Name: genetic engineering of an unconventional yeast for renewable biofuel and biochemical production
Uploaded: 2016-09-20T08:00:00
Description: Watch this Scientific Journal Video about Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production at JoVE.com

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...

<ol>
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Current research, technology and education topics in applied microbiology and microbial biotechnology. , 930-940 (2010).</li><li>Martinez-Vazquez, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+transcription+factor+Znc1p,+which+regulates+the+yeast-to-hypha+transition+in+the+dimorphic+yeast+Yarrowia+lipolytica.">Identification of the transcription factor Znc1p, which regulates the yeast-to-hypha transition in the dimorphic yeast Yarrowia lipolytica.</a> Plos One. 8 (6), e66790 (2013).</li><li>Richard, M., Quijano, R. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Food-related+applications+of+Yarrowia+lipolytica.">Food-related applications of Yarrowia lipolytica.</a> Food Chem. 152, 1-10 (2014).</li><li>Finogenova, T., Morgunov, I., Kamzolova, S., Chernyavskaya, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organic+acid+production+by+the+yeast+Yarrowia+lipolytica:+a+review+of+prospects.">Organic acid production by the yeast Yarrowia lipolytica: a review of prospects.</a> Appl. Biochem. 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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 ...

The overall goal of this procedure is to develop effective genetic engineering methods for an unconventional yeast species, including construction of transforming DNA, DNA transformation, transformant selection, and targeted gene deletion by homologous recombination. We've established a highly vision gene deletion technique in the yarrowia lipolytica PO1G strain with potential applications in bio fuel and bio chemical production. The main advantage of this technique over existing methods like PCR-mediated gene disruption is that large amounts of gene deletion crescents and the wide range of selectable markers are available.In this study, we report the genetics transformation protocol for the yarrowia lipolytica PO1G strain that is efficient, easy, reputed, and reproducible. The yarrowia lipolytica PO1G KU70 deletion strain constructed in this study show a very efficient homologous recombination frequency which enables faster and easier screenings of targeted gene deletions. The first step in this procedure is to amplify, by PCR, the LEU2 expression cassette from a Y.lipolytica expression vector and introduce LoxP sites into the 5-prime and 3-prime ends of the LEU2 cassette.A BamHI restriction site is also introduced in primer one for subsequent steps of the cloning process. After purifying the LoxP promoter LEU2 terminator LoxP cassette, three prime-A overhangs are added to the purified PCR product. The A-tailed PCR product is then ligated into a TA cloning vector to yield the plasma T-LEU2.The second PCR step is to amplify the one kilobase 5-prime upstream sequence of the KU70 gene using primers three and four from the Y.lipolytica PO1G genomic DNA. After that, the double digest both the purified PCR product and T-LEU2 plasmid with SacII and BamHI enzymes and ligate the purified and digested PCR product to the SacII BamHI sites of T-LEU2 to yield the plasmid T-LEU2-5E. The third PCR step is to amplify the one kilobase 3-prime downstream sequence of the KU70 gene using primers five and six from the Y.lipolytica PO1G genomic DNA.Then double digest both the purified PCR product and the T-LEU2-53 plasmid with NotI and NdeI enzymes and ligate the purified and digested PCR product to the NotI NdeI sites of T-LEU2-5E to yield the T-KO plasmid. Lastly, digest the T-KO plasmid with SacII and NdeI enzymes to produce the disruption cassette. Begin this procedure by preparing competent Y.lipolytica PO1G cells.Inoculate a colony of Y.lipolytica PO1G strain from a fresh yeast extract peptone dextrose or YPD plate in 10 milliliters of YPD medium in a 100 milliliter flask. Incubate in a 30 degree Celsius shaking incubator at 225 RPM for 20 hours until saturation. On the following day, pellet the cells by cetrifuging for five minutes at 5000 times G at room temperature.Wash the cells with 20 milliliters of TE buffer and pellet the cells again. After removing the supernatant, resuspend the cells in one milliliter of 0.1 molar lithium acetate and incubate for 10 minutes at room temperature. Divide the competent cells in to 100 microliter aliquots in sterile 1.5 milliliter tubes.Proceed immediately to the transformation procedure by gently mixing 10 microliters of denatured salmon sperm DNA and one to five micrograms of the purified disruption cassette together with 100 microliters of competent cells. Incubate at 30 degrees Celsius for 15 minutes. Add 700 microliters of 40%polyethylene glycol for 1000.Mix well, and incubate in a 30 degree Celsius shaking incubator at 225 RPM for 60 minutes. Next, heat chuck the transformation mixture by placing the tube in a 39 degree Celsius water bath for 60 minutes. After 60 minutes, add one milliliter of YPD medium to the tube and allow the cells to recover for two hours at 30 degrees Celsius at 225 RPM.Centrifuge at 9000 times G for one minute at room temperature. Remove the supernatant and resuspend the pellet in one milliliter of TE buffer. Centrifuge the cells again and discard the supernatant.Resuspend the pellet in 100 microliters of TE buffer and plate on to leucine-deficient plate. Incubate the plate at 30 degrees Celsius for two to three days. Prior to performing CRE recombinase mediated marker rescue, the CRE expression plasmid is constructed as described in the text protocol.Some of the features of this episomally replicating plasmid include a CRE recombinase gene, a hygromycin B-resistance marker, and an ampicillin-resistance gene. To begin this procedure transform the CRE expression plasmid into competent cells of the KU70 knockout strain following the transformation protocol demonstrated earlier. Plate the transformed cells on to YPD plus hygromycin B plates and incubate at 30 degrees Celsius for two to three days.After performing colony PCR, as described in the text protocol to identify positive colonies, pick a positive clone to inoculate two milliliters of YPDH medium and incubate in a 30 degree Celsius shaking incubator at 225 RPM overnight to saturation. On the following day, measure the OD 600 of the culture with a spectrophotometer. When the cells are at an OD 600 of about 15, harvest by centrifugation at 5000 times G for five minutes at room temperature.Remove the supernatant, add two milliliters of sterile water and spin again. Remove the supernatant and resuspend the cells in YPD medium Reinoculate the cells in to two milliliters of YPD medium with an initial OD 600 of 0.1. And allow the cells to grow in a 30 degree Celsius incubator at 225 RPM overnight.On the following day, streak the overnight cell culture on to YPD plates to isolate single colonies. Incubate at 30 Celsius until colonies appear. Replica plate colonies on to YPD, YPDH, and Leucine-deficient plates.Incubate the plates at 30 degrees Celsius for two days. A schematic of the KU70 knockout plasmid is shown. Digest of the plasmid with SacII and NdeI enzymes produces the disruption Cassette.Gel electrophoresis confirms the presence of a 4.3 kilobase band, which is the expected size of the disruption cassette. The KU70 gene deletion in Y.lipolytica PO1G is confirmed by PCR. Lanes one to 10 contain PCR products amplified from the genomic DNA of transformants and lane 11 contains PCR products from the wild type strain.Lane seven illustrates a positive knockout while lane one shows a false positive. The selection marker gene in the KU70 knockout strain was eventually removed by the expression of CRE recombinase. Positive marker-free transformants were verified by their growth on only a YPD plate.To verify the increase of the gene deletion rate, after deletion of the KU70 gene, 11 target genes were individually deleted using the same procedure in the KU70 knockout strain. Each gene disruption was confirmed by PCR and a representative result is shown here with lanes one, two and four identified as positive knockouts. The yarrowia lipolytica PO1G KU70 deletion platinum strain provides a more efficient system and a better choice for applications in pathway engineering and strain optimization when constructing other chromosomal deletion mutants.After watching this video, you should have a comprehensive understanding of the targeted single-gene deletion strategy through homologous recombination in the yarrowia lipolytica PO1G cells.

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