1. Synthetic design of S. cerevisiae ChrVII: 1 048 595 – 1 078 595
Frederico Magalhães, Kristoffer Krogerus

2. Region The right arm of chromosome VII (1 048 595 – 1 078 595)
ORFs in this region:
SCW4
ZUO1
MAL1x locus
PXR1
BIO2
MAL11
MAL12
YOR1
IMA1
MAL13
BGL2
YGR290W
YGR283C
YGR291C
ERV29

3. MAL1x locus One of many loci that enables maltose utilization
Three gene complex
MAL11: Maltose permease
MAL12: Maltase
MAL13: MAL activator
Essential phenotype for many industrial applications:
Beer fermentation
Bioethanol fermentation
Baking industry
Maltose
Maltose
Glucose
Maltase

4. Region Essential ORFs in this region: SCW4 BIO2 PXR1 IMA1 YOR1
MAL1x locus
BGL2
MAL11
YGR283C
MAL12
MAL13
ERV29
(YGR290W)
ZUO1
YGR291C
To maintain:
-PXR1: Essential protein involved in rRNA and snoRNA maturation
-BGL2: Endo-beta-1,3-glucanase; major protein of the cell wall, involved in cell wall maintenance
-ERV29: Protein localized to COPII-coated vesicles; involved in vesicle formation and incorporation of specific secretory cargo
-ZUO1: Ribosome-associated chaperone; zuotin functions in ribosome biogenesis and as a chaperone for nascent polypeptide chains in partnership with Ssz1p and SSb1/2
-BIO2:Biotin synthase; catalyzes the conversion of dethiobiotin to biotin, which is the last step of the biotin biosynthesis pathway
-IMA1:Major isomaltase (alpha-1,6-glucosidase/alpha-methylglucosidase); required for isomaltose utilization
-MAL
Effect of deletion must be clarified:
-SCW4:Cell wall protein with similarity to glucanases; scw4 scw10 double mutants exhibit defects in mating
-YGR283C:Putative methyltransferase; may interact with ribosomes
Can be deleted:
-YOR1: Plasma membrane ATP-binding cassette (ABC) transporter
-YGR290W*: Dubious open reading frame; unlikely to encode a functional protein. *partially overlaps with MAL11
-YGR291C: Dubious open reading frame; unlikely to encode a functional protein
Removing the ORFs (maltose-rich media)
No effect
Slightly reduced growth and viability
Reduced growth and viability

5. Alterations Removal of non-essential genes
Critera: no effect on growth or viability in industrial settings
ORFs did not contain any introns
Replace stop codons (TAG > TAA)
Add PCRtags to ORFs
Add loxP sites to 3’ UTR of SCW4 and YGR283C, and in place of YOR1 and YGR291C

6. Workflow Retrieve sequences from SGD (www.yeastgenome.org)
Replace stop codon in all ORFs (TAG > TAA)
Insert PCRTags using ’Codon Juggle’ and ’most different’-algorithm from GeneDesign tool (
Design primers and check specificity (e.g. Primer3;
Insert loxP sites
Design ~10 kB megachunks
Linkers on both side (allow integration by recombination)
Potential antibiotic resistance marker for selection

7. Example of PCRTag addition
Switch codons in the ORF, such that the amino acid sequence stays the same, but the DNA sequence is different (at least two locations of ~ 20 bp)
PCR primer pairs can then be designed for these locations such that the synthetic and wild type sequences can be selectively differentiated.
Target areas with serine, arginine, and leucine
E.g. MAL13:
Wild-type:
Altered:

8. Example of loxP site addition
Add in the 34 bp sequence for the wild-type loxP site in the 3’ untranslated region (i.e. 3 bp following the stop codon)
Recognition region
Spacer region
ATAACTTCGTATA
ATGTATGC
TATACGAAGTTAT
ORF
… NNNTAANNNNN …
… NNNTAANNNATAACTTCGTATAATGTATGCTATACGAAGTTATNN …

9. Construction – 3 megachunks
loxP site

10. Construction
YPD +
hygromycin
hphMX
Hygromycin resistance
Linker
hphMX

11. Construction YPD + YPD + Nourseothricin resistance Linker hygromycin
hphMX
YPD +
hygromycin
YPD +
nourseothricin
natMX4
Nourseothricin resistance
Linker
natMX4

12. Construction
natMX4
YPD +
nourseothricin
YPD
Linker

13. Final synthetic region
Size: bp
4 loxP sites
2 ORFs removed
PCRtags to distinguish wild-type and synthetic genes
ORFs (1-10) WT
strain
loxP site
SynVII
strain
(adapted from Dymond et al. 2011, Nature)

14. Suggestions for future work
Additional deletions:
The whole MAL locus could be eliminated if a maltase that is secreted was introduced:
Bacillus subtilis
Bacillus brevis
Maltose
Glucose
Maltase