis, Pichki guilliermondii, Rhodotonda gram is, Trichosporon fermentans, Debaryomyces occi entalis_m Myrotheciu verrucarkL Pseudomoncis ψ. , Rhod porkii m kmdokks, Rhodotonda graminis, Saccha omycopsis fibtriigera, and Trichosporon cutanewn.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the cells are selected from the group consisting of the cells depicted in figure 4.

in certain embodiments, the invention relates to any one of the aforementioned methods, wherein the cells are fungi cells. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the cells arc filamentous fungi.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the genera of the cells is selected from the group consisting of Cryptococcus, Asperg llus, and Neurospom.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the cells are selected from the group consisting of Cryptococcus rteoforma , Aspergillus nig&\ and Neurospam crassa.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the cells are mammalian ceils.

in certain embodiments, the invention relates to any one of the aforementioned methods, wherein the cells are algae cells.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the ceils are plant cells. Exemplary Genetically Engineered Cells

in certain embodiments, the invention relates to a genetically engineered cell made by any one of the aforementioned methods.

EXEMPLIFICATION

The following examples are provided to illustrate the invention. It will be understood, however, that the specific details gi ven in each example have been selected for purpose of illustration and are not to be construed as limiting the scope of the invention. Generally, the experiments were conducted under similar conditions unless noted.

Overvi w

DNA integration in Yammia tipol tica prefers the NHEJ pathway over MR. As a result, targeted insertions or gene knockouts are very inefficient due to random integration of the construct into the genome. In other species, HR is found to be upreguiated in relation to NHEJ in S phase when sister chromatids are avatiafaie to be used as tempiates for DNA repair. We explored the possibility that transforming Yarrowia lipolytics cells synchronized in S phase shifts integration in favor of HR. The ribonucleotide reductase inhibitor hydroxyurea (HU) was used to arrest cells in S phase. A standard yeast transformation protocol was then applied to transform cells with liner DNA encoding the nourseothricirt resistance gene (NA ^'I) flanked by sequences homologous to fee regions upstream and downstream of the Yarrowia lipolytics gene YAU0D2I384g, Antibiotic resistant transformants were analyzed b PC to determine whether the NAT gene successfully integrated into the chromosome replacing YAU0D2i384g. Accurate targeting of the antibiotic cassette was observed in at least 30% of transformants pre-treated with HU but not in untreated cells.

Methods

Cell cycle synchronization and transformation; NSIS, the Yarrowia lipolytics strain to be transformed was grown overnight on a Y^'PD plate at 30 ^ύ€,. Water (1 niL) was applied to the plate to collect the cells using art L-shaped spreader and pipet. Cells were diluted to Ο-¾» ~ 0.5 in 2 flasks containing 25 mL YPD and shaken at 30 "C for 30 min to acclimate the cells to liquid culture. 95 rag HU was added to one flask to a final concentration of 50 mM. Shaking was continued for 2 hours to allow for cell cycle arrest as determined by microscopy (ail ceils were arrested at the budded stage). Ceils were collected by centrifugation, washed with water and resuspeeded in a pellet volume of water. 50 pL was aliquoted per transformation, cells were collected by centrifugation and the supernatant was discarded. The cells were resuspended in 100 p^'L transformation mix (80 pL 60% PEG 4000., filter sterile: 5 ,uL 2 M Lithium acetate, pH adjusted to 6.0, filter sterile; 5 itL 2 M dithiothreiiol (DTT)„ fitter sterile; 10 pL 2 mg mL salmon sperm DNA„ boiled 10 mm prior to use), t pg Linearized DMA was added. The ceils were subjected to heat shock at 39 X,^' for 1 , collected by centrifugation, resuspended in 1 mL YPD and cultured overnight at 30 °C to allow NAT gene expression. 100 pL was spread onto dry selective plates (YPD/agar containing 500 pg mL noitrseothriein) the next day and transformants were analyzed by PGR a day later.

Overview

GUT2 was deleted from Y. Upo!yiica wild- type strain HS 18 (obtained from NRLL# YB-392). The Y. lipofytim (ΆΓΓ2 gene (YAU0Bi3970g, SEQ ID NO: 2) was deleted as follows: A two-fragment deletion cassette was amplified by PCR from a plasmid containing the hygromyein resistance gene ("hph " SEQ I.D NO: 8) using primer pairs NPI 563-NP656 and NP655-NP1 S O (SEQ ID NOs: 3, 9, 10, and 4, respectively). The resulting PCR fragments (SEQ ID NOs: 5 & 6) were co-transformed into NS.1 . The omission of a promoter and terminator in the hph cassette and the splitting of the hph coding sequence into two PCR fragments reduce the probability that random integration of these pieces will confer hygromyein resistance. The hph gene should only be expressed if it integrates at the GUT2 locus by homologous recombination so that the GIJT2 promoter and terminator can direct its transcription. Hygromyein resistant colonies from each condition were patched onto minimal media containing glucose or glycerol- to screen for isolates that have lost the ability to grow on glycerol- due to loss of GUT2 function. No successful targeted integration was obtained when hydroxyrea was not used in the transformation protocol (0 out of 99 colonies screened,). Seven correct integrations were obtained when the transformed cells were first arrested in S phase with hydroxyurea (48 colonies screened). This corresponds to an increase of targeted integration efficiency form 0 to 15%. Methods

Ceil cycle synchronization and transformation; the transformation protocol was the same as described in Example ! above. ^'NSi8, the Yatrowia Hpofyiic strain to be transformed was grown overnight on a YPD plate at 30 °C. Water (1 ml) was applied to the plate to collect the cells using an L-shaped spreader and pipet. Cells were diluted to OD_(i«,^:::: 0.5 in 2 flasks containing 25 ml YPD and shaken at 30 "C for 30 min to acclimate the cells to liquid culture. 95 mg Htj was added to one flask to a final concentration of 50 niM. Shaking was continued for 2 hours to allow for cell cycle arrest as determined by microscopy fall cells were arrested at the budded stage). Cells were collected by eennitugation, washed with water and resuspended in a pellet volume of water. 50 gL was aliqiioted per transformation, cells were collected by centrifugation and the supernatant was discarded. The cells were resuspended in 100 itL transformation mix (SO μΕ 60% PEG 4000, filter sterile; 5 gL 2 M Lithium acetate, pH adjusted to 6.0, filter sterile; 5 μί, 2 dithiothreitol (DTT), filter sterile; 10 Τ 2 mg/mL salmon sperm DNA, boiled 10 min prior to use). 9 gL itnpurified PCR product for each of SEQ ID NOs: 5 & 6 was added. The cells were subjected to heat shock at 39 °C for 1 h, collected by centrifugation, resuspended in I mL YPD and cultured overnight at 30 ^!'C to allow hph gene expression. 100 pL was spread onto dry selective plates YPD/agar containing 300 gg mL hygromycin) the next day and iransfbrmants were screened for growth on glycerol a day later.

Overview

TGL3 was deleted fr m F. lipofylica wild-type strain NS18 (obtained from N.RLL# YB-392). The F tipolytica TGU gene (YALIODI ?534_8j SEQ ID NO; 12) was deleted as follows: A fcvo-fragment deletion cassette was amplified by PCR from a pl smid containing the hygromycin resistance gene ("hph " SEQ ID NO: 8) using primer pairs NPI 798-NP656 and ΝΡ655-ΝΡ.Π99 (SEQ ID NOs: 13, 9, 10, and 14, respectively). The resulting PCR fragments (SEQ ID NOs: 15 & 16) were co-transformed into NS18. The omission of a promoter and terminator in the hph cassette and the splitting of the hph coding sequence into two PCR fragments reduce the probability that random integration of these pieces will confer hygromycin resistance. The hph gene should only be expressed if it integrates at the TGI locus by homologous recombination so that the TGU p moter and terminator can direct its transcription. 48 hygromycin resistant colonies from each condition were screened by PCR to confirm the presence of a fgl3::hyg specific product (product of NP1033 and NP656_f SEQ ID Os; 17 & 9, 990 bp). No successful targeted integration was obtained when hydroxyrea w s not used in the transformation protocol. Two correct integrations were obtained when the transformed cells were first arrested in S phase with hydroxyurea. This correspond to an increase of targeted integration efficiency form 0 to

Methods

Ceil cycle synchronization and transformation: the transformation protocol was the same as described in examples 1 and 2 above. MSI 8, the Yarrvwia lipofytiea strain to be transformed was grown overnight on a YPD plate at 30 "€. Water (1 mL) was applied to the plate to collect the cells using an L-shaped spreader and pipet Cells were diluted to OD600 ^::: 0.5 in 2 flasks containing 25 mL YPD and shaken at 30 °C for 30 roin to acclimate the cells to liquid culture. 95 mg HU was added to one flask to a final concentration of 50 iii . Shaking was continued for 2 hours to allow for eel! cycle arrest as determined by microscopy (ail ceils were arrested at the budded stage). Ceils were collected by centrifugation, washed with water and resuspended in a pellet volume of water. 50 μ.1... was aliquoted per transformation, ceils were collected by centrifugation and the supernatant was discarded. The cells were resuspended in i OO μΐ, transformation mix (80 ,uL 60% PEG 4000., filter sterile; 5 μΐ, 2 M lithium acetate, pH adjusted to 6.0, filter sterile; 5 ,uL 2 M ditlnothreito! (DTT), filter sterile: 10

2 nig niL salmon sperm DNA, boiled 10 min prior to use). 9 p^'L unpurified PCR product for each of SEQ ID NOs: 15 & 16 was added. The ceils were subjected to heat shock at 39 "C for 1 h, collected by centrifugation. resuspended in ! mL YPD and cultured overnight at 30 X to allow- hph gene expression., 100 pL was spread onto dry selective plates (YPD/agar containing 300 g/roL hygromyem) the next day, and transfonnaats were analyzed by PCR a day later.

Example 4 /: x piarv S quenc s ' of the invention

Sequence 1 is the amino acid sequence of the GUT2 protein from Y. lipofytiea.

Sequence 2 is the DNA sequence of the GUT2 gene from Hpolyfic .

Sequence 3 is the D A sequence of primer NPl 563.

Sequence 4 is the DNA sequence of primer NPl 800, Sequence 5 is the DMA sequence of a 5^! deletion cassette For knocking out the GUT2 gene m F. lipolytics

Sequence 6 is the DNA sequence of a 3^* deletion cassette for knocking out the GUT2 gene in Y. Uptylylica.

Sequence 7 is the amino acid sequence of the phosphotransferase protein from E. coii that confers hygromycin resistance- Sequence 8 is the DNA sequence of the hph gene .from E. coli that confers hygromycin resistance.

Sequ ence 9 is the DMA sequence of primer NP656.

Sequence 10 is the DNA sequence of primer P655.

Sequence 11 is the amino acid sequence of the TGL3 protein from Y. lipolytica. Sequence 12 is the DNA sequence of the TGL3 gene from F. lipolytica.

Sequence 13 is the DNA sequence of primer ΝΡΪ 798.

Sequence 14 is the DNA sequence of primer P 1 799.

Sequence 15 is the DNA sequence of a 5^' deletion cassette for knocking out the TGL3 gene in Y. lipolytica.

Sequence 16 is the DNA sequence of a 3' deletion cassette for knocking out the TGL3 gene in F. lipolytica.

Sequence 17 is the DNA sequence of primer N 1033.

INCORPORATION BY REFERENCE

All of the U.S. patents and U.S. published patent applications cited herein are hereby incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or he able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

We claim:

1. A method, comprising the steps of:

providing a plurality of cells;

arresting the ceil cycle of the plurality of cells, thereby forming a first mixture comprising a plurality of arrested cells; and

subjecting the plurality of arrested cells to transformation conditions, thereby forming a plurality of genetically engineered cells comprising a first traction of genetically engineered cells and a second fraction of genetically engineered cells, wherein the first fraction of genetically engineered cells comprises the desired transformation and the second fraction of genetically engineered cells does not,

2. The method of claim 1 , wherein arresting the cell cycle of the plurality of cells comprises (i) clutriation, (ii) utilizing cell cycle mutants, (iii) exposing the plurality of cells to a chemical, or (tv) limiting the nutrition of the ceils.

3. The method of claim. 1 or 2, wherein the first fraction of genetically engineered cells is larger than it would have been if the plurality of cells had not been arrested prior to being subjected to transformation conditions.

4. A method, comprising the steps of:

providing a plurality of cells:

contacting the plurality of ceils with a ribonucleotide reductase inhibitor at a first temperature for a first period of time, thereby forming a. first mixture comprising a plurality of arrested cells; and

subjecting the plurality of arrested ceils to transformation conditions, thereby forming a plurality of genetically engineered cells comprising a first fraction of genetically engineered cells and a second fraction of genetically engineered cells, wherein the first fraction of genetically engineered cells comprises the desired transformation and the second fraction of geneticall engineered cells does not,

5. The method of claim 4, wherein

the first fraction of genetically engineered cells is larger than it would have been if the plurality of cells had not been contacted wish the ribonucleotide reductase inhibitor at the first temperature for the first period of time prior to being subjected to transformation conditions.

6. The method of any one of claims 1 -5. wherein, gene targeting efficiency is greater than m a method consisting of subjecting the plurality of ceils to transformation conditions.

S ?, The method of claim 6, wherein the gene targeting efficiency is about 1 % to about

99%.

8. The method of any one of claims 1- 7, farther comprising the step of: selecting the first fraction of genetically engineered cells.

9. The method of any one of claims 1-8, wherein the plurality of ceils are native or0 wild-type cells.

10. The method of any one of claims 1 -8, wherein the plurality of cells have not been genetically altered to increase Homologous Recombination ("HR") or decrease Non Homologous End Joining ("NHEJ").

1 1. The method of any one of claims 1-10, wherein the ceils are prokaryotie.

5 12. The method of any one of claims i- ! 0, wherein the cells are eukaryotic.

13. The method of claim 12, wherein the cells are higher eukaryotes.

14. The method of any one of claims 1 - 10, wherein the cells are eukaryotic; and the cells, m their native state, predominantly repair double stranded D A breaks by NHEJ.

15. Th method of an one of claims 1-10, wherein the cells are yeast cells.

0 1 . The method of any one of claims I - 10„ wherein the cells are selected from the group consisting of Arxida, Candida, Crypiococcm, Debaryomyees, Hai ida, Kla ekera, Kluyveromyces, Lipomyces, Myrothecittm, Phaffia, Pichia, Pseudomonas, Rhvdasporidium, Sac haro yces, Sckkosaccharomyces, Sek wiitimomyees, Rhodotond , Trick sp r n, and Yarrtmia.

5 17. The method of any one of claims J -10, wherein the cells are selected from the group consisting of Yarroma tipolylica, Saccharamyces cerevisiae, Saccharamyces bulderi, Sac h myces b rnefti, Saccharamyces exigum, Saccharmnyces uvarum, Saccharomyces diastaH us, Kluyveromyces la iis, Kluyveromyces marximms, Kluyveromyces fragile, Candida albicans, Pichia pasiotis, Pichia stiptiw, Hcmsenu!a polymorpha, Phaffia0 rhodozym , Candida utilis, Arxula adenimvomns, Debaryomyees hamenii, Candida 3 . gl brala, JJ b ryomyces polymorphic Schteosaccharomyces pombe, Schwanniomyces accidentally Rhodosporidiu toruloid s, Cryptococcus curvai Upomyces starkeyi, Rhodoiortda giutinis, Pichi guilliermondii, Rhodoiorula gr mmis, Trichosporon fementans, Debatyomyees occidenfalis, Myrothecium verr caria, Pseudomonas sp. , Rhodosporidium toruloides, Rhodotond gramln , Saccharomycopsis fibulig m, and Trichosporon cwlaneum ,

1 . The method of any one of claims 1 -10, wherein the cells are selected from the group consisting of the cells depicted in Figure 4.

] 9. The method of any one of claims 1 - 10, wherein the cells are fungi ceils.

20. The method of any one of claims i-10, wherein the ceils are filamentous fungi.

21. The method of any one of claims 1 -10. wherein the cells are selected from the group consisting of Cnpiococeus, Aspergillus, and Neurospom.

22. The method of any one of claims -10, wherein the cells are selected from the group consisting of Cryptococc s neoformans, Aspergillus niger, and N rospora crassa,

23. The method of any one of claims i-10, wherein the cells are mammalian ceils,

24. The method of any one of claims 1-10, wherein the ceils are algae ceils.

25. The method of any one of claims 1 -10, wherein the cells are plant cells,

26. A genetically engineered cell made by a method of any one of claims 1 -25.