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Genetics

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

Published: September 20th, 2016

DOI:

10.3791/54371

1Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 2NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of Singapore, 3Food Science and Chemical Engineering, Singapore Institute of Technology

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 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.

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 am....

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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.......

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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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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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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&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).

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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
Name Company Catalog Number Comments
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

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