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Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 926985 9 pageshttpdxdoiorg1011552013926985

Research ArticleCombinatorial Mutagenesis and Selection to Understand andImprove Yeast Promoters

Laila Berg1 Trine Aakvik Strand2 Svein Valla1 and Trygve Brautaset2

1 Department of Biotechnology Norwegian University of Science and Technology Sem Saeliglands vei 6 7491 Trondheim Norway2Department of Molecular Biology SINTEF Materials and Chemistry Sem Saeliglands vei 2 7465 Trondheim Norway

Correspondence should be addressed to Trygve Brautaset trygvebrautasetsintefno

Received 14 March 2013 Revised 3 May 2013 Accepted 8 May 2013

Academic Editor Dimitrios Karpouzas

Copyright copy 2013 Laila Berg et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Microbial promoters are important targets both for understanding the global gene expression and developing genetic tools forheterologous expression of proteins and complex biosynthetic pathways Previously we have developed and used combinatorialmutagenesis methods to analyse and improve bacterial expression systems Here we present for the first time an analogous strategyfor yeast Our model promoter is the strong and inducible 119875

1198601198741198831promoter in methylotrophic Pichia pastoris The Zeocin resistance

gene was applied as a valuable reporter for mutant 1198751198601198741198831

promoter activity and we used an episomal plasmid vector to ensure aconstant reporter gene dosage in the yeast host cells This novel design enabled direct selection for colonies of recombinant cellswith altered Zeocin tolerance levels originating solely from randomly introduced point mutations in the 119875

1198601198741198831promoter DNA

sequence We demonstrate that this approach can be used to select for 1198751198601198741198831

promoter variants with abolished glucose repressionin large mutant libraries We also selected 119875

1198601198741198831promoter variants with elevated expression level under induced conditions The

properties of the selected1198751198601198741198831

promoter variants were confirmed by expressing luciferase as an alternative reporter geneThe toolsdeveloped here should be useful for effective screening characterization and improvement of any yeast promoters

1 Introduction

Microorganisms are widely used in biotechnology as cellfactories for sustainable production of proteins biopoly-mers and chemicals for medical pharmaceutical and indus-trial applications Today bioprospecting programs exploringmicrobial diversity around theworld lead to the identificationof new species new genes and new products As themajorityof natural microorganisms are not cultivable under labora-tory conditions the application of robust microbial hosts forheterologous expression of genes and gene clusters is crucialboth to understand microbial genetic diversity as well as todevelop new value-added products Access to well-definedand specialized promoter systems is important to enablefunctional expression and concomitant verification of thecollected genes and pathways We have previously developedcombinatorialmutagenesis and selectionmethods to improvebacterial promoter systems for diverse purposes In partic-ular we have worked with the strong and flexible xylSPmregulatorpromoter system originating from TOL plasmid

pWWOof Pseudomonas putida [1] XylS belongs to the AraC-XylS family of transcription factors and XylS can upon bind-ing of effector molecules (alkylbenzoates) bind upstream ofPm and activate transcription (for a review see [2]) We haveconstructed expression vectors by combining xylSPm withthe minimal replicon trfAoriV of the natural broad hostrange plasmid RK2 and demonstrated industrial level pro-duction of several medical recombinant proteins in Escher-ichia coli under high-cell-density cultivations [3 4] By usingcombinatorial mutagenesis and selection methods we haveselected variants of the Pm promoter with improved expres-sion properties in E coli and we have also shown that suchmethods are highly useful to generate new basic knowledgeof bacterial gene expression [5ndash7] The major principleof this approach was on one hand to generate large andmutant promoter sequence libraries and on the other hand toestablish a direct selection method enabling efficient screen-ing of the libraries for promoter variants with the desiredimproved properties The selection system was based onusing the bla gene (encoding the ampicillin-inactivating

2 BioMed Research International

enzyme 120573-lactamase) as reporter placed under the transcrip-tional control of the Pm promoter on a plasmid enablingdirect selection of recombinant E coli host cells with alteredampicillin tolerance levels

Besides bacteria yeast is an important and sometimesa preferred host for recombinant expression of genes andpathways The methylotrophic yeast Pichia pastoris hasbecome an extensively used expression host and the strongand inducible 119875

1198601198741198831promoter is one of the most frequently

applied promoters for gene expression in this organism [8ndash11] Expression from the 119875

1198601198741198831promoter is tightly regulated

by the present carbon source involving strong repression inthe presence of glucose or glycerol and full induction bymethanol when the repressive carbon sources are absent [1213] Yeast promoters are in general longer and more complexthan bacterial promoters and the understanding of the about950 base pairs long P pastoris 119875

1198601198741198831promoter DNA region is

limited both with regard to its cis-acting elements and themolecular mechanisms underlying its regulation In recentyears several attempts have been made to understand andimprove the 119875

1198601198741198831promoter by using rational design and

site-directed mutagenesis [14ndash16] In spite of considerableefforts the details about the cis-acting elements involved inthe glucose regulation of the 119875

1198601198741198831promoter and the under-

lying molecular mechanisms are still not known Also theimprovements of the induced expression level in these reportswere moderate This emphasizes the need for effective meth-ods to analyse and improve yeast promoter systems

In the present study we introduce a novel combinatorialmutagenesis and selection strategy in yeast by usingP pastorisand the119875

1198601198741198831promoter asmodel systemWedemonstrate for

the first time that this approach also works in yeast and that itcan be used both to alter the regulation properties as well asto improve the maximized expression level of the 119875

1198601198741198831pro-

moter The methodology presented here should be valuableboth to manipulate and improve yeast promoters for pro-moter probe approaches and for applied perspectives as wellas to generate basic understanding of microbial promoters

2 Materials and Methods

21 Microbial Strains and Growth Media Strains and vectorsused in this study are listed in Table 1 and primers used forthe vector constructions are listed in Table 2 E coli cells weregenerally grown at 37∘C in L broth (1 tryptone 05 yeastextract and 05 NaCl) or on L agar (L broth with 2 agar)When Zeocin was used for selection the pH in the L brothand agar was adjusted to 75 Ampicillin (100 120583gmL) kana-mycin (50 120583gmL) and Zeocin (25 120583gmL) were added asappropriate

P pastoris cells were generally grown at 30∘C in richmediawith glucose (YPD 2 peptone 1 yeast extract and 2 glu-cose) or on YPD agar (YPD broth with 2 agar) YPDS agar(YPD agar with 1M sorbitol) was used for transformationpurposesTheminimalmedia contained 134 yeast nitrogenbase 4 sdot 10minus5 biotin 0004 histidine and 05 methanol2 agar was included for agar media preparation Methanol(05) was added once a day to minimal media containing

methanol to maintain the induction for agar media thiswas obtained by adding 150 120583L (9 cm plates) or 500120583L(14 cm plates) methanol to the lid of inverted plates G418(500120583gmL) was added as appropriate

22 Standard DNA Manipulations A RbCl protocol (httpwwwpromegacom) was used for transformation of E coliPlasmids were isolated from E coli by using WizardPlus SVminipreps DNA purification kit (Promega) or Qiafilter maxiplasmid purification kit (Qiagen) and enzymatic manipu-lations were performed as described by the manufacturersPCR reactions were performed using the Expand high-fidel-ity PCR system kit (Boehringer Mannheim) for cloningpurposes and the PfuUltra II Fusion HS DNA polymer-ase (Strategene) for site-directed mutagenesis applicationsSequencing analyses were purchased from Eurofins MWGoperon (httpwwweurofinsdnacom)

For transformation of yeast in general 10120583L of circularplasmid preparation was transformed to P pastoris in accor-dance with the electroporation protocol from Invitrogen(httpwwwInvitrogencom) Plasmids were isolated fromP pastoris by using the Easy Yeast Plasmid Isolation kit(Clontech)

23 Agar Plate Assay for Determination of theMaximal ZeocinTolerance Levels of the P pastoris Host Cells An agar plateassay was used to indirectly determine the yeast promoteractivity as the maximal Zeocin tolerance levels of the Ppastoris host cellsThis is a modified version of the previouslyused agar plate assay developed for the indirect determi-nation of the Pm promoter activity in E coli as maximalampicillin resistance levels of the host cells [17] In detailfreshly transformed P pastoris GS115 strains were inoculatedinto 100120583L YPD broth in 96 micro-well plates and incubatedat 30∘C with shaking overnight Next the cells were dilutedwith a 96-pin replicator in 09 NaCl (100120583L) and plated onYPD agar or minimal agar media with methanol containingvarious Zeocin concentrations Usually the cells were alsoplated on YPD agar containing G418 The plates were incu-bated at 30∘C for three days The maximal Zeocin tolerancelevel determinations were carried out with minimum fourtechnical recurrences

24 Construction of Promoter Mutant Libraries The mutantlibraries were established and stored as plasmid libraries in EcoliDH5120572 and circular plasmids were transformed to P pas-torisGS115 (see below) In total two differentmutant librarieswere made in the 119875

1198601198741198831promoter with the 119885119888119877 gene as the

promoter reporterThe presence of both a kanamycin- and anampicillin resistance gene allows for selection of the plasmidlibraries in E coli and the kanamycin resistance gene alsoconfers resistance against the antibiotic G418 in P pastorisavoiding that 119875

1198601198741198831expression levels (Zeocin resistance)

could affect the composition of the libraries in any wayPlasmid pPPE20 was used when constructing the mutant

library in the promoter core region of the 1198751198601198741198831

promoter(plasmid library LC) The construction of the LC plasmidlibrary is similar to the procedure previously described for

BioMed Research International 3

Table 1 Strains and vectors used in this study

Strain or plasmid Descriptionabc Reference orsource

Ecoli strainsDH5120572 General cloning host BRLER2925 Host used for cloning experiments involving methylation-sensitive restriction enzymes NEB

P pastoris strainGS115 Host used for all of the P pastoris screening and growth experiments Invitrogen

Vectors

pIB11 Bacterial RK2-based expression vector harboring PmxylS regulatory promoter system with thebla gene as reporter KmR [6]

pGL3-controlvector Firefly luciferase reporter vector ApR Promega

pBGP1 P pastoris episomal expression vector harbouring the 119875119866119860119875

promoter E coli-P pastoris shuttlevector ZcR ApR [19]

pPICZ A P pastoris expressional vector harbouring the 1198751198601198741198831

promoter E coli-P pastoris shuttle vectorZcR Invitrogen

pPPE3 pBGP1 derivative in which the three BspHI restriction sites are deleted including one situated inthe 119875119866119860119875

promoter by using site-directed mutagenesis with primer pair 1 2 and 3 ZcR ApR This study

pPPE8 pPPE3 derivative in which a BspHI restriction enzyme site was inserted in the start of thezeocin-resistance gene by using site-directed mutagenesis with primer pair 4 ApR This study

pPPE10

pPPE8 derivative in which the zeocin-resistance gene was substituted with thekanamycin-resistance gene from plasmid pIB11 The 119870119898119877 gene was amplified from pIB11 withprimer pair 5 and end digested with BspHI and EcoRV prior to ligation into the correspondingsites of pPPE8 ApR KmR G418R

This study

pPPE11 pPPE10 derivative in which a NcoI restriction enzyme site was inserted in the start of the Alphasignal by using site-directed mutagenesis with primer pair 6 ApR KmR G418R This study

pPPE12

pPPE11 derivative in which the zeocin-resistance gene was inserted as reporter for the modified119875119866119860119875

promoter The zeocin-resistance gene was amplified from plasmid pBGP1 with primer pair 7and end digested with NcoI and NotI prior to ligation into the corresponding sites of pPPE11ApR KmR G418R

This study

pPPE17pPPE12 derivative in which the modified 119875

119866119860119875promoter was substituted with the 119875

1198601198741198831 The

1198751198601198741198831

promoter was amplified from pPICZ A with primer pair 8 and end digested with SpeI andNcoI prior to ligation into the corresponding sites of pPPE12 ApR KmR G418R

This study

pIPA1 pPICZ A derivative in which the BspHI restriction enzyme site in the 1198751198601198741198831

promoter is deletedby using site directed mutagenesis with primer pair 9 ZcR This study

pIPA4pIPA1 derivative in which an EcoRI restriction enzyme site is inserted directly upstream of theputative TATA-box in the 119875

1198601198741198831promoter by using site-directed mutagenesis with primer pair 10

ZcRThis study

pIPA5pIPA4 derivative in which a NheI restriction enzyme site is inserted downstream of thetranscriptional start site in the 119875

1198601198741198831promoter by using site-directed mutagenesis with primer

pair 11 ZcRThis study

pPPE20

Similar to pPPE17 except for that the 1198751198601198741198831

promoter was substituted with the modified 1198751198601198741198831

promoter from plasmid pIPA5 The 1198751198601198741198831

promoter was amplified from plasmid pIPA5 withprimer pair 12 and end digested with SpeI and NcoI prior to ligation into the corresponding sitesof pPPE12 ApR KmR G418R

This study

pPPE22Similar to pPPE17 except for that the 119875

1198601198741198831promoter was substituted with the wild-type 119875

119866119860119875

promoter from plasmid pBGP1 by subcloning the SpeIMfeI fragment from plasmid pBGP1 intothe corresponding sites of plasmid pPPE12 ApR KmR G418R

This study

pPPE35

Similar to pPPE17 except for that the 119885119888119877 gene is substituted with the luc+ gene (encoding fireflyluciferase) The luc+ gene was amplified from plasmid pGL3-control vector with primer pair 13and end digested with NcoI and NotI prior to ligation into the corresponding sites of pPPE17ApR KmR G418R

This study

pPPE50Similar to pPPE20 except for that a KpnI restriction enzyme site is inserted in the 119875

1198601198741198831promoter

about 90 base pairs upstream of the EcoRI restriction enzyme site by using site-directedmutagenesis with primer pair 14 ApR KmR G418R

This study

aAp ampicillin Km kanamycin Zc Zeocin G418 aminoglycoside antibioticbSequences of the primer pairs are listed in Table 2cThe target region was subcloned and verified by sequencing for all of the site-directed mutagenesis applications Also all cloned regions amplified by PCRwere verified by sequencing


Table 2 Primers used in genetic modifications

Primerpairnumber

Sequencea

1 51015840-TGTATCCGCTAATGAGACAAT-31015840 and51015840-TTGTCTCATTAGCGGATACA-31015840

2 51015840-GATTTTGGTAATGAGATTAT-31015840 and51015840-ATAATCTCATTACCAAAATC-31015840

3 51015840-GGACGCATGTAATGAGATTATT-31015840 and51015840-AATAATCTCATTACATGCGTCC-31015840

4 51015840-AGGAACTAAATCATGACCAAGTTGAC-31015840 and51015840-GTCAACTTGGTCATGATTTAGTTCCT-31015840

5 51015840-TAATAATCATGAGCCATATTCAAC-31015840 and51015840-TAGATATCATTAGAAAAACTCATC-31015840

6 51015840-ATTTCGAAACCATGGGATTTCCTTC-31015840 and51015840-GAAGGAAATCCCATGGTTTCGAAAT-31015840

7 51015840-TAATAACCATGGCCAAGTTGACC-31015840 and51015840-TAATAAGCGGCCGCATCAGTCCTGCTCCTC-31015840

8 51015840-TAATAAACTAGTAGATCTAACATCCAA-31015840 and51015840-TAATAACCATGGTTTCGAATAATTAG-31015840

9 51015840-AGCCTAACGTTAATGATCAAAATT-31015840 and51015840-AATTTTGATCATTAACGTTAGGCT-31015840

1051015840-AAAATTTAACTGTTCGAATTCCTACTTGACAGCAA-31015840and51015840-TTGCTGTCAAGTAGGAATTCGAACAGTTAAATTTT-31015840

1151015840-TCATAATTGCGACTGCTAGCAATTGACAAGCTTT-31015840and51015840-AAAGCTTGTCAATTGCTAGCAGTCGCAATTATGA-31015840


13 51015840-TAATAACCATGGAAGACGCCAAAAACA-31015840 and51015840-TAATAAGCGGCCGCATTACACGGCGATCTTTC-31015840

14 51015840-ACCCGCTTTTTGGTACCTTATGCATTGT-31015840 and51015840-ACAATGCATAAGGTACCAAAAAGCGGGT-31015840

aThe restriction enzyme site is underlined

the Pm promoter libraries [5 6] The oligonucleotides weredesigned to constitute a double-stranded DNA fragmentcontaining the 119875

1198601198741198831promoter core region with EcoRI- and

NheI-compatible ends when annealed for subsequent easycloning into the pPPE20 vector One of the oligonucleotidescorresponded to the wild-type sequence (51015840-CTAGCA-GTCGCAATTATGAAAGTAAGCTAATAATGATGA-TAAAAAAAAAGGTTTAAGACAGGGCAGCTTCCT-TCTGTTTATATATTGCTGTCAAGTAGG-31015840) and theoligonucleotide corresponding to the complementary strandwas randomly mutagenized by the use of a doped oligonu-cleotide solution (51015840-AATTC1231224313413323232333134-334433412411124212233311222222222321321322322341223-12221323322414312G-31015840 where the numbers in the sequenceindicate the doping percentages of the nucleotides 1 = 79C 7A 7 T and 7G 2 = 79 T 7A 7C and 7G 3= 79 A 7 C 7 T and 7 G 4 = 79 G 7 A 7 T and7 C) Approximately 410000 E coli DH5120572 transformantswere obtained constituting the LC plasmid library

Plasmid pPPE50 was used when constructing the plas-mid library LU and the construction of this library is identicalto that of the LC plasmid library except that the oligonucleot-ides were designed to constitute a double-stranded DNAfragment containing the 119875

1198601198741198831promoter core upstream

region with KpnI- and EcoRI-compatible ends whenannealed for subsequent easy cloning into the pPPE50 vectorAs before one of the oligonucleotides corresponded to thewild-type sequence (51015840-AATTCGAACAGTTAAATT-TTGATCATTAACGTTAGGCTATCAGCAGTATTC-CCACCAGAATCTTGGAAGCATACAATGTGGAGA-CAATGCATAAGGTAC-31015840) and the oligonucleotidecorresponding to the complementary strand was randomlymutagenized by the use of a doped oligonucleotide solution(51015840-C22324132242121131322423241221133432212442444332-312412432341123314223324321333322233124221G-31015840 wherethe numbers in the sequence indicate the doping percentagesof the nucleotides 1 = 88 C 4A 4 T and 4G 2 = 88T 4 A 4 C and 4 G 3 = 88 A 4 C 4 T and 4 G4 = 88 G 4 A 4 T and 4 C) Approximately 1 millionE coli DH5120572 transformants were obtained constituting theLU plasmid library

25 Screening for 1198751198601198741198831

Promoter Variants The Qiafiltermaxi plasmid isolation kit (Qiagen) was used for high-yield purification of the plasmid libraries from E coli DH5120572according to the procedure described by themanufacturers inorder to obtain sufficient transformation efficiencies of theselibraries into P pastoris Circular plasmids were transformedto P pastoris GS115 and the transformation suspensions wereplated on rich agar media with glucose or minimal agarmedia with methanol containing increasing concentrationsof Zeocin to directly select for the transformants containingthe 1198751198601198741198831

promoter variants leading to stimulated expressioncompared to that of the wild-type Normally 100 120583L of thetransformation suspension was plated on 9 cm agar platesexcept for the big-scale screening experiment of the LC plas-mid library on rich agar media with glucose (plating above100000 transformants) in which 21mL of the transformationsuspension was plated on square Corning bioassay plates(245 times 245 cm) To determine an approximate number ofthe transformants containing the plasmid that were platedthe transformation suspension was also plated on YPDS agarcontaining G418

26 Luciferase Activity Assay Freshly transformed P pastorisGS115 strains were grown according to the protocols forinduced expression of the 119875

1198601198741198831promoter by Invitrogen

(httpwwwinvitrogencom) For repressed expressionfreshly prepared P pastoris GS115 transformants wereinoculated to 10mL YPD in 125mL bottles and incubated at225 rpm and 30∘C overnight Next overnight cultures wereinoculated to 50mL YPD in 250mL baffled bottles to a finaloptical density at 600 nm of 0008 and incubated at 225 rpmand 30∘C for 3 days Samples were collected as indicated inthe results Lysis of the cell samples was carried out accordingto the protocols by Invitrogen and the luciferase activityassay was performed as described by the manufacturers(httpwwwpromegacom) The luciferase activity analyseswere repeated at least twice and measurements were carriedout with minimum three technical recurrences


GGATGATTATGCATTGTCTCCACATTGTATGCTTCCAAGATTCTGGTGGGAATACTGCTG

ATAGCCTAACGTTCATGATCAAAATTTAACTGTTCTAACCCCTACTTGACAGCAATATATAAACAGAAGGAAG

CTGCCCTGTCTTAAACCTTTTTTTTTATCATCATTATTAGCTTACTTTCATAATTGCGACTCCAATTAATTGAC

AAGCTTTTGATTTTAACGACTTTTAACGACAACTTGAGAAGATCAAAAAACAACTAATTATTCGAAACCATGG

NotI

Putative TATA-box

Transcriptional start site

middot middot middot

ZcR

KmR

ApR

NcoI

NcoI

Spel

pPPE17

(5498 bps)

PARS1

pUC origin

AOX1 TT

PEM7

PAOX1

PTEF1

Figure 1 Physical map of the E coli-P pastoris shuttle plasmid pPPE17 and its derivatives The restriction enzyme sites shown are uniqueAbbreviations 119875

1198601198741198831 P pastoris AOX1 promoter 119885119888119877 Zeocin resistance gene AOX1 TT P pastoris AOX1 transcription termination region

1198751198791198641198651

yeast promoter TEF11198751198641198727

bacterial promoter EM7119870119898119877 kanamycin resistance gene (confers resistance against kanamycin in bacteriaandG418 in yeast) PARS1 P pastoris autonomous replication sequence pUCorigin bacterial replication sequence119860119901119877 ampicillin resistancegene (bla) DNA sequence of the 31015840-part of the wild-type 119875

1198601198741198831promoter (base pairs 670 to 950) is displayed above the plasmid map

The promoter core region is shown in bold (see also text) The transcriptional start site (A) and the putative TATA-box (TATATAAA) arehighlighted in grey The BspHI (TCATGA) restriction enzyme site is written in bold and underlined and is deleted by the introduction of apointmutation (TAATGA) in plasmids pPPE20 andpPPE50GGATGA ismutated to theKpnI (GGTACC) restriction enzyme site in plasmidspPPE50 TAACCC is mutated to the EcoRI (GAATTC) restriction enzyme site in plasmids pPPE20 and pPPE50 CCAATT is mutated to theNheI (GCTAGC) restriction enzyme site in plasmid pPPE20 and pPPE50

3 Results and Discussion

31 Design and Construction of the Cassette Expression Plas-mid pPPE17 to Enable Direct Selection of Yeast PromoterVariants with Altered Expression Properties It has been welldocumented that xylSPm is a strong and flexible promotersystem useful for high-level as well as for controlled recom-binant expression of genes and pathways in Gram-negativebacteria (see Section 1) Still Pm promoter variants withnew and improved properties have been generated by usingcombinatorial mutagenesis and in such approaches we havetaken advantage of the correlation between the expressionlevel of the bla gene (encoding 120573-lactamase) and the corre-sponding ampicillin tolerance level of the host cells [5 6 18]We here chose to apply the Zeocin resistance gene 119885119888119877 asreporter due to its documented usefulness in identifyingyeast transformants with multicopy chromosomal insertionsbased on the Zeocin tolerance levels of the host cells (httpwwwinvitrogencom) When using this strategy to directlyselect recombinant yeast strains with altered Zeocin tolerance

levels it is critical to ensure a constant 119885119888119877 gene dosage inthe yeast host cells Therefore we chose to use the P pastorisepisomal (i e autonomously replicating) plasmid vectorpBGP1 [19] as the carrier for the expression cassette in favourof using chromosomally integrated vectors whichmay lead todifferent expression levels depending on the location of theintegration and possible multicopy insertions of the expres-sion cassette Plasmid pBGP1was used as basis to generate thepPPE17 plasmid which contains the 119885119888119877 gene under controlof the 119875

1198601198741198831promoter This design enables the substitution

of the yeast promoter andor gene of interest in a one-stepcloning procedure (Figure 1) In addition plasmid pPPE22was constructed by substituting the 119875

1198601198741198831promoter in

pPPE17 with the constitutive P pastoris 119875119866119860119875

promoter (seeFigure 1) and it was included as a valuable control systemThe latter promoter is reported to give strong expressionunder both the induced (presence ofmethanol and absence ofrepressive carbon sources) and repressed (presence of glu-cose) growth conditions for the 119875

1198601198741198831promoter [20]


Both plasmids pPPE17 and pPPE22 were transformedinto host strain P pastoris GS115 and the maximal Zeocintolerance levels were determined under 119875

1198601198741198831-promoter-

repressed conditions for strains GS115 GS115 (pPPE17) andGS115 (pPPE22) We chose to use rich agar media withglucose (YPD) for the repressed growth condition purposeRecombinant strains GS115 (pPPE17) and GS115 (pPE22)displayed about 5-fold and 400-fold higher Zeocin tolerancelevels (25 120583gmL and 2000120583gmL resp) compared to that ofhost GS115 (up to 5120583gmL)The results show that the expres-sion level from the 119875

1198601198741198831promoter is low under the glucose-

repressed conditions tested as expected Further the resultsdemonstrate a correlation between the expected expressionlevels of the 119885119888119877 gene from the two promoters and the toler-ance levels of the corresponding host cells

Plasmid pPPE17 was further modified into a promoter-cloning cassette system by deletion of a BspHI restrictionenzyme recognition site and insertion of two unique restric-tion enzyme recognition sites (EcoRI and NheI) in the 119875

1198601198741198831

promoter generating plasmid pPPE20 (Figure 1)Thesemod-ifications did not affect the expression properties of theplasmid compared to pPPE17 (data not shown) and pPPE20was used for screening of119875

1198601198741198831promotermutant libraries for

variants with altered expression properties as described next

32 Selection of 1198751198601198741198831

Promoter Variants with AbolishedGlucose Repression The 119875

1198601198741198831cis-acting promoter elements

involved in the glucose repression are not yet identified (seeSection 1)We here initially selected for119875

1198601198741198831promoter vari-

ants with altered glucose repression to establish and assess thecombinatorial mutagenesis approach Previous mutagenesisstrategies aiming at identifying 119875

1198601198741198831promoter variants with

altered glucose repression have focused on the promoterregion upstream of the putative TATA-box [10 12] We heredecided to explore the impact on the glucose regulation oftwo distinct regions of the 119875

1198601198741198831promoter First the 119875

1198601198741198831

promoter core region was here defined as the region fromabout 15 base pairs upstream of the putative TATA-box toabout 35 base pairs downstream of the transcriptional startsite (see Figure 1) A mutant library was constructed in this1198751198601198741198831

promoter core region by the use of randomly mutatedsynthetic oligonucleotides (designated plasmid library LC)cloned into the NheIEcoRI sites of pPPE20 (Figure 1) andtransformed into P pastoris GS115 Second we constructed apromoter mutant library (designated plasmid library LU) byrandomizing the 90 base pairs region directly upstream of the1198751198601198741198831

promoter core region corresponding to the region thatwas extensively studied byHartner et al [14] by using rationaldesign To accomplish this plasmid pPPE20 was modified bythe insertion of a unique restriction enzyme site (KpnI) in the1198751198601198741198831

promoter generating plasmid pPPE50 In the latterplasmid the promoter upstream region can be easily sub-stituted by a one-step cloning procedure using the uniqueEcoRIKpnI restriction sites (Figure 1) As for pPPE20 (seeabove) this modification did not affect the expression prop-erties of pPPE50 compared to pPPE17 (data not shown)

The LC and LU plasmid libraries were then screened forincreased Zeocin tolerance levels relative to the control strain

P pastoris GS115 (pPPE17) under glucose-repressed condi-tions Over 100000 transformants representing library LCand over 5000 transformants representing library LU werescreened and five totally different 119875

1198601198741198831promoter variants

(named LC-1 LC-2 LU-1 LU-2 and LU-3) with strongly ele-vated Zeocin tolerance levels were isolated The selected pro-moter variants provided significantly higher maximal Zeocintolerance levels of the GS115 cells under repressed conditions(up to 2000120583gmL) compared to that of the wild-type 119875

1198601198741198831

promoter (25120583gmL)Thefive selected promoter variants hadbetween 2 (LU-2) and 18 (LC-1) point mutations (Figure 2)The LU-2 and the LC-2 promoter variants have only twoand three pointmutations respectively suggesting that singlenucleotides directly upstream of the TATA box are importantfor glucose repression of this promoter The LC-1 LU-1 andLU-3 promoter variants on the other hand have multiple(between 11 and 19) point mutations distributed over theentire randomized promoter regions and thus both upstreamand downstream of the TATA-box Together these data maysuggest the existence of several cis-acting promoter elementsinvolved in the glucose repression of the 119875

1198601198741198831promoter

These results demonstrated that the combinatorial mutagen-esis and selection strategy can be used to efficiently select foraltered promoter variants and they also show that the 119875

1198601198741198831

promoter core region and the 90 base pairs upstream regioncan have high impact on the glucose repression under theconditions tested

33 Selected 1198751198601198741198831

Promoter Variants Retain Their Charac-teristics When Expressing an Alternative Reporter Protein theFirefly Luciferase Previous combinatorial mutagenesis workwith the bacterial Pm promoter has shown that there can bea strong context dependency between the promoter sequenceand the coding region of the reporter gene which often affectsthe expression level in an unpredictable manner [5 18] Twoof the selected119875

1198601198741198831promoter variants (LC-2 andLU-2)were

therefore chosen for expression studies in liquid P pastoriscultures with the alternative reporter gene luc+ encodingfirefly luciferase For this purpose the119885119888119877 gene in the pPPE17plasmids containing the wild-type 119875

1198601198741198831promoter and each

of the LC-2 and LU-2 promoter variants was substitutedwith the luc+ gene The luciferase activities of GS115 cellsharbouring these plasmids were quantified under repressedconditions and during the glucose-depletion phase in shakeflasks (Figure 3) Both promoter variants LC-2 and LU-2 gavestrongly enhanced luciferase activities at the first time pointcompared to that of the wild-type (about 300-fold and 600-fold resp) These differences decrease to around 10-fold forthe remaining time points for both of the promoter variants(the second time point represents the transition from expo-nential to stationary growth phase) Thus the increase inactivity after the first time point is much larger for the wild-type which shows that the LC-2 and LU-2 promoter variantsare far less affected by the depletion of glucose comparedto that of the wild-type promoter (Figure 3) Taken togetherthese results confirmed that LC-2 and LU-2 are 119875

1198601198741198831pro-

moter variants with abolished glucose repression irrespectiveof the context of the gene to be expressed


ggatgattatgcattgtctccacattgtatgcttccaagattctggtgggaatactgctgatagcctaacgttcatgatcaaaatttaactgttctaacccctacttgacagcaa

ggatgattatgcattgtctccacattgtatgcttccaagattctggtgggaatactgctgatagcctaacgttaatgatcaaaatttaactgttcGAATT ctgctcagtcgcga

ggatgattatgcattgtctccacattgtatgcttccaagattctggtgggaatactgctgatagcctaacgttaatgatcaaaatttaactgttcGAATT cttcgcgacagcaa

GGTACC-tatgcattgtcgcgacattgcatgcctccacgattctggtgggaattctgcttatggaccaacgttcatgatcaaaatttaactgttcGAATT ctacttgacagcaa

GGTACC-tatgcattgtctccacattgtatgcttccaagattctggtgggaatactgctgatagcctaacgttaatgatctaaatttaactgctcGAATTCctacttgacagcaa

GGTACC-tatgcattgtctccacattgtaaactcccacgactcttgtgggaatacggcttacagccgaccgttaatgctcaaaatttaaccgttcGAATT ctacttgacagcaa

GGTACC-taaccattgtgtccacattgtatgctttcaagcttctggtgggaatactgctaataccctaacgttaatgatcaaaattttactgctcGAATT ctacttgacagcaa

GGTACC-tctgcattgtctccacattgtatgcttccaagattctggtgggaatttcgctgatagcataacgttaatgatcaaaatttaactgttcGAATT ctacttgacagcaa

GGTACC-tatgcgttgacttcacattccatgcttaccagattctggtgggcatactgctgatagcataacgttaatggtcaaaatttcactgctcGAATT ctacttgacagcaa

GCTAGCTATATAACcagaaggaagctgctctcccttaaatcttgttttttCtcatcactatttacctactttcataattgccact

GCTAGCTATATAAAcagaaggaagctgccctgtcttaaacctttttttttAtcatcattattagcttactttcataattgcgact







TATATAAAcagaaggaagctgccctgtcttaaacctttttttttAtcatcattattagcttactttcataattgcgactggttcc

Wild-type

LC-1

LC-2

LU-1

LU-2

LU-3

LU-4

LU-5

LU-6

Wild-type

LC-1

LC-2

LU-1

LU-2

LU-3

LU-4

LU-5

LU-6

Promoter variant phenotype

40-fold wild-typeR

80-fold wild-typeR

40-fold wild-typeR

40-fold wild-typeR

40-fold wild-typeR

gt3-fold wild-typeI

gt3-fold wild-typeI

gt3-fold wild-typeI

C

C

C

C

C

C

C

+1

-158

-158

-158

-158

-158

-158

-159

-159

-159

-44

-44

-44

-44

-44

-44

-44

-44

-44

Figure 2 DNA sequence of region between base pairs-159 to +41 (promoter core region and the about 90 base pairs region directly upstreamof the promoter core region the transcriptional start site is set as +1) of the wild-type 119875

1198601198741198831promoter and variants LC-1 LC-2 and LU-1 to

LU-6 Mutations are shown in bold and underlined Deletion mutations are indicated by short horizontal lines The transcriptional start site(A) and the putative TATA-box (TATATAAA) are written in upper case and highlighted in grey Mutations leading to restriction-enzyme-site insertions or deletions without affecting the promoter activity are written in grey The KpnI (GGTACC) EcoRI (GAATTC) and NheI(GCTAGC) restriction enzyme sites are written in upper case and underlined The relative Zeocin tolerance levels of the promoter variantscompared to the wild-type promoter under repressed (wild-typeR) and induced (wild-typeI) growth conditions are shown

1

10

100

1000

10000

0 20 40 60 80 100 120Time (hours)

Relat

ive l

ucife

rase

activ

ities

Figure 3 Relative luciferase activities under repressed growthconditions and during the glucose-depletion phase for the wild-type P pastoris 119875

1198601198741198831promoter (black squares) and variants LC-

2 (grey triangles) and LU-2 (black circles dashed line) in plasmidpPPE35 expressed in P pastoris GS115 Enzyme activities are shownin logarithmic scale The values are the average of two biologicalreplicas and the error bars show the standard deviations Theactivities are relative to the wild-type for which the value at the firsttime point is arbitrarily set to 1

34 Selection of 1198751198601198741198831

Promoter Variants with IncreasedExpression Level under Induced Conditions Finally wewanted to test if it was possible to select 119875

1198601198741198831promoter

variants with increased expression levels under induced con-ditions As an initial test the maximum Zeocin tolerancelevels under induced conditions (minimal agar media withmethanol) of strains GS115 and GS115 (pPPE17) were deter-mined and found to be 25 120583gmL and 2000120583gmL respec-tively Next over 5000 transformants representing each of theLC and LU promoter libraries (see above) were screened withrespect to increased Zeocin tolerance levels relative to GS115cells harbouring original plasmid pPPE17 under 119875

1198601198741198831-

induced conditions In total three promoter variants (LU-4LU-5 and LU-6)were identified that caused increased Zeocintolerance levels of the GS115 cells under induced conditions(7000120583gmL or more) compared to that of the wild-typepromoter (2000120583gmL) The three selected promoter vari-ants contained between 5 and 12 point mutations (Figure 2)These promoter variants contain multiple (between 5 and12) point mutations distributed over the randomized regionanalogous to the LU-1 and LU-3 variants that were screenedfor increased activity under glucose-repressed conditions (seeabove) The biological reason for these results is unclear Onepotential explanation might be that there are cis-acting pro-moter elements important for both methanol induction andglucose repression located in the same region of the 119875

1198601198741198831

promoterFor practical reasons we found that using Zeocin concen-

trations higher than 7000120583gmL was undesirable and due tothis the identified promoter variants were not further ranked


at this stage Instead luciferase expression studies with GS115cells under induced growth conditions were performed inshake flasks for two of these 119875

1198601198741198831promoter variants (LU-

4 and LU-5) Analyses of samples withdrawn at the timepoints for transition from exponential to stationary growthphase (about 72 hours) showed a similar activity for theLU-5 promoter variant and the wild-type promoter whereasvariant LU-4 displayed a 2-fold increase in luciferase activitycompared to that of the wild-type (data not shown) Thisdemonstrates that the combinatorial mutagenesis and selec-tion strategy can be applied to also generate improved 119875

1198601198741198831

promoter variants under induced conditionsThe potential is(further) likely to be greater than indicated here consideringthat only a small fraction of themutant libraries was screenedand a narrow selection window was used (a concentration ofZeocin ranging from 2000 to 7000120583gmL)

4 Conclusions

Mutational analysis is an efficient approach to map specificpromoter functions and properties to defined sequencemotifs and nucleotides Rational methods including site-directed mutagenesis are preferred but such approaches relyon a detailed preinsight into the relation between the se-quence and function which is typically not the case for mostpromoters Accordingly the need for creating libraries withrandommutations distributed over defined regions might bepreferred however such strategies are dependent on efficientselection and screening methods to identify mutations lead-ing to the desired phenotypes The current study demon-strates for the first time a combinatorial mutagenesis andselection approach for generating new understanding of theregulation of promoter systems and to develop improvedexpression systems in yeast The tools and technology devel-oped here should be valuable for investigations and improve-ment of any yeast promoter system leading to new basicknowledge as well as to useful tools for heterologous expres-sion of single genes and complex biosynthetic pathways inyeast Understanding promoters is crucial to generate newbasic knowledge of microbial gene expression and regula-tion at all levels and development of new and specializedexpression systems is important to experimentally analyzemicrobial diversity including metagenome libraries for newfunctions and products Finally the tools and technologydeveloped here should also be useful as a promoter probesystem for efficient screening and characterization of naturalpromoter libraries in yeast

Conflict of Interests

Theauthors of this paper have no conflict of interests and theyhave no direct financial relation with the commercial identi-ties mentioned in this paper

Acknowledgment

This work was supported by a grant from the Research Coun-cil of Norway

References

[1] J L Ramos S Marques and K N Timmis ldquoTranscriptionalcontrol of the Pseudomonas TOL plasmid catabolic operonsis achieved through an interplay of host factors and plasmid-encoded regulatorsrdquo Annual Review of Microbiology vol 51 pp341ndash373 1997

[2] T Brautaset R Lale and S Valla ldquoPositively regulated bacterialexpression systemsrdquo Microbial Biotechnology vol 2 pp 15ndash302009

[3] H Sletta A Nedal T E V Aune et al ldquoBroad-host-rangeplasmid pJB658 can be used for industrial-level production of asecreted host-toxic single-chain antibody fragment in Escher-ichia colirdquo Applied and Environmental Microbiology vol 70 no12 pp 7033ndash7039 2004

[4] H Sletta A Toslashndervik S Hakvag et al ldquoThe presence of N-terminal secretion signal sequences leads to strong stimulationof the total expression levels of three testedmedically importantproteins during high-cell-density cultivations of EscherichiacolirdquoApplied and Environmental Microbiology vol 73 no 3 pp906ndash912 2007

[5] I Bakke L Berg T E V Aune et al ldquoRandom mutagenesisof the Pm promoter as a powerful strategy for improvement ofrecombinant-gene expressionrdquo Applied and EnvironmentalMicrobiology vol 75 no 7 pp 2002ndash2011 2009

[6] L Berg R Lale I Bakke N Burroughs and S Valla ldquoTheexpression of recombinant genes in Escherichia coli can bestrongly stimulated at the transcript production level by mutat-ing the DNA-region corresponding to the 51015840-untranslated partof mRNArdquo Microbial Biotechnology vol 2 no 3 pp 379ndash3892009

[7] R Lale L Berg F Stuttgen et al ldquoContinuous control of theflow in biochemical pathways through 51015840 untranslated regionsequence modifications in mRNA expressed from the broad-host-range promoter Pmrdquo Applied and Environmental Microbi-ology vol 77 pp 2648ndash2655 2011

[8] C P Hollenberg and G Gellissen ldquoProduction of recombinantproteins by methylotrophic yeastsrdquo Current Opinion in Biotech-nology vol 8 no 5 pp 554ndash560 1997

[9] L M Damasceno C Huang and C A Batt ldquoProtein secretionin Pichia pastoris and advances in protein productionrdquo AppliedMicrobiology and Biotechnology vol 93 pp 31ndash39 2012

[10] J M Cregg K R Madden K J Barringer G P Thill andC A Stillman ldquoFunctional characterization of the two alcoholoxidase genes from the yeast Pichia pastorisrdquoMolecular andCel-lular Biology vol 9 no 3 pp 1316ndash1323 1989

[11] S B Ellis P F Brust andP J Koutz ldquoIsolation of alcohol oxidaseand two other methanol regulatable genes from the yeast Pichiapastorisrdquo Molecular and Cellular Biology vol 5 no 5 pp 1111ndash1121 1985

[12] F S Hartner andAGlieder ldquoRegulation ofmethanol utilisationpathway genes in yeastsrdquoMicrobial Cell Factories vol 5 pp 39ndash59 2006

[13] J F Tschopp P F Brust J M Cregg C A Stillman and TR Gingeras ldquoExpression of the lacZ gene from two methanol-regulated promoters in Pichia pastorisrdquo Nucleic Acids Researchvol 15 no 9 pp 3859ndash3876 1987

[14] S F SHartner C RuthD Langenegger et al ldquoPromoter librarydesigned for fine-tuned gene expression in Pichia pastorisrdquoNucleic Acids Research vol 36 no 12 article e76 2008

[15] C A Staley A Huang M Nattestad et al ldquoAnalysis of the 51015840-untranslated region (51015840 UTR) of the alcohol oxidase 1 (AOX1)


gene in recombinant protein expression in Pichia pastorisrdquoGene vol 496 pp 118ndash227 2012

[16] Y Xuan X Zhou W Zhang X Zhang Z Song and Y ZhangldquoAn upstream activation sequence controls the expression ofAOX1 gene in Pichia pastorisrdquo FEMS Yeast Research vol 9 no8 pp 1271ndash1282 2009

[17] H C Winther-Larsen J M Blatny B Valand T Brautaset andS Valla ldquoPm promoter expression mutants and their use inbroad-host-range RK2 plasmid vectorsrdquoMetabolic Engineeringvol 2 no 2 pp 92ndash103 2000

[18] L Berg V Kucharova I Bakke S Valla and T BrautasetldquoExploring the 51015840-UTR DNA region as a target for optimizingrecombinant gene expression from the strong and inducible Pmpromoter in Escherichia colirdquo Journal of Biotechnology vol 158pp 224ndash230 2012

[19] C C Lee T G Williams D W S Wong and G H RobertsonldquoAn episomal expression vector for screening mutant genelibraries in Pichia pastorisrdquo Plasmid vol 54 no 1 pp 80ndash852005

[20] H R Waterham M E Digan P J Koutz S V Lair and JM Cregg ldquoIsolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of itspromoterrdquo Gene vol 186 no 1 pp 37ndash44 1997

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


enzyme 120573-lactamase) as reporter placed under the transcrip-tional control of the Pm promoter on a plasmid enablingdirect selection of recombinant E coli host cells with alteredampicillin tolerance levels

Besides bacteria yeast is an important and sometimesa preferred host for recombinant expression of genes andpathways The methylotrophic yeast Pichia pastoris hasbecome an extensively used expression host and the strongand inducible 119875

1198601198741198831promoter is one of the most frequently

applied promoters for gene expression in this organism [8ndash11] Expression from the 119875

1198601198741198831promoter is tightly regulated

by the present carbon source involving strong repression inthe presence of glucose or glycerol and full induction bymethanol when the repressive carbon sources are absent [1213] Yeast promoters are in general longer and more complexthan bacterial promoters and the understanding of the about950 base pairs long P pastoris 119875

1198601198741198831promoter DNA region is

limited both with regard to its cis-acting elements and themolecular mechanisms underlying its regulation In recentyears several attempts have been made to understand andimprove the 119875

1198601198741198831promoter by using rational design and

site-directed mutagenesis [14ndash16] In spite of considerableefforts the details about the cis-acting elements involved inthe glucose regulation of the 119875

1198601198741198831promoter and the under-

lying molecular mechanisms are still not known Also theimprovements of the induced expression level in these reportswere moderate This emphasizes the need for effective meth-ods to analyse and improve yeast promoter systems

In the present study we introduce a novel combinatorialmutagenesis and selection strategy in yeast by usingP pastorisand the119875

1198601198741198831promoter asmodel systemWedemonstrate for

the first time that this approach also works in yeast and that itcan be used both to alter the regulation properties as well asto improve the maximized expression level of the 119875

1198601198741198831pro-

moter The methodology presented here should be valuableboth to manipulate and improve yeast promoters for pro-moter probe approaches and for applied perspectives as wellas to generate basic understanding of microbial promoters

2 Materials and Methods

21 Microbial Strains and Growth Media Strains and vectorsused in this study are listed in Table 1 and primers used forthe vector constructions are listed in Table 2 E coli cells weregenerally grown at 37∘C in L broth (1 tryptone 05 yeastextract and 05 NaCl) or on L agar (L broth with 2 agar)When Zeocin was used for selection the pH in the L brothand agar was adjusted to 75 Ampicillin (100 120583gmL) kana-mycin (50 120583gmL) and Zeocin (25 120583gmL) were added asappropriate

P pastoris cells were generally grown at 30∘C in richmediawith glucose (YPD 2 peptone 1 yeast extract and 2 glu-cose) or on YPD agar (YPD broth with 2 agar) YPDS agar(YPD agar with 1M sorbitol) was used for transformationpurposesTheminimalmedia contained 134 yeast nitrogenbase 4 sdot 10minus5 biotin 0004 histidine and 05 methanol2 agar was included for agar media preparation Methanol(05) was added once a day to minimal media containing

methanol to maintain the induction for agar media thiswas obtained by adding 150 120583L (9 cm plates) or 500120583L(14 cm plates) methanol to the lid of inverted plates G418(500120583gmL) was added as appropriate

22 Standard DNA Manipulations A RbCl protocol (httpwwwpromegacom) was used for transformation of E coliPlasmids were isolated from E coli by using WizardPlus SVminipreps DNA purification kit (Promega) or Qiafilter maxiplasmid purification kit (Qiagen) and enzymatic manipu-lations were performed as described by the manufacturersPCR reactions were performed using the Expand high-fidel-ity PCR system kit (Boehringer Mannheim) for cloningpurposes and the PfuUltra II Fusion HS DNA polymer-ase (Strategene) for site-directed mutagenesis applicationsSequencing analyses were purchased from Eurofins MWGoperon (httpwwweurofinsdnacom)

For transformation of yeast in general 10120583L of circularplasmid preparation was transformed to P pastoris in accor-dance with the electroporation protocol from Invitrogen(httpwwwInvitrogencom) Plasmids were isolated fromP pastoris by using the Easy Yeast Plasmid Isolation kit(Clontech)

23 Agar Plate Assay for Determination of theMaximal ZeocinTolerance Levels of the P pastoris Host Cells An agar plateassay was used to indirectly determine the yeast promoteractivity as the maximal Zeocin tolerance levels of the Ppastoris host cellsThis is a modified version of the previouslyused agar plate assay developed for the indirect determi-nation of the Pm promoter activity in E coli as maximalampicillin resistance levels of the host cells [17] In detailfreshly transformed P pastoris GS115 strains were inoculatedinto 100120583L YPD broth in 96 micro-well plates and incubatedat 30∘C with shaking overnight Next the cells were dilutedwith a 96-pin replicator in 09 NaCl (100120583L) and plated onYPD agar or minimal agar media with methanol containingvarious Zeocin concentrations Usually the cells were alsoplated on YPD agar containing G418 The plates were incu-bated at 30∘C for three days The maximal Zeocin tolerancelevel determinations were carried out with minimum fourtechnical recurrences

24 Construction of Promoter Mutant Libraries The mutantlibraries were established and stored as plasmid libraries in EcoliDH5120572 and circular plasmids were transformed to P pas-torisGS115 (see below) In total two differentmutant librarieswere made in the 119875

1198601198741198831promoter with the 119885119888119877 gene as the

promoter reporterThe presence of both a kanamycin- and anampicillin resistance gene allows for selection of the plasmidlibraries in E coli and the kanamycin resistance gene alsoconfers resistance against the antibiotic G418 in P pastorisavoiding that 119875

1198601198741198831expression levels (Zeocin resistance)

could affect the composition of the libraries in any wayPlasmid pPPE20 was used when constructing the mutant

library in the promoter core region of the 1198751198601198741198831

promoter(plasmid library LC) The construction of the LC plasmidlibrary is similar to the procedure previously described for






Vectors










pPPE10


This study


pPPE12



This study



1198601198741198831 The

1198751198601198741198831


This study





ZcRThis study




pPPE20





This study



119866119860119875


This study

pPPE35


This study


1198601198741198831promoter


This study




Primerpairnumber

Sequencea






















25 Screening for 1198751198601198741198831











NotI

Putative TATA-box



ZcR

KmR

ApR

NcoI

NcoI

Spel

pPPE17

(5498 bps)

PARS1

pUC origin

AOX1 TT

PEM7

PAOX1

PTEF1



1198751198791198641198651










1198601198741198831promoter in



1198601198741198831promoter [20]



1198601198741198831-promoter-





1198601198741198831




32 Selection of 1198751198601198741198831




1198601198741198831promoter vari-





1198601198741198831









1198601198741198831


1198601198741198831promoter


1198601198741198831


33 Selected 1198751198601198741198831






1198601198741198831pro-





















Wild-type

LC-1

LC-2

LU-1

LU-2

LU-3

LU-4

LU-5

LU-6

Wild-type

LC-1

LC-2

LU-1

LU-2

LU-3

LU-4

LU-5

LU-6


40-fold wild-typeR

80-fold wild-typeR

40-fold wild-typeR

40-fold wild-typeR

40-fold wild-typeR

gt3-fold wild-typeI

gt3-fold wild-typeI

gt3-fold wild-typeI

C

C

C

C

C

C

C

+1

-158

-158

-158

-158

-158

-158

-159

-159

-159

-44

-44

-44

-44

-44

-44

-44

-44

-44




1

10

100

1000

10000

0 20 40 60 80 100 120Time (hours)

Relat

ive l

ucife

rase

activ

ities




34 Selection of 1198751198601198741198831


1198601198741198831promoter


1198601198741198831-


1198601198741198831







1198601198741198831


4 Conclusions




Acknowledgment


References






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






Vectors










pPPE10


This study


pPPE12



This study



1198601198741198831 The

1198751198601198741198831


This study





ZcRThis study




pPPE20





This study



119866119860119875


This study

pPPE35


This study


1198601198741198831promoter


This study




Primerpairnumber

Sequencea






















25 Screening for 1198751198601198741198831











NotI

Putative TATA-box



ZcR

KmR

ApR

NcoI

NcoI

Spel

pPPE17

(5498 bps)

PARS1

pUC origin

AOX1 TT

PEM7

PAOX1

PTEF1



1198751198791198641198651










1198601198741198831promoter in



1198601198741198831promoter [20]



1198601198741198831-promoter-





1198601198741198831




32 Selection of 1198751198601198741198831




1198601198741198831promoter vari-





1198601198741198831









1198601198741198831


1198601198741198831promoter


1198601198741198831


33 Selected 1198751198601198741198831






1198601198741198831pro-





















Wild-type

LC-1

LC-2

LU-1

LU-2

LU-3

LU-4

LU-5

LU-6

Wild-type

LC-1

LC-2

LU-1

LU-2

LU-3

LU-4

LU-5

LU-6


40-fold wild-typeR

80-fold wild-typeR

40-fold wild-typeR

40-fold wild-typeR

40-fold wild-typeR

gt3-fold wild-typeI

gt3-fold wild-typeI

gt3-fold wild-typeI

C

C

C

C

C

C

C

+1

-158

-158

-158

-158

-158

-158

-159

-159

-159

-44

-44

-44

-44

-44

-44

-44

-44

-44




1

10

100

1000

10000

0 20 40 60 80 100 120Time (hours)

Relat

ive l

ucife

rase

activ

ities




34 Selection of 1198751198601198741198831


1198601198741198831promoter


1198601198741198831-


1198601198741198831







1198601198741198831


4 Conclusions




Acknowledgment


References






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Primerpairnumber

Sequencea






















25 Screening for 1198751198601198741198831











NotI

Putative TATA-box



ZcR

KmR

ApR

NcoI

NcoI

Spel

pPPE17

(5498 bps)

PARS1

pUC origin

AOX1 TT

PEM7

PAOX1

PTEF1



1198751198791198641198651










1198601198741198831promoter in



1198601198741198831promoter [20]



1198601198741198831-promoter-





1198601198741198831




32 Selection of 1198751198601198741198831




1198601198741198831promoter vari-





1198601198741198831









1198601198741198831


1198601198741198831promoter


1198601198741198831


33 Selected 1198751198601198741198831






1198601198741198831pro-





















Wild-type

LC-1

LC-2

LU-1

LU-2

LU-3

LU-4

LU-5

LU-6

Wild-type

LC-1

LC-2

LU-1

LU-2

LU-3

LU-4

LU-5

LU-6


40-fold wild-typeR

80-fold wild-typeR

40-fold wild-typeR

40-fold wild-typeR

40-fold wild-typeR

gt3-fold wild-typeI

gt3-fold wild-typeI

gt3-fold wild-typeI

C

C

C

C

C

C

C

+1

-158

-158

-158

-158

-158

-158

-159

-159

-159

-44

-44

-44

-44

-44

-44

-44

-44

-44




1

10

100

1000

10000

0 20 40 60 80 100 120Time (hours)

Relat

ive l

ucife

rase

activ

ities




34 Selection of 1198751198601198741198831


1198601198741198831promoter


1198601198741198831-


1198601198741198831







1198601198741198831


4 Conclusions




Acknowledgment


References






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






NotI

Putative TATA-box



ZcR

KmR

ApR

NcoI

NcoI

Spel

pPPE17

(5498 bps)

PARS1

pUC origin

AOX1 TT

PEM7

PAOX1

PTEF1



1198751198791198641198651










1198601198741198831promoter in



1198601198741198831promoter [20]



1198601198741198831-promoter-





1198601198741198831




32 Selection of 1198751198601198741198831




1198601198741198831promoter vari-





1198601198741198831









1198601198741198831


1198601198741198831promoter


1198601198741198831


33 Selected 1198751198601198741198831






1198601198741198831pro-





















Wild-type

LC-1

LC-2

LU-1

LU-2

LU-3

LU-4

LU-5

LU-6

Wild-type

LC-1

LC-2

LU-1

LU-2

LU-3

LU-4

LU-5

LU-6


40-fold wild-typeR

80-fold wild-typeR

40-fold wild-typeR

40-fold wild-typeR

40-fold wild-typeR

gt3-fold wild-typeI

gt3-fold wild-typeI

gt3-fold wild-typeI

C

C

C

C

C

C

C

+1

-158

-158

-158

-158

-158

-158

-159

-159

-159

-44

-44

-44

-44

-44

-44

-44

-44

-44




1

10

100

1000

10000

0 20 40 60 80 100 120Time (hours)

Relat

ive l

ucife

rase

activ

ities




34 Selection of 1198751198601198741198831


1198601198741198831promoter


1198601198741198831-


1198601198741198831







1198601198741198831


4 Conclusions




Acknowledgment


References






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



1198601198741198831-promoter-





1198601198741198831




32 Selection of 1198751198601198741198831




1198601198741198831promoter vari-





1198601198741198831









1198601198741198831


1198601198741198831promoter


1198601198741198831


33 Selected 1198751198601198741198831






1198601198741198831pro-





















Wild-type

LC-1

LC-2

LU-1

LU-2

LU-3

LU-4

LU-5

LU-6

Wild-type

LC-1

LC-2

LU-1

LU-2

LU-3

LU-4

LU-5

LU-6


40-fold wild-typeR

80-fold wild-typeR

40-fold wild-typeR

40-fold wild-typeR

40-fold wild-typeR

gt3-fold wild-typeI

gt3-fold wild-typeI

gt3-fold wild-typeI

C

C

C

C

C

C

C

+1

-158

-158

-158

-158

-158

-158

-159

-159

-159

-44

-44

-44

-44

-44

-44

-44

-44

-44




1

10

100

1000

10000

0 20 40 60 80 100 120Time (hours)

Relat

ive l

ucife

rase

activ

ities




34 Selection of 1198751198601198741198831


1198601198741198831promoter


1198601198741198831-


1198601198741198831







1198601198741198831


4 Conclusions




Acknowledgment


References






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















Wild-type

LC-1

LC-2

LU-1

LU-2

LU-3

LU-4

LU-5

LU-6

Wild-type

LC-1

LC-2

LU-1

LU-2

LU-3

LU-4

LU-5

LU-6


40-fold wild-typeR

80-fold wild-typeR

40-fold wild-typeR

40-fold wild-typeR

40-fold wild-typeR

gt3-fold wild-typeI

gt3-fold wild-typeI

gt3-fold wild-typeI

C

C

C

C

C

C

C

+1

-158

-158

-158

-158

-158

-158

-159

-159

-159

-44

-44

-44

-44

-44

-44

-44

-44

-44




1

10

100

1000

10000

0 20 40 60 80 100 120Time (hours)

Relat

ive l

ucife

rase

activ

ities




34 Selection of 1198751198601198741198831


1198601198741198831promoter


1198601198741198831-


1198601198741198831







1198601198741198831


4 Conclusions




Acknowledgment


References






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





1198601198741198831


4 Conclusions




Acknowledgment


References






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Combinatorial Mutagenesis and Selection ...downloads.hindawi.com/journals/bmri/2013/926985.pdf · combinatorial mutagenesis and selection methods we have selected