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Table S1 : Detailed overview of strategies used to assemble the different constructs.
Overview of all constructions that were compared. The molecular strategy and applied primer sequences are summed in the table. In the primer
sequences, uppercase Italic letters are part of the secretion signal sequence, while uppercase bold letters are used for TrCel6A+His-tag DNAsequence.
Construct description
Codon optimized TrCel61A
preceded by alpha-mating
factor (EKR-EA-EA)
TrCel61A with native
codon usage + alphamating factor (EKR-EA-EA)
Trichoderma reesei Cel61A
preceded by alpha-Mating
factor ending in
Enterokinase cleavage site
Strategy
1. Include XhoI restriction site at 5’ and NotI at 3’ end of the
sequence, starting from a pMAT vector including TrCel61A
(primers FW1+REV1, restriction sites underlined)
2. Restriction digest from the PCR product and pPpT4
backbone with XhoI-NotI followed by ligation
1. Include XhoI restriction site at 5’ and NotI at 3’ end of the
sequence, starting from a pJET vector including TrCel61A
(primers FW1 +REV1)
2. Restriction digest from the PCR product and pPpT4
backbone with XhoI-NotI followed by ligation
One-piece Gibson assembly (1) removing E-A-E-A from the alphamating factor and changing it for D-D-D-D-R amino acid sequence
(underlined) (Primers FW1 + REV2)
Primer sequences
FW1 : 5’- AAACTCGAGAAGAGAGAGGCCGAAGCT
CACGGTCACATTAACGACATCGTTATC -3’
REV1 : 5’tagcggccgcCTAGTGATGGTGATGGTGATGGTTCAAAC
ACTGAGCGTAGTAAGGG -3’
FW1 : 5’AAACTCGAGAAAAGAGAGGCTGAAGCTCATGGACATA
TTAA -3’
REV1 : 5’aatgcggccgcTCAATGATGATGATGATGATGGTCGTCGT
T -3’
FW1: 5’GGTGTCTCTCTCGAGAAGAGAGATGACGATGACAGAC
ACGGTCACATTAACGACATCG -3’
REV1: 5’-
(EKR-DDDDR-)
TrCel61A with native
codon usage +
CGATGTCGTTAATGTGACCGTGTCTGTCATCGTCATCTC
TCTTCTCGAGAGAGACACC -3’
Method as described by Sanchis et al (2008) (2) to remove aminoacids E-A-E-A at the 3’ end of the alpha-mating factor starting from
the complete vector with alpha-mating factor ending in EKR-E-A-EA (Primers FW1 +REV1)
FW1: 5’GGTGTCTCTCTCGAGAAGAGACACGGTCACATTAACGA
CATCG -3’
REV1: 5’- gaagaggagtgggaaatacc -3’
1. Amplify TrCel61A with the DDDK-protein secretion signal in
front via PCR amplification and including the sequence into
the primer (primers FW1+REV1)
2. Gibson assembly (1) to insert gene and secretion signal
into pPpT4 vector (insert amplification primers: FW2
+REV1, backbone primers: FW3 + REV2)
FW1: 5’CTACTTTGGCTTCCATTGCTGTTGCTCACGGTCACATTA
ACGACATCGTTATC -3’
REV1: 5’cttgagcggccgcctAGTGATGGTGATGGTGATGGTTCAAA
CACTGAGCGTAGTAAGG -3’
alpha- mating factor (EKR)
DDDK protein native
secretion signal
FW2: 5’GTTTAACTTGAAGACTATTTTGATTTCTACTTTGGCTTCC
ATTGCTGTTGCT -3’
Phanerochaete
chrysosporium GH61D
Gibson assembly (1) to exchange TrCel61A sequence to pPpT4
vector containing already Phanerochaete chrysosporium GH61D
secretion signal (insert amplification primers: FW1 +REV1,
backbone primers: FW2 + REV2)
FW3: 5’CCTTACTACGCTCAGTGTTTGAACCATCACCATCACCAT
CACTAGgcggccgctcaag -3’
REV2: 5’CAAAATAGTCTTCAAGTTAAACATcgtttcggaattctttcaat
aattag -3’
FW1: 5’GTTGTCTCTGCTCCATTTGTCTTGGGTCACGGTCACATT
AACGACATCG -3’
REV1: 5’ccgctcAATGATGATGATGATGATGGTTCAAACACTGAG
CGTAGTAAGG -3’
Trichoderma reesei Cel61A
preceded by its native
secretion signal sequence
1. Precede TrCel61A by its native secretion signal sequence
via PCR primers and include EcoRI restriction site at the 5’
and NotI site at 3’ end of the sequence (primers FW1 +
REV1, restriction sites underlined)
2. Restriction digest from the PCR product and pPpT4
backbone with EcoRI-NotI followed by ligation
FW2: 5’CCTTACTACGCTCAGTGTTTGAACCATCATCATCATCAT
CATTGAgcgg -3’
REV2: 5’CGATGTCGTTAATGTGACCGTGACCCAAGACAAATGGA
GCAGAGACAAC -3’
FW1 : 5’aaagaattccgaaacgATGATTCAAAAATTGTCTAACTTACT
TGTTACTGCTTTGGCAGTTGCTACTGGTGTTGTGGGAC
ACGGTCACATTAACGACATCGTTATCAAC - 3’
REV1 : 5’tagcggccgcCTAGTGATGGTGATGGTGATGGTTCAAAC
ACTGAGCGTAGTAAGGG -3’
1.
D.G. Gibson, L. Young, R. Chuang, et al. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. 6, 12–16.
2.
J. Sanchis, L. Fernández, J.D. Carballeira, et al. (2008) Improved PCR method for the creation of saturation mutagenesis libraries in
directed evolution: application to difficult-to-amplify templates. Applied microbiology and biotechnology. 81, 387–97.