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

Summary

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

Abstract

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.

Introduction

Directed molecular evolution is a robust, fast and reliable method to design enzymes^{1, 2}. Through iterative rounds of random mutation, recombination and screening, improved versions of enzymes can be generated that act on new substrates, in novel reactions, in non-natural environments, or even to assist the cell to achieve new metabolic goals^3-5. Among the hosts used in directed evolution, the brewer's yeast Saccharomyces cerevisiae offers a repertoire of solutions for the functional expression of complex eukaryotic proteins that are not otherwise available in prokaryotic counterparts^6,7.

Used e....

Protocol

1. Mutant Library Construction

1. Choose the regions to be subjected to MORPHING with the help of computational algorithms based on the available crystal structure or homology models¹⁸.
   1. Here, target two regions of AAO from Pleurotus eryngii for random mutagenesis and recombination (Met[α1]-Val109, Phe392-Gln566), while amplifying the remainder of the gene (844 bp) by high-fidelity PCR (Figure 1).
      Note: Several segments can be studied by MORPHING in an independent or combined manner¹⁶.
2. Amplify the targeted areas by mutagenic PCR. Create overlapping....

Results

AAO from P. eryngii is an extracellular flavooxidase that supplies fungal peroxidases with H₂O₂ to start attacking lignin. Two segments of AAO were subjected to focused-directed evolution by MORPHING in order to enhance its activity and its expression in S. cerevisiae ¹⁹. Irrespective of the foreign enzymes harbored by S. cerevisiae, the most critical issue when constructing mutant libraries in yeast concerns the engineering of s.......

Discussion

In this article, we have summarized most of the tips and tricks employed in our laboratory to engineer enzymes by directed evolution in S. cerevisiae (using AAO as an example) so that they can be adapted for use with many other eukaryotic enzyme systems by simply following the common approach described here.

In terms of library creation, MORPHING is a fast one-pot method to introduce and recombine random mutations in small protein stretches while leaving the remaining regions of the p.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1. Culture media
Ampicillin sodium salt	Sigma-Aldrich	A0166	CAS Nº 69-52-3 M.W. 371.39
Bacto Agar	Difco	214010
Cloramphenicol	Sigma-Aldrich	C0378	CAS Nº 56-75-7 M.W. 323.13
D-(+)-Galactose	Sigma-Aldrich	G0750	CAS Nº 59-23-4 M.W. 180.16
D-(+)-Glucose	Sigma-Aldrich	G5767	CAS Nº 50-99-7 M.W. 180.16
D-(+)-Raffinose pentahydrate	Sigma-Aldrich	83400	CAS Nº 17629-30-0 M.W. 594.51
Peptone	Difco	211677
Potassium phosphate monobasic	Sigma-Aldrich	P0662	CAS Nº 7778-77-0 M.W. 136.09
Uracil	Sigma Aldrich	U1128
Yeast Extract	Difco	212750
Yeast Nitrogen Base without Amino Acids	Difco	291940
Yeast Synthetic Drop-out Medium Supplements without uracil	Sigma-Aldrich	Y1501
Name	Company	Catalog Number	Comments
2. PCR Reactions
dNTP Mix	Agilent genomics	200415-51	25 mM each
iProof High-Fidelity DNA polymerase	Bio-rad	172-5301
Manganese(II) chloride tetrahydrate	Sigma-Aldrich	M8054	CAS Nº 13446-34-9 M.W. 197.91
Taq DNA Polymerase	Sigma-Aldrich	D4545	For error prone PCR
Name	Company	Catalog Number	Comments
3. Plasmid linearization
BamHI restriction enzyme	New England Biolabs	R0136S
Bovine Serum Albumin	New England Biolabs	B9001S
XhoI restriction enzyme	New England Biolabs	R0146S
Not I restriction enzyme	New England Biolabs	R0189S
Gel Red	Biotium	41003	For staining DNA
Name	Company	Catalog Number	Comments
4. FOX assays
Ammonium iron(II) sulfate hexahydrate	Sigma-Aldrich	F3754	CAS Nº 7783-85-9 M.W. 392.14
Anysil Alcohol	Sigma Aldrich	W209902	CAS Nº 105-13-5 M.W. 138.16
D-Sorbitol	Sigma-Aldrich	S1876	CAS Nº 50-70-4 M.W. 182.17
Hydrogen peroxide 30%	Merck Millipore	1072090250	FOX standard curve
Xylenol Orange disodium salt	Sigma-Aldrich	52097	CAS Nº 1611-35-4 M.W. 716.62
Name	Company	Catalog Number	Comments
5. Agarose gel stuff
Agarose	Norgen	28035	CAS Nº 9012-36-6
Gel Red	Biotium	41003	DNA analysis dye
GeneRuler 1kb Ladder	Thermo Scientific	SM0311	DNA M.W. standard
Loading Dye 6x	Thermo Scientific	R0611
Low-melting temperature agarose	Bio-rad	161-3112	CAS Nº 39346-81-1
Name	Company	Catalog Number	Comments
6. Kits and cells
S. cerevisiae strain BJ5465	LGC Promochem	ATTC 208289	Protease deficient strain with genotype: MATα ura3-52 trp1 leu2-delta1 his3-delta200 pep4::HIS3 prb1-delta1.6R can1 GAL
E. coli XL2-Blue competent cells	Agilent genomics	200150	For plasmid purification and amplification
NucleoSpin Gel and PCR Clean-up Kit	Macherey-Nagel	740,609,250	DNA gel extraction
NucleoSpin Plasmid Kit	Macherey-Nagel	740,588,250	Column miniprep Kit
Yeast Transformation Kit	Sigma-Aldrich	YEAST1-1KT	Included DNA carrier (Salmon testes)
Zymoprep yeast plasmid miniprep I	Zymo research	D2001	Plasmid extraction from yeast
Name	Company	Catalog Number	Comments
7. Plates
96-well plates	Greiner Bio-One	655101	Clear, non-sterile, polystyrene (for activity measurements)
96-well plates	Greiner Bio-One	655161	Clear, sterile, polystyrene (for microfermentations)
96-well plate lid	Greiner Bio-One	656171	Clear, sterile, polystyrene (for microfermentations)