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Chapter 20 : Genetic Engineering Tools for

Authors: Verena Siewers, Uffe H. Mortensen, Jens Nielsen
Content Type: Reference
Publication Year: 2010

Category: Applied and Industrial Microbiology

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Abstract:

The yeast is a widely used cell factory. Genetic engineering requires efficient transformation techniques, and many protocols for transformation of have been developed. These include methods involving spheroplast generation, electroporation, and biolistics with DNA coated microprojectiles. For expression of heterologous genes or overexpression of homologous genes in , plasmid vectors are usually employed. Most are shuttle vectors that allow cloning and amplification in . Yeast plasmid vectors can be divided into different types. Probably the most widely used type of selectable markers are ones that confer prototrophy to auxotrophic strains containing mutations in amino acid and/or nucleotide biosynthetic pathways, e.g., and . Several of the prototrophic markers offer this possibility, the most widely used being the marker, which can be selected against using 5-fluoroorotic acid (5FOA). Proper transcription termination is a requirement for mRNA stability and thus also for high expression levels. Protein secretion requires the presence of an N-terminal signal sequence. Gene targeting is particularly efficient in because DNA double-strand breaks are preferentially repaired by homologous recombination as opposed to nonhomologous end joining. Since the codon composition at the gene’s 5’ end seems to be most important, it may be sufficient only to optimize the first 14 to 32 codons to achieve good expression.

Citation: Siewers V, Mortensen U, Nielsen J. 2010. Genetic Engineering Tools for , p 287-301. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch20
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Genetic Engineering Tools for Saccharomyces cerevisiae
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Citation: Siewers V, Mortensen U, Nielsen J. 2010. Genetic Engineering Tools for , p 287-301. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch20
/content/10.1128/9781555816827.ch20.ch20fig01
FIGURE 1
Genomic integration using an integrative plas-mid. and a selective marker, in this case , are cloned into an plasmid. The plasmid is linearized at a site within prior to transformation into a strain. After integration, the gene is duplicated, typically resulting in one functional and one nonfunctional copy. Chromosomal DNA is indicated by the presence of a gray oval representing the centromere.
10.1128/9781555816827/f0293-01_thmb.gif
10.1128/9781555816827/f0293-01.gif
Image of FIGURE 1
FIGURE 1

Genomic integration using an integrative plas-mid. and a selective marker, in this case , are cloned into an plasmid. The plasmid is linearized at a site within prior to transformation into a strain. After integration, the gene is duplicated, typically resulting in one functional and one nonfunctional copy. Chromosomal DNA is indicated by the presence of a gray oval representing the centromere.

Citation: Siewers V, Mortensen U, Nielsen J. 2010. Genetic Engineering Tools for , p 287-301. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch20
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FIGURE 2
Substrates for disruption and deletion of (A) Disruption of is achieved by using a linear substrate composed of a selectable marker () flanked by sequences up- and downstream of the integration site in . (B) Deletion of is achieved by using a linear substrate composed of a selectable marker () flanked by sequences up- and downstream of . Original sequence between the up- and downstream sequences will be deleted. (C) Construction of a gene deletion substrate by PCR. In a first round of PCR, fragments containing the up- and downstream sequences of and the marker gene () are generated in three independent PCR reactions. Note that one of the primers in the primer pair used to generate the upstream fragment is extended by a sequence identical to the extreme 5′-upstream section of as indicated by a black line added to the gray primer. The sequence of the extension is complementary to the 5′-primer used to amplify . Similarly, one of the primers used to amplify the downstream fragment is extended by a sequence that is complementary to the 3′-primer used to amplify . The two primer extensions produce sequence overlaps that allow all three fragments to be fused and amplified in a second round of PCR. (D) Construction of a bipartite gene deletion substrate. The up- and downstream sections are generated as shown in panel C. In a first round of PCR, two fragments consisting of the 2/3 upstream and 2/3 downstream parts of URA3 are generated in two individual PCR reactions. The primer extensions added to primers used to generate the up- and downstream sequences allow the up- and downstream fragments to be individually fused to the 5′-2/3 and 3′-2/3 fragments in separate PCR reactions.
10.1128/9781555816827/f0294-01_thmb.gif
10.1128/9781555816827/f0294-01.gif
Image of FIGURE 2
FIGURE 2

Substrates for disruption and deletion of (A) Disruption of is achieved by using a linear substrate composed of a selectable marker () flanked by sequences up- and downstream of the integration site in . (B) Deletion of is achieved by using a linear substrate composed of a selectable marker () flanked by sequences up- and downstream of . Original sequence between the up- and downstream sequences will be deleted. (C) Construction of a gene deletion substrate by PCR. In a first round of PCR, fragments containing the up- and downstream sequences of and the marker gene () are generated in three independent PCR reactions. Note that one of the primers in the primer pair used to generate the upstream fragment is extended by a sequence identical to the extreme 5′-upstream section of as indicated by a black line added to the gray primer. The sequence of the extension is complementary to the 5′-primer used to amplify . Similarly, one of the primers used to amplify the downstream fragment is extended by a sequence that is complementary to the 3′-primer used to amplify . The two primer extensions produce sequence overlaps that allow all three fragments to be fused and amplified in a second round of PCR. (D) Construction of a bipartite gene deletion substrate. The up- and downstream sections are generated as shown in panel C. In a first round of PCR, two fragments consisting of the 2/3 upstream and 2/3 downstream parts of URA3 are generated in two individual PCR reactions. The primer extensions added to primers used to generate the up- and downstream sequences allow the up- and downstream fragments to be individually fused to the 5′-2/3 and 3′-2/3 fragments in separate PCR reactions.

Citation: Siewers V, Mortensen U, Nielsen J. 2010. Genetic Engineering Tools for , p 287-301. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch20
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FIGURE 3
Substrates for iterative gene deletion. Direct repeats flanking the selectable marker allow the marker to be eliminated from the genome by recombination. If the selectable marker is , recombinants can be selected using 5-FOA. (A) Construction of a bipartite gene deletion substrate with the marker flanked by a direct repeat (labeled DR). The template for the marker fragments is a plasmid that contains direct repeats flanking the marker. The remaining steps are similar to the legend for Fig. 2D . (B) Genomic integration of a bipartite gene-targeting substrate. Note that homologous recombination completes the selectable marker after cotransforming the two substrate fragments into the cell. (C) Direct repeat recombination eliminates the selectable marker from the genome. Note that one repeat remains in the genome. (D) By varying the sequence content of the DR repeats flanking the marker in the template plasmid, different useful sequences may be introduced between defined up and down sequences, as indicated; see text for details.
10.1128/9781555816827/f0295-01_thmb.gif
10.1128/9781555816827/f0295-01.gif
Image of FIGURE 3
FIGURE 3

Substrates for iterative gene deletion. Direct repeats flanking the selectable marker allow the marker to be eliminated from the genome by recombination. If the selectable marker is , recombinants can be selected using 5-FOA. (A) Construction of a bipartite gene deletion substrate with the marker flanked by a direct repeat (labeled DR). The template for the marker fragments is a plasmid that contains direct repeats flanking the marker. The remaining steps are similar to the legend for Fig. 2D . (B) Genomic integration of a bipartite gene-targeting substrate. Note that homologous recombination completes the selectable marker after cotransforming the two substrate fragments into the cell. (C) Direct repeat recombination eliminates the selectable marker from the genome. Note that one repeat remains in the genome. (D) By varying the sequence content of the DR repeats flanking the marker in the template plasmid, different useful sequences may be introduced between defined up and down sequences, as indicated; see text for details.

Citation: Siewers V, Mortensen U, Nielsen J. 2010. Genetic Engineering Tools for , p 287-301. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch20
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FIGURE 4
Incorporation of GFP DNA sequence into the genome. (A) Universal vector set for incorporating the GFP DNA sequence at any location in the genome using a bipartite gene-targeting substrate. Positions of the primers are indicated. (B) Strategy to extend the protein encoded by C-terminally with GFP. The position of the primer pairs used to amplify the two targeting sequences is indicated. Each fragment will terminate with a sequence tag matching either the 5′ - or the 3′ -end of GFP as indicated. Note that a sequence complementary to a stop codon, shown as an open circle, must be incorporated in the forward primer used to amplify the downstream targeting fragment. This codon is positioned right after the GFP segment of this primer as indicated.
10.1128/9781555816827/f0296-01_thmb.gif
10.1128/9781555816827/f0296-01.gif
Image of FIGURE 4
FIGURE 4

Incorporation of GFP DNA sequence into the genome. (A) Universal vector set for incorporating the GFP DNA sequence at any location in the genome using a bipartite gene-targeting substrate. Positions of the primers are indicated. (B) Strategy to extend the protein encoded by C-terminally with GFP. The position of the primer pairs used to amplify the two targeting sequences is indicated. Each fragment will terminate with a sequence tag matching either the 5′ - or the 3′ -end of GFP as indicated. Note that a sequence complementary to a stop codon, shown as an open circle, must be incorporated in the forward primer used to amplify the downstream targeting fragment. This codon is positioned right after the GFP segment of this primer as indicated.

Citation: Siewers V, Mortensen U, Nielsen J. 2010. Genetic Engineering Tools for , p 287-301. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch20
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FIGURE 5
Substrates for introduction of point mutations. Identical direct repeats of sequences containing the relevant mutation flank a selectable marker (). The substrate is shown as a bipartite gene-targeting substrate. Direct repeat recombination between mutant repeats leaves only the mutation in the genome.
10.1128/9781555816827/f0297-01_thmb.gif
10.1128/9781555816827/f0297-01.gif
Image of FIGURE 5
FIGURE 5

Substrates for introduction of point mutations. Identical direct repeats of sequences containing the relevant mutation flank a selectable marker (). The substrate is shown as a bipartite gene-targeting substrate. Direct repeat recombination between mutant repeats leaves only the mutation in the genome.

Citation: Siewers V, Mortensen U, Nielsen J. 2010. Genetic Engineering Tools for , p 287-301. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch20
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Tables

Genetic Engineering Tools for Saccharomyces cerevisiae
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Citation: Siewers V, Mortensen U, Nielsen J. 2010. Genetic Engineering Tools for , p 287-301. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch20
/content/10.1128/9781555816827.ch20.ch20tab01
TABLE 1
Marker genes
Generic image for table
TABLE 1

Marker genes

Citation: Siewers V, Mortensen U, Nielsen J. 2010. Genetic Engineering Tools for , p 287-301. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch20
/content/10.1128/9781555816827.ch20.ch20tab02
TABLE 2
Promoters for heterologous gene expression
Generic image for table
TABLE 2

Promoters for heterologous gene expression

Citation: Siewers V, Mortensen U, Nielsen J. 2010. Genetic Engineering Tools for , p 287-301. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch20
/content/10.1128/9781555816827.ch20.ch20tab03
TABLE 3
Available plasmid expression vector
Generic image for table
TABLE 3

Available plasmid expression vector

Citation: Siewers V, Mortensen U, Nielsen J. 2010. Genetic Engineering Tools for , p 287-301. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch20

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