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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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 ....

Results

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 flanked by.......

Discussion

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.......

Materials

List of materials used in this article

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