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

Podsumowanie

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.

Streszczenie

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 KU70Δ. 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.

Wprowadzenie

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 KU70Δ 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.

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Protokół

1. Generation of the Y. lipolytica KU70 Deletion Strain

1. Construction of the disruption cassette
   Note: See Table 1 for all primers used in polymerase chain reaction (PCR) amplifications.
   1. 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' and 3' 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.
   2. Perform PCR using a high-fidelity DNA polymerase as detailed in Table 2.
   3. Purify the PCR product LoxP-promoter-LEU2-terminator-LoxP cassette using a PCR purification kit according to the manufacturer's instructions. Add 3'-A overhangs to the purified PCR product according to the manufacturer'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.
   4. PCR amplify the 1 kb 5' 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's instructions.
      1. 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'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.
   5. PCR amplify the 1 kb 3' 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's instructions. Double digest both the purified PCR product and T-LEU2-5E plasmid with NotI and NdeI enzymes as per Table 4.
      1. Purify the digestion mixture using a PCR purification kit as per manufacturer'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.
   6. 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's instructions.
2. Competent cell preparation and transformation of Y. lipolytica Po1g strain
   1. Competent cell preparation
      1. 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 °C shaking incubator at 225 rpm for 20 hr until saturation (an OD₆₀₀ of about 15, measured using a spectrophotometer).
      2. 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.
      3. Aliquot the competent cells (100 µl) 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 °C for long-term storage.
   2. Transformation
      1. Gently mix 10 µl of denatured salmon sperm DNA (10 mg/ml) and 1-5 µg of the purified disruption cassette together with 100 µl of competent cells, and incubate at 30 °C for 15 min.
      2. Add 700 µl of 40% polyethylene glycol (PEG)-4000 (dissolved in 0.1 M lithium acetate pH 6.0), mix well and incubate in a 30 °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).
      3. Heat shock the transformation mixture by placing the tube in a 39 °C water bath for 60 min.
      4. Add 1 ml YPD medium and recover for 2 hr at 30 °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.
      5. 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 µl of TE buffer and plate onto selective plates (e.g., leucine-deficient plates), and incubate at 30 °C for 2-3 days.
      6. Pick 4 to 10 single colonies and inoculate separately into 2 ml of YPD medium. Incubate overnight in a 30 °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 µg plasmid DNA used ²³.

2. Marker Rescue

1. Construction of the Cre expression plasmid
   1. Construction of pYLEX1-CRE and pYLEX1-HPH
      1. 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.
      2. Purify both cre and hph fragments using a PCR purification kit according to the manufacturer'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's instructions.
      3. 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.
   2. Construction of pSL16-CRE-HPH
      1. 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's instructions.
         1. 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'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.
      2. 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's instructions.
         1. 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'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).
      3. 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.
2. Cre recombinase-mediated marker rescue
   1. 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 µg/ml hygromycin B), and incubate at 30 °C for 2-3 days.
   2. 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.
   3. Pick a positive clone and inoculate into 2 ml of YPDH medium, and incubate in a 30 °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.
   4. 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 °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).

3. Deletion of Alcohol Dehydrogenase and Alcohol Oxidase Genes

1. 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&db=yli&seqid=aa).
   Note: The BLAST search tool returns the most similar protein sequences in the Y. lipolytica genome database.
2. 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.
3. 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.

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Wyniki

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 >100 cfu/µg DNA. A knockout cassette flan...

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Dyskusje

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...

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Materiały

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