We gratefully acknowledge the funding support from the National Environment Agency of Singapore (ETRP 1201102), the Competitive Research Program of the National Research Foundation of Singapore (NRF-CRP5-2009-03), the Agency for Science, Technology and Research of Singapore (1324004108), Global R&#38;D Project Program, the Ministry of Knowledge Economy, the Republic of Korea (N0000677), the Defense Threat Reduction Agency (DTRA, HDTRA1-13-1-0037) and the Synthetic Biology Initiative of the National University of Singapore (DPRT/943/09/14).

1. Generation of the Y. lipolytica KU70 Deletion Strain
<ol>
	<li>Construction of the disruption cassette 
	Note: See Table 1 for all primers used in polymerase chain reaction (PCR) amplifications.
	<ol>
		<li>Design primers 20 to PCR amplify the LEU2 expression cassette (see Table 1) from a Y. lipolytica expression vector and introduce LoxP sites into the 5&#39; and 3&#39; ends of the LEU2 cassette with a long forward primer (# 1; Table 1) and a long reverse primer (# 2; Table 1), respectively. To introduce an additional restriction site for subsequent steps of the cloning process, add a BamHI restriction site in primer # 1.</li>
		<li>Perform PCR using a high-fidelity DNA polymerase as detailed in Table 2.</li>
		<li>Purify the PCR product LoxP-promoter-LEU2-terminator-LoxP cassette using a PCR purification kit according to the manufacturer&#39;s instructions. Add 3&#39;-A overhangs to the purified PCR product according to the manufacturer&#39;s instructions 21. Use T4 DNA ligase to ligate the A-tailed PCR product into a TA cloning vector to yield the plasmid T-LEU2 as per Table 3.</li>
		<li>PCR amplify the 1 kb 5&#39; upstream sequence of the KU70 gene using primers # 3/# 4 from the Y. lipolytica Po1g genomic DNA 22 as per Table 2. Purify the PCR product using a PCR purification kit according to the manufacturer&#39;s instructions.
		<ol>
			<li>Double digest both the purified PCR product and T-LEU2 plasmid with SacII and BamHI enzymes as per Table 4. Purify the digestion mixture using a PCR purification kit as per manufacturer&#39;s instructions. Use T4 DNA ligase to ligate the purified and digested PCR product to the SacII/BamHI sites of T-LEU2 to yield the plasmid T-LEU2-5E as per Table 3.</li>
		</ol>
		</li>
		<li>PCR amplify the 1 kb 3&#39; downstream sequence of the KU70 gene using primers # 5/# 6 from the Y. lipolytica Po1g genomic DNA 22 as per Table 2. Purify the PCR product using a PCR purification kit according to the manufacturer&#39;s instructions. Double digest both the purified PCR product and T-LEU2-5E plasmid with NotI and NdeI enzymes as per Table 4.
		<ol>
			<li>Purify the digestion mixture using a PCR purification kit as per manufacturer&#39;s instructions. Then, ligate the purified and digested PCR product to the NotI/NdeI sites of T-LEU2-5E to yield the plasmid T-KO using T4 DNA ligase as per Table 3.</li>
		</ol>
		</li>
		<li>Digest the plasmid T-KO with SacII and NdeI enzymes as per Table 4 to produce the disruption cassette (Figure 1). Purify the digested DNA fragments using a PCR purification kit as per manufacturer&#39;s instructions.</li>
	</ol>
	</li>
	<li>Competent cell preparation and transformation of Y. lipolytica Po1g strain
	<ol>
		<li>Competent cell preparation
		<ol>
			<li>Inoculate a colony of Y. lipolytica Po1g strain from a fresh yeast extract-peptone-dextrose (YPD) plate in 10 ml of YPD medium (1% yeast extract, 2% peptone, 2% dextrose and 50 mM citrate buffer pH 4.0) in a 100 ml flask. Incubate in a 30 &#176;C shaking incubator at 225 rpm for 20 hr until saturation (an OD600 of about 15, measured using a spectrophotometer).</li>
			<li>Pellet the cells by centrifuging for 5 min at 5,000 x g at room temperature. Wash the cells with 20 ml Tris-EDTA (TE) buffer (10 mM Tris, 1 mM EDTA, pH 7.5), and pellet the cells as described in step 1.2.1.2. Resuspend the cells in 1 ml of 0.1 M lithium acetate (pH 6.0, adjusted with acetic acid), and incubate for 10 min at room temperature.</li>
			<li>Aliquot the competent cells (100 &#181;l) into sterile 1.5 ml tubes. Proceed to the transformation steps below immediately, or add glycerol to a final concentration of 25% (v/v) and store at -80 &#176;C for long-term storage.</li>
		</ol>
		</li>
		<li>Transformation
		<ol>
			<li>Gently mix 10 &#181;l of denatured salmon sperm DNA (10 mg/ml) and 1-5 &#181;g of the purified disruption cassette together with 100 &#181;l of competent cells, and incubate at 30 &#176;C for 15 min.</li>
			<li>Add 700 &#181;l of 40% polyethylene glycol (PEG)-4000 (dissolved in 0.1 M lithium acetate pH 6.0), mix well and incubate in a 30 &#176;C shaking incubator at 225 rpm for 60 min. 
			Note: It is important to use PEG with average molecular weight of 4000, instead of PEG-3350 (typically used in the transformation of conventional yeast).</li>
			<li>Heat shock the transformation mixture by placing the tube in a 39 &#176;C water bath for 60 min.</li>
			<li>Add 1 ml YPD medium and recover for 2 hr at 30 &#176;C and 225 rpm. Centrifuge at 9,000 x g for 1 min at room temperature, remove the supernatant and resuspend the pellet in 1 ml of TE buffer.</li>
			<li>Repellet the cells by centrifugation at 9,000 x g for 1 min at room temperature and discard the supernatant. Resuspend the pellet in 100 &#181;l of TE buffer and plate onto selective plates (e.g., leucine-deficient plates), and incubate at 30 &#176;C for 2-3 days.</li>
			<li>Pick 4 to 10 single colonies and inoculate separately into 2 ml of YPD medium. Incubate overnight in a 30 &#176;C shaking incubator at 225 rpm. Identify positive colonies by PCR analysis of genomic DNA from the transformants 22 as per Table 5 using two sets of primers # 55/# 56, and # 56/# 57, respectively. 
			Note: The NotI linearized pYLEX1 vector is transformed into Y. lipolytica Po1g competent cells in order to determine the transformation efficiency by counting the number of colony forming units (cfu) per &#181;g plasmid DNA used 23.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Marker Rescue
<ol>
	<li>Construction of the Cre expression plasmid
	<ol>
		<li>Construction of pYLEX1-CRE and pYLEX1-HPH
		<ol>
			<li>Design primers 20 to PCR amplify the open reading frames of cre and hph. Add an extra adenine nucleotide upstream of the start codons in the forward primers # 82 and # 84, and insert KpnI restriction sites downstream of the stop codons in the reverse primers # 83 and # 85. PCR amplify the open reading frames of cre and hph from pSH69 (accession number HQ412578) 24 as per Table 2.</li>
			<li>Purify both cre and hph fragments using a PCR purification kit according to the manufacturer&#39;s instructions. Digest both the purified cre and hph fragments with KpnI enzyme as per Table 4. Double digest the pYLEX1 plasmid with PmlI and KpnI enzymes as per Table 4. Purify the digestion mixture using a PCR purification kit as per manufacturer&#39;s instructions.</li>
			<li>Use T4 DNA ligase as per Table 3 to ligate the purified and digested cre and hph fragments to PmlI/KpnI sites of the Y. lipolytica expression vector, yielding pYLEX1-CRE and pYLEX1-HPH, respectively.</li>
		</ol>
		</li>
		<li>Construction of pSL16-CRE-HPH
		<ol>
			<li>PCR amplify the promoter-gene-terminator cassette of cre from pYLEX1-CRE using primers # 86/# 87 flanking the SalI/PstI restriction sites as per Table 2. Purify the PCR product using a PCR purification kit according to the manufacturer&#39;s instructions.
			<ol>
				<li>Double digest both the purified PCR product and pSL16-CEN1-1 (227) plasmid 25 with SalI and PstI enzymes as per Table 4. Purify the digestion mixture using a PCR purification kit as per manufacturer&#39;s instructions. Then, ligate the purified and digested PCR product into pSL16-CEN1-1 (227) at the corresponding sites to create pSL16-CRE using T4 DNA ligase as per Table 3.</li>
			</ol>
			</li>
			<li>PCR amplify the promoter-gene-terminator cassette of hph from pYLEX1-HPH using primers # 88/# 89 flanking the XhoI/BglII restriction sites as per Table 2. Purify the PCR product using a PCR purification kit according to the manufacturer&#39;s instructions.
			<ol>
				<li>Double digest both the purified PCR product and pSL16-CRE plasmid with XhoI and BglII enzymes as per Table 4. Purify the digestion mixture using a PCR purification kit as per manufacturer&#39;s instructions. Then, ligate the purified and digested PCR product into pSL16-CRE at the corresponding sites to generate pSL16-CRE-HPH using T4 DNA ligase as per Table 3. 
				Note: The resulting Cre expression plasmid, pSL16-CRE-HPH, harbors a selectable hygromycin B marker and is a centromeric and episomally replicating vector (Figure 2).</li>
			</ol>
			</li>
			<li>Verify all the constructs by digestion with appropriate restriction enzymes (e.g., SalI/PstI, to excise the insert from the vector) as per Table 4.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Cre recombinase-mediated marker rescue
	<ol>
		<li>Transform pSL16-CRE-HPH into competent cells of the KU70 knockout strain following the transformation protocol described in section 1.2 above. Plate transformed cells onto YPD plus hygromycin B (YPDH) plates (containing 400 &#181;g/ml hygromycin B), and incubate at 30 &#176;C for 2-3 days.</li>
		<li>Perform colony PCR as per Table 5 using primers # 84/# 85 to identify positive colonies containing pSL16-CRE-HPH plasmid after transformation. 
		Note: The forward primer # 84 and the reverse primer # 85 are located inside the coding region of hph gene (Table 1). Only positive clones generate a specific PCR product in the PCR reaction.</li>
		<li>Pick a positive clone and inoculate into 2 ml of YPDH medium, and incubate in a 30 &#176;C shaking incubator at 225 rpm overnight to saturation. Measure the cell OD600 with a spectrophotometer. Harvest the cells at an OD600 of ~15. Centrifuge at 5,000 x g for 5 min at room temperature, and wash once with 2 ml of sterile water.</li>
		<li>Re-inoculate cells into 2 ml of YPD medium with an initial OD600 of 0.1 and allow the cells to grow in a 30 &#176;C shaking incubator at 225 rpm overnight. Streak the overnight cell culture onto YPD plates to isolate single colonies 23. Replica plate colonies onto YPD, YPDH, and leucine-deficient plates 23. 
		Note: Colonies that have lost both LEU2 marker and pSL16-CRE-HPH plasmid can only grow on YPD plates (Figure 3).</li>
	</ol>
	</li>
</ol>
3. Deletion of Alcohol Dehydrogenase and Alcohol Oxidase Genes
<ol>
	<li>Perform a search on the Y. lipolytica genome database using the Basic Local Alignment Search Tool (BLAST) to identify the gene candidates for deletion 26. Input the protein sequence of S. cerevisiae alcohol dehydrogenase I into BLAST search tool (http://www.genome.jp/tools-bin/search_sequence?prog=blast&#38;db=yli&#38;seqid=aa). 
	Note: The BLAST search tool returns the most similar protein sequences in the Y. lipolytica genome database.</li>
	<li>Construct the deletion cassettes by PCR with primers # 7 to # 56 (Table 1), and perform restriction enzyme digestion and ligation reactions using the same methods as described in section 1.1 above.</li>
	<li>Prepare competent cells of the KU70 knockout strain, carry out transformations and perform PCR to identify positive colonies with primers # 55, # 58 to # 81 (Table 1) by following the protocol described in section 1.2 above. 
	Note: As an example, the procedure for the YALI0E17787g gene deletion is described in Figures 4 and 5. The effects of 1 kb and 2 kb long homologous region on homologous recombination rate are reported.</li>
</ol>

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The authors have nothing to disclose.

Our objective for this study is to enable quick and efficient generation of targeted gene knockouts in the Y. lipolytica Po1g strain. Several considerations need to be addressed to achieve this. First, a high transformation efficiency is required. Thus, an efficient and convenient chemical transformation protocol for the Y. lipolytica Po1g strain was described in this study. The use of PEG-4000 is a critical factor for the successful transformation of this strain. No transformants were obtained...

Unlike Saccharomyces cerevisiae, Yarrowia lipolytica, an unconventional yeast, can grow in the form of yeast or mycelium in response to changes in environmental conditions 1,2. Thus, this dimorphic yeast can be used as a good model for the study of fungal differentiation, morphogenesis and taxonomy 3,4,5. It is generally regarded as a safe (GRAS) yeast species, which is widely used to produce a variety of food additives such as organic acids, polyalcohols, aroma compounds, emulsifiers and surfactants 6,7,8,9. It is an obligate aerobe and a well-known oleaginous yeast capable of naturally accumulating lipids at high amounts, i.e., up to 70% of cell dry weight 10. It can also utilize a wide spectrum of carbon sources for growth, including different kinds of residues in waste resources as nutrients 11,12,13. All of these unique features make Y. lipolytica very attractive for various biotechnological applications.
Although the whole genome sequence of the Y. lipolytica has been published 14,15, genetic manipulation of this unconventional yeast is more complex than other yeast species. First, transformation of this yeast species is much less efficient due to the absence of a stable and efficient genetic transformation system 16,17. Second, laborious genomic integration of linear expression cassettes is commonly used for the expression of genes of interest as no natural episomal plasmid system has been found in this yeast 18. Third, generation of genetic knock-outs and knock-ins are limited because the gene targeting efficiency via accurate homologous recombination in this yeast is low and most integration events occur through non-homologous end joining (NHEJ) 19.
In this study, we report an optimized transformation protocol for the Y. lipolytica Po1g strain, which is easy, rapid, efficient and reproducible. To enhance the frequency of precise homologous recombination, we deleted the KU70 gene, which encodes a key enzyme in the NHEJ pathway. By using the optimized transformation protocol and transforming a linear knockout cassette containing flanking homology regions of 1 kb, the KU70 gene of the Y. lipolytica Po1g was successfully deleted. The robustness of this gene deletion methodology was then demonstrated by targeting alcohol dehydrogenase and alcohol oxidase genes in the Po1g KU70&#916; strain. It was observed that the KU70 deletion strain exhibited a considerably higher efficiency of homologous recombination-mediated gene targeting than that of the wild-type Po1g strain. In addition, a replicative Cre expression plasmid carrying the hygromycin B resistance marker was constructed to perform marker rescue. The marker rescue facilitates multiple rounds of gene targeting in the obtained gene deletion mutants. Besides gene deletion, our protocol for genetic transformation and gene deletion described here can be applied to insert genes to specific loci, and to introduce site-specific mutations into the Y. lipolytica genome.

<table border="0" cellpadding="0" cellspacing="0" width="605">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:140px;">Reagent/Material</td>
			<td colspan="3" style="width:465px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oligonucleotide primers</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>25 nmole&#160; DNA oligos</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Y. lipolytica strain Po1g</td>
			<td>Yeastern Biotech</td>
			<td></td>
			<td style="width:207px;">leucine auxotrophic derivative of the wild-type strain W29 (ATCC 20460)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vector pYLEX1</td>
			<td>Yeastern Biotech</td>
			<td>FYY203-5MG</td>
			<td>Y. lipolytica expression vector&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">E. coli TOP10&#160;</td>
			<td>Invitrogen</td>
			<td></td>
			<td>For cloning and propagation of plasmids</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pGEM-T vector&#160;</td>
			<td>Promega</td>
			<td>A3600</td>
			<td>TA cloning vector</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td>For plasmid isolation</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:140px;">Wizard SV Gel and 
			PCR Clean-Up System</td>
			<td>Promega</td>
			<td>A9282</td>
			<td style="width:207px;">Extract DNA fragments from agarose gels 
			and purify PCR products from an amplification reaction</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:140px;">The iProof high-fidelity 
			DNA polymerase</td>
			<td>Bio-Rad</td>
			<td>172-5302</td>
			<td>High-fidelity DNA polymerase</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BamHI&#160;</td>
			<td>New England Biolabs</td>
			<td>R0136S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BglII&#160;</td>
			<td>New England Biolabs</td>
			<td>R0144L</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KpnI</td>
			<td>New England Biolabs</td>
			<td>R0142S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NdeI&#160;</td>
			<td>New England Biolabs</td>
			<td>R0111S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NotI</td>
			<td>New England Biolabs</td>
			<td>R0189L</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PmlI</td>
			<td>New England Biolabs</td>
			<td>R0532S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PstI&#160;</td>
			<td>New England Biolabs</td>
			<td>R0140S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SacII</td>
			<td>New England Biolabs</td>
			<td>R0157S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SalI</td>
			<td>New England Biolabs</td>
			<td>R0138S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">XhoI</td>
			<td>New England Biolabs</td>
			<td>R0146L</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T4 DNA ligase</td>
			<td>New England Biolabs</td>
			<td>M0202L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Taq DNA polymerase&#160;</td>
			<td>Bio-Rad</td>
			<td>M0267L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ampicillin</td>
			<td>Gibco-Life Technologies</td>
			<td>11593-027</td>
			<td>Antibiotics</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hygromycin B</td>
			<td>PAA</td>
			<td>P21-014</td>
			<td>Antibiotics</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GeneRuler 1 kb DNA ladder</td>
			<td>Thermo Scientific</td>
			<td>SM0312</td>
			<td>1 kb DNA ladder</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PEG4000</td>
			<td>Sigma</td>
			<td>95904-F</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris</td>
			<td>Promega</td>
			<td>H5135</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Bio-Rad</td>
			<td>161-0729</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Salmon Sperm DNA</td>
			<td>Invitrogen</td>
			<td>15-632-011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lithium Acetate</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic acid</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass beads (425-600 &#181;m)</td>
			<td>Sigma</td>
			<td>G8772</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNAse A</td>
			<td>Thermo Scientific</td>
			<td>EN0531</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNA Loading Dye</td>
			<td>Thermo Scientific</td>
			<td>R0611</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bacto Yeast Extract</td>
			<td>BD</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bacto Peptone</td>
			<td>BD</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D-Glucose</td>
			<td>1st Base</td>
			<td>BIO-1101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">YNB without amino acids</td>
			<td>Sigma</td>
			<td>Y0626</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DO Supplement-Leu</td>
			<td>Clontech</td>
			<td>630414</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Difco LB Broth</td>
			<td>BD</td>
			<td>244620</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Difco LB Agar</td>
			<td>BD</td>
			<td>244520</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bacto Agar</td>
			<td>BD</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PCR machine</td>
			<td>Biorad</td>
			<td>T100 Thermal Cycler</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water bath</td>
			<td>Memmert</td>
			<td>WNB 14</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stationary/Shaking Incubator</td>
			<td>Yihder</td>
			<td>LM-570RD</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermo-shaker</td>
			<td>Allsheng</td>
			<td>MS-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro centrifuge</td>
			<td>Eppendorf</td>
			<td>5424R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spectrophotometer</td>
			<td>Eppendorf&#160;</td>
			<td>BioPhotometer plus</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gel imager</td>
			<td>GE</td>
			<td colspan="2">Amersham Imager 600</td>
		</tr>
	</tbody>
</table>

genetic engineering of an unconventional yeast for renewable biofuel and biochemical production

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic characteristics, this unconventional yeast is not only a good model for the study of the fundamental nature of fungal differentiation but is also a promising microbial platform for biochemical production and various biotechnological applications, which require extensive genetic manipulations. However, genetic manipulations of Y. lipolytica have been limited due to the lack of an efficient and stable genetic transformation system as well as very high rates of non-homologous recombination that can be mainly attributed to the KU70 gene. Here, we report an easy and rapid protocol for the efficient genetic transformation and for gene deletion in Y. lipolytica Po1g. First, a protocol for the efficient transformation of exogenous DNA into Y. lipolytica Po1g was established. Second, to achieve the enhanced double-crossover homologous recombination rate for further deletion of target genes, the KU70 gene was deleted by transforming a disruption cassette carrying 1 kb homology arms. Third, to demonstrate the enhanced gene deletion efficiency after deletion of the KU70 gene, we individually deleted 11 target genes encoding alcohol dehydrogenase and alcohol oxidase using the same procedures on the KU70 knockout platform strain. It was observed that the rate of precise homologous recombination increased substantially from less than 0.5% for deletion of the KU70 gene in Po1g to 33%-71% for the single gene deletion of the 11 target genes in Po1g KU70&#916;. A replicative plasmid carrying the hygromycin B resistance marker and the Cre/LoxP system was constructed, and the selection marker gene in the yeast knockout strains was eventually removed by expression of Cre recombinase to facilitate multiple rounds of targeted genetic manipulations. The resulting single-gene deletion mutants have potential applications in biofuel and biochemical production.

The linearized Y. lipolytica expression vector was inserted into the pBR docking platform in the genome of Y. lipolytica Po1g strain by performing a single crossover recombination 27. By using the rapid chemical transformation procedure established in this study, the linearized Y. lipolytica expression vector was successfully transformed into the wild-type Po1g strain at a transformation efficiency of &#62;100 cfu/&#181;g DNA. A knockout cassette flan...

Watch this Scientific Journal Video about Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production at JoVE.com

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

We herein report methods on the molecular genetic manipulation of the Yarrowia lipolytica Po1g strain for improved gene deletion efficiency. The resulting engineered Y. lipolytica strains have potential applications in biofuel and biochemical production.

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic ...

genetic-engineering-an-unconventional-yeast-for-renewable-biofuel

Research

JoVE Journal

Genetics

13.5K Views.  National University of Singapore. We herein report methods on the molecular genetic manipulation of the Yarrowia lipolytica Po1g strain for improved gene deletion efficiency. The resulting engineered Y. lipolytica strains have potential applications in biofuel and biochemical production.

Video: Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment. 
To date, gRNA sequences for the construction of libraries have been almost exclusively generated by oligonucleotide synthesis, which limits the achievable complexity of sequences in the library and is relatively cost-intensive. Here, a detailed protocol for CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a simple and cost-effective method for the generation of highly complex gRNA libraries based on enzymatic digestion of input DNA, is described. Since CORALINA libraries can be generated from any source of DNA, plenty of options for customization exist, enabling a large variety of CRISPR-based screens.

Methods for generating large-scale gRNA libraries should be simple, efficient and cost-effective. We describe a protocol for the production of gRNA libraries based on enzymatic digestion of target DNA. This method, CORALINA (comprehensive gRNA library generation through controlled nuclease activity) presents an alternative to costly custom oligonucleotide synthesis.

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

Production and Purification of Baculovirus for Gene Therapy Application

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a promising vector for gene therapy application. Here, baculovirus is produced by transient transfection of the baculovirus plasmid DNA (bacmid) in an adherent culture of Sf9 cells. Baculovirus is subsequently expanded in Sf9 cells in a serum-free suspension culture until the desired volume is obtained. It is then purified from the culture supernatant using heparin affinity chromatography. Virus supernatant is loaded onto the heparin column which binds baculovirus particles in the supernatant due to the affinity of heparin for baculovirus envelop glycoprotein. The column is washed with a buffer to remove contaminants and baculovirus is eluted from the column with a high-salt buffer. The eluate is diluted to an isotonic salt concentration and baculovirus particles are further concentrated using ultracentrifugation. Using this method, baculovirus can be concentrated up to 500-fold with a 25% recovery of infectious particles. Although the protocol described here demonstrates the production and purification of the baculovirus from cultures up to 1 L, the method can be scaled-up in a closed-system suspension culture to produce a clinical-grade vector for gene therapy application.

In this protocol, baculovirus is produced by transient transfection of baculovirus plasmid into Sf9 cells and amplified in a serum-free suspension culture. The supernatant is purified by heparin affinity chromatography and further concentrated by ultracentrifugation. This protocol is useful for the production and purification of baculovirus for gene therapy application.

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a ...

High-Resolution Comparison of Bacterial Conjugation Frequencies

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons of conjugation frequency under different conditions are required to understand how the conjugative element spreads in nature. However, conventional methods for comparing conjugation frequency are not appropriate for in-depth comparisons because of the high background caused by the occurrence of additional conjugation events on the selective plate. We successfully reduced the background by introducing a most probable number (MPN) method and a higher concentration of antibiotics to prevent further conjugation in selective liquid medium. In addition, we developed a protocol for estimating the probability of how often donor cells initiate conjugation by sorting single donor cells into recipient pools by fluorescence-activated cell sorting (FACS). Using two plasmids, pBP136 and pCAR1, the differences in conjugation frequency in Pseudomonas putida cells could be detected in liquid medium at different stirring rates. The frequencies of conjugation initiation were higher for pBP136 than for pCAR1. Using these results, we can better understand the conjugation features in these two plasmids.

With an aim to understand the behaviors of various bacterial conjugative DNA elements under different conditions, we describe a protocol for detecting differences in conjugation frequency, with high resolution, to estimate how efficiently the donor bacterium initiates conjugation.

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons ...

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies.
The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.

This protocol describes the transient genetic engineering of dental stem cells extracted from the human dental follicle. The applied non-viral modification strategy may become a basis for the improvement of therapeutic stem cell products.

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely related biochemical pathways. Previous methods such as the Synthetic Genetic Array (SGA) analysis, and random mutagenesis techniques using ultraviolet (UV) or chemicals like ethyl methanesulfonate (EMS) or N-ethyl-N- nitrosourea (ENU), have been widely used but are often costly and laborious. Also, these mutagen-based screening methods are frequently associated with severe side effects on the organism, inducing multiple mutations that add to the complexity of isolating the suppressors. Here, we present a simple and effective protocol to identify suppressor mutations in mutants which confer a growth defect in Schizosaccharomyces pombe. The fitness of cells with a growth deficiency in standard rich liquid media or synthetic liquid media can be monitored for recovery using an automated 96-well plate reader over an extended period. Once a cell acquires a suppressor mutation in the culture, its descendants outcompete those of the parental cells. The recovered cells that have a competitive growth advantage over the parental cells can then be isolated and backcrossed with the parental cells. The suppressor mutations are then identified using whole-genome sequencing. Using this approach, we have successfully isolated multiple suppressors that alleviate the severe growth defects caused by loss of Elf1, an AAA+ family ATPase that is important in nuclear mRNA transport and maintenance of genomic stability. There are currently over 400 genes in S. pombe with mutants conferring a growth defect. As many of these genes are uncharacterized, we propose that our method will hasten the identification of novel functional interactions with this user-friendly, high-throughput approach.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and&#160; selective&#160; for&#160; mutations that&#160; often&#160; occur at&#160; &#160;a&#160; single&#160; genomic&#160; locus.&#160; The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented in human embryos. Preimplantation genetic testing for aneuploidy (PGT-A) is a genetic test that significantly improves reproductive outcomes by detecting chromosomal abnormalities of embryos. Next-generation sequencing (NGS) provides a high-throughput and cost-effective approach for genetic analysis and has shown clinical applicability in PGT-A. Here, we present a rapid and low-cost semiconductor sequencing-based NGS method for screening of aneuploidy in embryos. The first step of the workflow is whole genome amplification (WGA) of the biopsied embryo specimen, followed by construction of sequencing library, and subsequent sequencing on the semiconductor sequencing system. Generally, for a PGT-A application, 24 samples can be loaded and sequenced on each chip generating 60&#8722;80 million reads at an average read length of 150 base pairs. The method provides a refined protocol for performing template amplification and enrichment of sequencing library, making the PGT-A detection reproducible, high-throughput, cost-efficient, and timesaving. The running time of this semiconductor sequencer is only 2&#8722;4 hours, shortening the turnaround time from receiving samples to issuing reports into 5 days. All these advantages make this assay an ideal method to detect chromosomal aneuploidies from embryos and thus, facilitate its wide application in PGT-A.

Here, we introduce a semiconductor sequencing method for preimplantation genetic testing for aneuploidy (PGT-A) with the advantages of short turnaround time, low cost, and high throughput.

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented ...

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian cells, this technology represents a novel means to perform genome-wide genetic screens for functional genomics studies. Libraries of guide RNAs (sgRNA) targeting all open reading frames permit the facile generation of thousands of genetic perturbations in a single pool of cells that can be screened for specific phenotypes to implicate gene function and cellular processes in an unbiased and systematic way. CRISPR-Cas screens provide researchers with a simple, efficient, and inexpensive method to uncover the genetic blueprints for cellular phenotypes. Furthermore, differential analysis of screens performed in various cell lines and from different cancer types can identify genes that are contextually essential in tumor cells, revealing potential targets for specific anticancer therapies. Performing genome-wide screens in human cells can be daunting, as this involves the handling of tens of millions of cells and requires analysis of large sets of data. The details of these screens, such as cell line characterization, CRISPR library considerations, and understanding the limitations and capabilities of CRISPR technology during analysis, are often overlooked. Provided here is a detailed protocol for the successful performance of pooled genome-wide CRISPR-Cas9 based screens.

CRISPR-Cas9 technology provides an efficient method to precisely edit the mammalian genome in any cell type and represents a novel means to perform genome-wide genetic screens. A detailed protocol discussing the steps required for the successful performance of pooled genome-wide CRISPR-Cas9 screens is provided here.

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian ...

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the only known non-legume lineage able to establish a nitrogen-fixing nodule symbiosis with rhizobium. Comparative studies between legumes and P. andersonii could provide valuable insight into the genetic networks underlying root nodule formation. To facilitate comparative studies, we recently sequenced the P. andersonii genome and established Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing. Here, we provide a detailed description of the transformation and genome editing procedures developed for P. andersonii. In addition, we describe procedures for the seed germination and characterization of symbiotic phenotypes. Using this protocol, stable transgenic mutant lines can be generated in a period of 2-3 months. Vegetative in vitro propagation of T0 transgenic lines allows phenotyping experiments to be initiated at 4 months after A. tumefaciens co-cultivation. Therefore, this protocol takes only marginally longer than the transient Agrobacterium rhizogenes-based root transformation method available for P. andersonii, though offers several clear advantages. Together, the procedures described here permit P. andersonii to be used as a research model for studies aimed at understanding symbiotic associations as well as potentially other aspects of the biology of this tropical tree.

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae) and can form nitrogen-fixing root nodules in association with the rhizobium. Here, we describe a detailed protocol for reverse genetic analyses in P. andersonii based on Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing.

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the ...