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

13.6K 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 ...

In Vivo Monitoring of Transcriptional Activity During Metabolic Transition Using a Bioluminescent Reporter in Yeast

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is particularly relevant for survival in fluctuating environments such as those found in beer fermentation. However, sugar transitions are an environmental variable that is challenging to predict and detect, impacting the outcome of beer fermentations. This protocol describes an in vivo system to monitor transcriptional activation associated with the glucose-to-maltose metabolic shift in Saccharomyces eubayanus that applies to different wild Saccharomyces yeast strains. 
The system employs an episomal bioluminescent transcriptional reporter for maltose metabolism, focusing on MAL32, since it provides a good readout for metabolic shifts, as studied in S. cerevisiae. For this, yeast strains were transformed with plasmids containing the MAL32 regulatory region from S. eubayanus, controlling the expression of a gene encoding for a destabilized version of firefly luciferase1, and a hygromycin resistance gene used exclusively during transformation to ensure plasmid acquisition. Following selection, transformed yeast cells can be cultured under non-selective conditions, as the episomal plasmid remains stable in culture conditions for up to 7 days. 
This system was validated under a complex sugar environment in microfermentation assays, confirming the effectiveness of the luciferase reporter in informing metabolic transitions. Samples were collected regularly and analyzed with a luminometer, providing continuous insights into yeast responses. While broadly applicable, this protocol is particularly valuable for assessing yeast performance under fermentation conditions, where metabolic changes pose a significant challenge. Additionally, this methodology can be adapted by selecting alternative promoters to explore a broader range of responses to environmental changes, allowing characterization as well as optimization of wild yeast strains for diverse industrial applications.

This protocol employs a bioluminescent reporter, allowing measurements of transcriptional activity in Saccharomyces eubayanus to monitor the glucose-to-maltose transition, enabling real-time analysis of metabolic adaptations and supporting strain optimization for industrial fermentation under diverse conditions.

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is ...

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme saccharification processes release sugars that can be fermented by yeast. Traditional industrial yeasts do not ferment xylose (comprising up to 40% of plant sugars) and are not able to function in concentrated hydrolyzates. Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment. While detoxification methods can render hydrolyzates fermentable, they are costly and generate waste disposal liabilities. Here, adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of the native Scheffersomyces stipitis strain NRRL Y-7124 that are able to efficiently convert hydrolyzates to economically recoverable ethanol despite adverse culture conditions. Improved individuals are enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and mutations induced by exposures to two diverse hydrolyzates, ethanol or UV radiation. Final evolution cultures are dilution plated to harvest predominant isolates, while intermediate populations, frozen in glycerol at various stages of evolution, are enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data. Using the RPI statistical parameter, an overall relative performance average is calculated to rank isolates based on multiple factors, including culture conditions (varying in nutrients and inhibitors) and kinetic characteristics. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.

Adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of Scheffersomyces stipitis strain NRRL Y-7124 that are able to rapidly consume hexose and pentose mixed sugars in enzyme saccharified undetoxified hydrolyzates and to accumulate over 40 g/L ethanol.

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme ...

Bioengineering

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular processes. There is a need for culturing and measuring systems that incorporate programmed illumination and stimulation of optogenetic systems. We present a protocol for building and using a continuous culturing apparatus to illuminate microbial cells with programmed doses of light, and automatically acquire and analyze images of cells in the effluent. The operation of this apparatus as a chemostat allows the growth rate and the cellular environment to be tightly controlled. The effluent of the continuous cell culture is regularly sampled and the cells are imaged by multi-channel microscopy. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses in the fluorescence intensity and cellular morphology of cells sampled from the culture effluent are measured over multiple days without user input. We demonstrate the utility of this culturing apparatus by dynamically inducing protein production in a strain of Saccharomyces cerevisiae engineered with an optogenetic system that activates transcription.

We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days.

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular ...

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

Immunology and Infection

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

A bottleneck in the &#8216;design-build-test&#8217; cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

Biology

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, ...

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that ...

Biofuels: Producing Ethanol from Cellulosic Material

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University
In this experiment, cellulosic material (such as corn stalks, leaves, grasses, etc.) will be used as a feedstock for the production of ethanol.&#160;The cellulosic material is first pretreated (ground and heated), digested with enzymes, and then fermented with yeast.&#160;Ethanol production is monitored using an ethanol probe.&#160;The experiment can be extended to optimize ethanol production by varying the feedstock used, pretreatment conditions, enzyme variation, yeast variation, etc.&#160;An alternative method of monitoring the reaction is to measure the carbon dioxide produced (using a gas sensor) instead of the ethanol.&#160;As a low-tech alternative, glucose meters (found in any drug store) can be used to monitor the glucose during the process, if an ethanol probe or carbon dioxide gas sensor is not available.
With an increased emphasis on &#8216;inquiry-based learning&#8221;, scientific probes are becoming more popular.&#160;Handheld devices like the Vernier Lab Quest used in conjunction with a variety of probes (such as those for conductivity, dissolved oxygen, voltage, and more) allow for less focus on collecting data and/or making graphs and more on analyzing the data and making predictions.&#160;Another advantage is that these are small and lightweight and can be taken into the field for measurements.

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University
In this experiment, cellulosic material (such as corn stalks, leaves, ...

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

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