Yeast Or E. Coli ?

Yeast or E. coli ?

PichiaExpress!

Novel&gene&co*expression&strategies&by& synthetic&biology& & Thomas&Vogl& !

2nd Applied Synthetic Biology in Europe 25-27.11.2013, Malaga $ “Costs&of&biocatalyst&is&a& key&factor&for&the&feasibility& of&commercial&applica4ons”& ? Protein expression key for success

selec3vity*

Extended diversity Industry& adapted for industrial needs

solubility* stability*

Expression* Laboratory*evolu3on* Chemical*engineering* Nature* Structure*guided* sequence*guided* engineering* engineering* Bio$prospec*ng-–-natural-diversity- Industrial enzymes Global*industrial*enzymes*markets:*3.3*bn** Household,*beverages*&*food*&*feed,** (BCC*Res*Jan*2011:*Enzymes*in*industrial*applica3ons:*global*markets)* * 2*major*players*share*more*than*2/3*of*global*industrial* enzymes*business* * BASF/Verenium,*(Dyadic),* Advanced*Enzymes….*

Novozymes*report*2012* Microbial protein production ...... *is*a*general*boVleneck*in*industrial* biotechnology*!* * Major*produc3on*hosts:* Aspergillus,+Trichoderma,+C1,+E.+coli,+Bacillus,+(Yeasts,...... + * Any*host*which*provides*ac3ve*enzymes:* Vmax,+Pseudomonas,+yeasts,+extremophiles,+insect+cells,...... + + Z******** cheap&catalysts&and&protein&materials& Z&&&&&&&& correctly&folded&and&ac4ve&enzymes& Z&&&&&&&& balanced&biosynthe4c&pathways& * Novozymes enzymes from Sigma 35*catalogue*enzymes* Most*industrial*enzymes*are* produced*as*secreted*proteins* * bacterial* simple*and*eﬃcient*DSP*on*large* fungal* scale* * animal* cheap*enzymes* * mostly*for*large*applica3ons*

Most&industrial&enzymes&produced&by&recombinant&GRAS&organisms& Biocatalysis&for&pharma&is&diﬀerent:&higher&catalyst&costs&for&high&value&products& e.g.&transaminases,&keto&reductases,&P450s,&BVMOs,&esterases,&aldolases& mostly&intracellular&enzymes,&oJen&produced&by&E.&coli,&& E.+coli+ Yeast* Bacillus* doubling&4me & &&&&&20Q60&min &&&&&90Q120&min &40Q120&min& & Cheap&minimal&media & &+ &&&&&&&&&&&+ &&&&&& &&+& & Cheap&induc4on & &&+/Q &&&&&&&&&&&+ &&&&&& &+/Q& & Stable&strains & &&+/Q &&&&&&&&&&&&+ &&&&&& &+& & Typical&4me&for& CloningQexpress&experiment &&&&&&&&&&&&1&week &&&&2&weeks &1Q2&weeks& & Tool&set & &&&+ &&&&&&&&&&&+ &&&&&& &+/Q& & NQ,&OQglycosyla4on& &&Q &&&&&&&&&&&+ &&&&&& &Q& & Disulﬁde&bridges & &&+/Q &&&&&&&&&&&&+ &&&& &+/Q& & Inclusion&bodies & &&+ &&&&&&&&Q&(+) & &Q&(+)& & Secre4on& &&&Q &&&&&&&&&&&+ & &+& Microbial Cell Factories 2009, 8:9 http://www.microbialcellfactories.com/content/8/1/9 E. coli:periplasmatic production

Review:*Use*of*folding*modulators*to*improve*heterologous*protein*produc3on*in*Escherichia*coli* Olga*Kolaj*et*al.*Microbial*Cell*Factories*2009,*8:9*doi:10.1186/1475Z2859Z8Z9*

MembraneFigure 2 translocation and periplasmic folding in E. coli Membrane translocation and periplasmic folding in E. coli. Most polypeptides cross the cytoplasmic membrane in an unfolded conformation using the Sec translocase (1), following delivery to SecA at the inner surface of the membrane by DnaK or SecB chaperones. Polypeptides with highly hydrophobic signal sequences or transmembrane domains may, however, be rec- ognised by Ffh which, together with its FtsY receptor, can target the polypeptide to either the Sec machinery or to the YidC translocase (2). Alternatively, the twin-arginine translocation (Tat) machinery is responsible for the translocation of already folded proteins (3), typically with bound metal cofactors. After cleavage of the leader peptide upon crossing the membrane, partially folded proteins may (4) aggregate, (5) be degraded by periplasmic proteases, or fold into their native state, often with the assistance of periplasmic chaperones (6) and/or folding catalysts such as disulfide bond metabolising enzymes (7) or peptidyl-prolyl cis-trans isomerases (8).

expression of CorA and failed to prevent inclusion body tides in a non-aggregated, translocation-competent form formation [66]. in the cytoplasm or in avoidance of aggregation in the periplasm subsequent to membrane translocation. Overall, while E. coli strains that allow formation of disulfide bridges in the cytoplasm are now available, thus Folding in the periplasm negating the need for secretion of disulfide-containing Following membrane translocation, folding of the heter- recombinant proteins, there is little evidence that the ologous polypeptide takes place in the periplasmic space secretion process limits the production of most heterolo- (Figure 2). While disulfide bond formation and peptidyl- gous proteins. Instead, the bottleneck for production is prolyl cis-trans isomerisation can occur here, no general usually more likely to involve maintenance of polypep- molecular chaperones that prevent non-productive fold-

Page 9 of 17 (page number not for citation purposes) Microbial Cell Factories 2009, 8:9 http://www.microbialcellfactories.com/content/8/1/9

Tips and Tricks

StrategyFigureReview: 3 for selection*Use*of*folding*modulators*to*improve*heterologous*protein*produc3on*in*Escherichia*coli* of molecular chaperones and folding catalysts for co-production analyses StrategyOlga*Kolaj for selection*et*al.*Microbial*Cell*Factories*2009,*8:9*doi:10.1186/1475Z2859Z8Z9 of molecular chaperones and folding catalysts for co-production analyses. Following production of a recombinant protein in E. coli, analysis of cell growth, protein solubility and subcellular location, macromolecular* state and activity provide some insight into the limiting step in the folding and production process. This Figure shows the major bottlenecks typically encountered (in hexagons) during production of a difficult-to-express recombinant target and identifies the co-production strategies that have been most successful in overcoming these bottlenecks to date (corresponding ovals).

and a novel trigger factor from another psychrophile, Psy- from the Irish Research Council for Science, Engineering and Technology chrobacter frigidicola [188], suggest that these studies may (IRCSET; to SS). represent the beginning of a new era in chaperone-assisted production of recombinant proteins in E. coli. References 1. Rai M, Padh H: Expression systems for production of heterologous proteins. Curr Science 2001, 80:1121-8. Competing interests 2. Spada S, Walsh G: Directory of approved biopharmaceutical products The authors declare that they have no competing interests. Boca Raton: CRC Press, USA; 2005. 3. Wall JG, Plückthun A: The hierarchy of mutations influencing the folding of antibody domains in Escherichia coli. Protein Eng Authors' contributions 1999, 12:605-11. All authors contributed equally to this manuscript, and 4. Hoffmann F, Rinas U: Stress induced by recombinant protein production in Escherichia coli. Adv Biochem Eng Biotechnol 2004, read and approved the final version. 89:73-92. 5. Wall JG, Plückthun A: Effects of overexpressing folding modulators on the in vivo folding of heterologous proteins in Acknowledgements Escherichia coli. Curr Opin Biotechnol 1995, 6:507-16. The authors gratefully acknowledge the support of grants CFTD/04/106 (to 6. Goloubinoff P, Christeller JT, Gatenby AA, Lorimer GH: Reconsti- OK and SR) and PC/2007/021 (to SR) from Enterprise Ireland Science and tution of active dimeric ribulose bisphosphate carboxylase Technology Development agency and Postdoctoral Fellowship PD/2005/44

Page 12 of 17 (page number not for citation purposes) Enhanced intracellular folding of proteins with disulfide bridges

The*easy*way……..*

E.+Coli+Origami* E.+Coli+RoseVaZgami*

E.+Coli+Shuﬄe*/*NEB*

For*overview:*hVp://wolfson.huji.ac.il/expression/bacZstrainsZprotZexp.html* Novagen Transfor m atio n/Transfe ctio n Competent Cells

Strain Descriptions continued 1

Features and Applications of Novagen’s Competent Cell Strains continued I S 2 c s I S a y c s L L I S y a c p p s L L ) ) ) I S I y a S p p 3 3 3 c s ) ) ) c L L s I S E E E y a 3 3 3 y a p p c s ) ) ) L L D D D E E E L L ™ y a ( ( ( 3 3 3 p p p p i i i i D D D ) ) ) L L ™ ) ) ) E E E ( ( ( 3 3 3 p p ) 3 3 3 m m m m e e e e D D D ) ) E E E ( ( ( 3 E E E a a a a u u u u 3 3 l l l l E D D D g g g g B B B B D D D ™ E E ( ( ( ™ - - - - ( ( ( B B B B i i i i i i i i D D D ( a a a a a a a a a a a a ™ ( ( t t t t t t t t t t t t m m m m m m m m r t t t t t t t t t r t t t r r n i a a a a a a a a e e e e e e e e e e e e e e e e a g g g g g g g g s s s s s s s s s s s s n n n n i i i i i i i i r 3 r r r r r r r r o o o o o o o o o o o o t u u u u S O O O O O O O O R R R R R R R R R T T R R R T T Strain background K-12 K-12 K-12 K-12 B B B B B B B B K-12 K-12 K-12 K-12 K-12 K-12 K-12 K-12 B B B B Protein expression: pET1 ✓2 ✓2 ✓2 ✓2 ✓ ✓ ✓ ✓ ✓2 ✓2 ✓ ✓ Protein expression: pETBlue™ ✓ ✓ ✓ ✓ ✓ ✓ Protein expression: pTriEx™ ✓ ✓ ✓ ✓ ✓ ✓ 4 Protein expression: non-T7 3 ✓ ✓ ✓ ✓ ✓ ✓ recA – 4 ✓ ✓ ✓ ✓ endA – 5 ✓ ✓ ✓ ✓ Blue/white screening6 ✓ 5 lacI q 7 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ F' episome8 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ompT – 9 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ lon – 10 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ trxB – 11 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ 6 gor – 12 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ lacY – 13 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Rare codon tRNAs14 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ pLysS15 ✓ ✓ ✓ ✓ ✓ ✓ pLacI16 ✓ ✓ ✓ ✓ ✓ ✓ 7 met – 17 dcm – 18 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Available as Singles™ ✓ ✓ ✓ ✓ Available as HT96™ 8 Chloramphenicol resistance ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Kanamycin resistance ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Tetracycline resistance ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓

10. Deficient in cytoplasmic lon protease. 11. Lacks thioredoxin reductase, thereby facilitating formation of disulfide bonds in the cytoplasm. 9 12. Lacks glutathione reductase, which, when combined with trxB mutation, greatly facilitates formation of disulfide bonds in the cytoplasm. 13. Lacks lac permease, which provides for homogeneous uptake of IPTG into all cells in the population, facilitating concentration-dependent induction of protein expression 14. Provides tRNAs for mammalian codons that rarely occur in E. coli, which increases the expression level of proteins otherwise limited by codon usage. 15. Provides T7 lysozyme to reduce basal expression of target genes and therefore stabilize plasmids that express proteins toxic to E. coli. Even greater stringency is provided by pLysE hosts; these are available separately as glycerol stocks. 16. Over produces lac repressor from a compatible plasmid, to suppress basal transcription of target genes controlled by appropriately placed lac operators. Designed for use with pETBlue and pTriEx constructs. 17. Methionine auxotroph facilitates metobolic labeling with met analogs. 10 18. Lacks methylation of internal cytosine residues in the sequences CCAGG and CCTGG at the C5 position.

11 Appendices Indices

Orders: www.novagen.com Shipping and Storage Information: begins on page 280 Novagen 2002–2003 Catalog 67 Technical Support: [email protected] Ordering and Bulk Order Information: see page 10 Test case: Horseradish Peroxidase (HRP) HRP isoenzymes

Reporter*in* diagnos3c*assays* and*histochemical* stainings* *

Increasing*interest*in* industrial* applica3ons,* chemical*synthesis*

Medicine* (ADEPT)*

John*Mann* Nature*Reviews*Cancer*2,*143Z148* (February*2002)* Pure HRP isoenzymes were not available 26*HRP*isoenzymes*

mRNA* Enzyme*analysis*

Expression*in* normalized*cDNA*library* P.-pastoris-or* ﬁssion*yeast*

454*sequencing* Bioinforma3c*analysis* Synthe3c*genes* Highly&pure&enzyme&–&homogenous&NQglycosyla4on&>100&mg/L& highest&space/4me&yields&by&cons4tu4ve&expression& Example HRP: Soluble/ IB production

Gundinger*&*Spadiut,*J.*Biotechnol.,* Volume*248*reactZtext:*68*,*/reactZtext*reactZtext:*69*20*April*2017*/reactZtext*reactZtext:*70*,*Pages*15Z24* disrupt&cells& Example HRP: Solubilize&(8M&urea):&dilute&(recycle!)& Soluble/ Refold:&DILUTE!& IB production

disrupt&cells& STY-?-

Gundinger*&*Spadiut,*J.*Biotechnol.,* Volume*248*reactZtext:*68*,*/reactZtext*reactZtext:*69*20*April*2017*/reactZtext*reactZtext:*70*,*Pages*15Z24* HNL – a successful history in Graz Biocatalyst - Enzyme (S)-Hydroxynitrile lyase – made by Pichia pastoris

Molecular biotechnology

Pichia pastoris 1993-1997

Nature Biocatalytic process >*20g/L*HNL* ( S ) H O H Cl O H H CN CN C OPh C OPh Pr O O Cl ( R ) ( S ) ( S ) Cypermethrin Fenvalerate Cl Product: 100 t / year M.Haslacher,*H.*Schwab,*S.*Kohlwein,*H.Griengl*et*al* 17 R-HNL made by Pichia pastoris RQCHO&to&& plant&gene& RQHC(OH)QCN& diﬃcult&to&express&in&E.-coli-

Prunus+amygdalus+RZHNL* 2000Q2003& *ACE*inhibitor* *cardiovascular*drug*

Glieder*et*al.,*Angew.*Chem.*Int.*Ed.*(2003)*42;*4815Z4818,*** Weis*et*al.,*Angew.*Chem.*Int.*Ed.*(2005)*44*(30),*4700Z4704* Pscheidt**et*al,*Adv.*Synth*Catal.*(2008)**350,*13,*1943Z1948**

H C OH H C CH 3 3 3 R-HNL/NaCN H C 3M Citrate- 3 CHO phosphate- CN buffer (pH 2.4) OH OH Hydroxypivalaldehyde R-Hydroxypivalaldehyde- cyanohydrine even&more&interes4ng&–&but&much&more&diﬃcult&to&engineer& In*collabora3on*with*DSM*Fine*Chemicals*Linz*and*the*Griengl*&*Kratky/Gruber*group* Komagataella phafﬁi (Pichia pastoris)

A preferred expression host: & *(diﬃcult)*protein*produc3on* *whole*cell*biocatalysis* *chemical*produc3on*by*synthe3c*biology*

RS1* GOI* RS2* RS1* GOI* RS1*

E.+coli+ Pta T* E_Sel* P.+pastoris+ c* Pta T* E_Sel* c*

& 3*kb*

P_Sel 8*kb* E.Coli*Ori* cut,*ligate,*transform* E.Coli*Ori* E.*coli,*isolate*plasmid* weeks*to*months*

(2Z3*days)* Ptac* GOI* T* E_Sel*

* >*10*kb*

P_Sel Ptac* GOI* T* E_Sel*

* E.Coli*Ori*

P_Sel

days& linearize*&*purify*(10*µg)*

E.Coli*Ori* weeksQmonths& P_Sel* Ptac* GOI* T* E_Sel* E.*Coli*Trafo*(1*day)* Pichia*trafo*(3*days)* grow/induce*–*harvest* auxotrophy*markers* –*lyseZmeasure*(12Z24h)* X* cul3va3on*&*assay* RQHNL&expression&anno&2001& 1*week* 21* E.+Coli:+ 2016+:+ simple*but*not* much*faster**&* for*heavy*loads* much*more*simple* than*other* eukaryotes* gene&to&product:&3Q4&days& +&less&4me&for&contracts& P.+pastoris+in+2000:+ gets*the*job*done*–*(if*there*is*no*hurry)** Appl Microbiol Biotechnol (2014) 98:5301–5317 DOI 10.1007/s00253-014-5732-5 Pichia&pastoris:&& DependenciesMINI-REVIEW and Resources

Protein expression in Pichia pastoris: recent achievements • Taxonomy& and perspectives for heterologous protein production

Mudassar Ahmad & Melanie Hirz & Harald Pichler & • Komagataella-phaﬃi- Helmut Schwab • Vendors*Major&available&host&and&vector&* Received: 27 January 2014 /Revised: 25 March 2014 /Accepted: 26 March 2014 /Published online: 18 April 2014 # The Author(s) 2014. This article is published with open access at Springerlink.com

Abstract Pichia pastoris is an established protein expression Introduction host mainly applied for the production of biopharmaceuticals systems& and industrial enzymes. This methylotrophic yeast is a distin- The methylotrophic yeast Pichia pastoris,currently guished production system for its growth to very high cell reclassified as Komagataella pastoris, has become a substan- densities, for the available strong and tightly regulated pro- tial workhorse for biotechnology, especially for heterologous moters, and for the options to produce gram amounts of protein production (Kurtzman 2009). It was introduced more • Methanol&independent&induc4on& recombinant protein per litre of culture both intracellularly than 40 years ago by Phillips Petroleum for commercial and in secretory fashion. However, not every protein of inter- production of single cell protein (SCP) as animal feed additive est is produced in or secreted by P. pastoris to such high titres. based on a high cell density fermentation process utilizing Frequently, protein yields are clearly lower, particularly if methanolRemote* as carbon source. However, the oil crisis in 1973 complex proteins are expressed that are hetero-oligomers, increased the price for methanol drastically and made SCP • Balanced&CoexpressionManufacturing** & membrane-attached or prone to proteolytic degradation. The production uneconomical. In the 1980s, P.pastoris was devel- last few years have been particularly fruitful because of nu- oped as aTeams heterologous protein* expression system using the merous activities in improving the expression of such com- strong and tightly regulated AOX1 promoter (Cregg et al. plex proteins with a focus on either protein engineering or on 1985). In combination with the already developed fermenta- • engineering the protein expression host P. pastoris. This re- tion process for SCP production, the AOX1 promoter provided Mul4&gene&expression& view refers to established tools in protein expression in exceptionally high levels of heterologous proteins. One of the P. pastoris and highlights novel developments in the areas of first large-scale industrial production processes established in expression vector design, host strain engineering and screen- the 1990s was the production of the plant-derived enzyme Project* ing for high-level expression strains. Breakthroughs in mem- hydroxynitrile lyase at >20 g of recombinant protein per litre • Genome&engineering& brane protein expression are discussed alongside numerous of culture volume (Hasslacher et al. 1997). This enzyme is commercial applications of P. pastoris derived proteins. used as biocatalyst for the production of enantiopure m- phenoxybenzaldehyde cyanohydrin — a building block of synthetic pyrethroids — on the multi-ton scale. Keywords Yeast . Pichia pastoris . Protein expression . Through a far-sighted decision this expression system, • Fast&cloning&and&engineering& Protein secretion . Protease-deficient strains . Chaperone initially patented by Phillips Petroleum, was made available to the scientific community for research purposes. A major breakthrough was the publication of detailed genome se- Mudassar Ahmad and Melanie Hirz contributed equally to this work. quences of the original SCP production strain CBS7435 (Küberl et al. 2011), the first host strain developed for heter- M. Ahmad : M. Hirz : H. Pichler : H. Schwab (*) Institute of Molecular Biotechnology, Graz University of ologous protein expression GS115 (De Schutter et al. 2009), Technology, Petersgasse 14/5, 8010 Graz, Austria as well as of the related P. pastoris DSMZ 70382 strain e-mail: [email protected] (Mattanovich et al. 2009b). Equally important breakthroughs for the commercial application of the P. pastoris cell factory H. Pichler : H. Schwab Austrian Centre of Industrial Biotechnology (ACIB), Petersgasse 14, were the Food and Drug Administration (FDA) GRAS (gen- 8010 Graz, Austria erally recognized as safe) status for a protein used in animal Engineering* Sales*

Appl*Microbiol*Biotechnol*(2014)*98:5301–5317* DOI*10.1007/s00253Z014Z5732Z5** 23* Pichia spp. Dependenciesfor enzyme and production Resources

Biotechnological&strains&of& Komagataella-(Pichia)&pastoris-are& Komagataella-phaYi-as&determined& from&mul4gene&sequence&analysis&* Cletus&Paul&Kurtzman&* J*Ind*Microbiol*Biotechnol*(2009)* 36:1435–1438*DOI*10.1007/ s10295Z009Z0638Z4** Pichia Hansenula+polymorpha+ Komagataella+pastoris+ (Hansenula+angusta,+ - Komagataella+phaﬃi+ Pichia+angusta)* Komagataella+pseudopastoris+ Ogataea*glucozyma+ * YBZ2185T** * Pichia+membranefaciens+YZ2026T** Cletus*P.*Kurtzman*(2005),*Int*J.*Systema3c*and*Evou3onary*Microbiology.*55.*973Z976* 24* DependenciesKomagataella and Resources- Biotechnological&strains&of& Komagataella-(Pichia)&pastoris-are& Komagataella-phaﬃi-as&determined& from&mul4gene&sequence&analysis&* Cletus&Paul&Kurtzman&* J*Ind*Microbiol*Biotechnol*(2009)* 36:1435–1438*DOI*10.1007/ s10295Z009Z0638Z4**

phaﬃi* pastoris* * YZ11430*and*YZ11431* described*in*patent*of*Wegner* US4414329,&published&1983* DSMZ*70382*

CBS7435* GS115*

25* PichiaDependencies-pastoris- and Resources (Komagataella-phaffi)- YQ11430& Eugene&Wegner:&US4414329& (priority&1981),&based&on& US4617274*/*Phillips*Petroleum* (first*filed:*1979,*April*12th)* Sibia,*Salk*Ins3tute* EP0017853D2* (first*filed:*1980,*April*2nd)*

Biogramma3cs* CBS7435& bisy* Life* Plarorm* Technologies* no*killer*plasmids* plarorm* Gras:&GRN540,&Impossible& no*killer*plasmids* foods& Gras:&GRN204,&Diversa& leghemoglobin& TU*Graz/VTU*FTO*plarorm*Phospholipase&C& genome*sequences*known*for*all*3*lines*Z*reﬁned*WT*sequence*by*Sturmberger*et*al*2016* 26* Retrofitting

Exis3ng*high*level*PAOX1* based**produc3on*strain* Ac3vator*plasmid*

P Ac3vator PAOX1+ GOI* + reg+ +

PAOX1+ GOI* Autoinduc3ve,* methanol*free*

P strain* = reg+ Ac3vator+ Pichia expression without methanol

1) Retroﬁung*strategy* autoinduced*overexpression*of** transcrip3on*factor*

Example:*fern*HNL*DtHNL1* No*methanol*&*fantas3c*STY* Methanol independent strong expression by Pichia pastoris

PDF* PDM* PDL* PDX1* PDX2* PDX3* hyperstrong*methanol*independent*expression* batch* Test*protein:*eGFP* glucose*deple3on* (low&energy!)& methanol*pulses* Speed up cloning for Pichia Vector + GOI new episomal vectors

SapIZcloning* ! Saves*3me*&* Reaction ! Recombinase*cloning* mix makes*undesired*bacterial* (Gibson)* sequences*on*vectors*unnecessary!* food&feed*applica3ons* Direct* transforma3on*of* Miniprep*and*reZtransforma3on* Ready&to&use& &“one&shot”&Pichia- No&liﬁca4on&by&E.-coli-& Chassis Engineering for improved NADH vailability

Enoate reductases X R' R' H X * * R'' Monooxygenases R R'' H R' OH

R R' R R'

Dehydrogenases X XH + NAD(P)H NAD(P) Kinases, R R' R R' ) ATP ADP Transferases 2- R-OH R-OPO3 metabolites, Central metabolism bulk chemicals

source(s Enzyme cascades - C

Optimized cell growth

high carbon efficiency! ONE EXAMPLE: ΔDAS-STRAINS IN BIOTRANSFORMATIONS

• Model reaction: 2,3-butanediol dehydrogenase High turnover number (98.000 min-1)

austrian centre of industrial biotechnology 32 Basic decisions: secre*on- or& intracellular-

strong/high©& integra4ve& weak/low©& cons4tu4ve& stable& extraQ& strong/high©& transient& chromosomal& weak/low©& stable& transient& regulated& (inducible)&

anton*glieder,*2017* promoter*1* strong* promoter*2* medium*

promoter*3* weak*

promoter*4* GOI* vector*backbone*1* GOI* vector* Backbone*2*

many*different*constructs* scale&up&?& to*be*tested*in*many*different*chassis* (protese*deficient,*supersecreters,*chaperone*overexpression,….)* One construct for all strategies?

anton*glieder,*2016* "One for all strain design

GOIZSynth1* HT/GAPZCAT1* GOIZSynth2*

Change&gears&to&adapt& to&the&road& & and&don’t&buy&a&new& car&if&weather&changes!&

P.Pastoris – S. cerevisiae – E. coli - CHO One strain–3 expression levels regulated*by*consecu3ve*induc3on*

GOIZSynth1* HT/GAPZCAT1* GOIZSynth2*

mRNA* of*GOI*

Batch FedBatch Methanol induction

1* 2* 3* New*bioreactor*protocols*and*procedures* &*alterna3ve*casseVe*copy*eﬀects* Synthetic bidirectional promoters

PDI&

Selec3on* marker* bidiPDI& plasmids& Consecu3ve*induc3on:* First*chaperon* Then*target*

Promoter& Direc4on&1&

Promoter& Direc4on&2&

stuffer fragment applications gene of interest 38* Optimized CBH II by DNA2.0 model

V10&&hiMe&&V3&&&V31& test&more&than&1&single&design!&

2Q3&4mes&more&CBH&II&with&DNA&2.0&model& op4mized&genes&(single©&strains)& Chassis strain development Genome engineering WT* 100 A 1) Use Δ KU70* 2) See*phenotype* 50 HRP*C1A* 3) Find*right*transformant* Δoch1*

relative HRP activity activity [%] relative HRP 0 Pichia+pastoris+ 0 20 40 60 80 100 120 volume [mL] B WT* Δoch1* 100 Δoch1* HRP*A2* 50 WT*

relative HRP activity [%] activity HRP relative 0 0 20 40 60 80 100 120 volume [mL] A SEC* B

Reduced N-glycosylation facilitates HRP purification

Florian*Krainer*et*al,*ScienNﬁc+Reports*3,*Ar3cle*number:*3279*(2013)* Polycistronic expression of at least 9 genes is possible! Simultaneous expression of the β-carotene and violacein pathway

Promoter 2A 2A 2A 2A 2A 2A 2A 2A Terminator

Promoter 2A 2A 2ApHTX1/pBZ6 2A 2A 2A 2A 2A Terminator

3,5

3,0

2,5

2,0 Relative protein 1,5 quantification Fold change 1,0 vio_crt vs. crt_vio 0,5

0,0 CrtE CrtB CrtI CrtY VioA VioB VioD VioC VioE

M. Geier et al., Chem. Commun., 2015 One for nine–multi gene expression

P.Pastoris S. cerevisiae 9&gene& pathways& made&within& 1&week& 20&genes?& Protein Process engineering optimization

weak, strong, constitutive inducible gRNA Cas9

Bidirectional promoters # medium, CRISPR-Cas9 derepressible Monodirectional promoters Expression Strain strategies engineering

43* Application „30 knockout strains in one month“ P. pastoris CYP1A2/CPR cell wall engnieering

3.000 2.500 2.000 31/A344 = 1.500 CYP1A2/CPR RFU/OD600 1.000 parental strain 500 0 14 1 4 6 2 7 3 9 18 8 5 26 31 28 27 10 24 22 25 23 21 30 13 11 17 20 12 19 15 29 16 Screening landscapes of 21 transformants/construct (not sequenced)

450 1.5 – fold 400 350 300 250 200

RFU/OD600 150 100 50 0

A.*Weninger,*unpublished* Shake flask cultivation of selected transformants 44* Conclusions !Speed*and*throughput*are*important** !Think*about*scalability* !FTO*situa3on?* !Access*to*expression*tools*

start new adventure:

TUG*and*acib*spin*oﬀ:* ROBOX:* This*work*received*funding*from*the*EU*project*ROBOX*(grant*agreement*n°*635734)*under*EU’s* H2020*Programme*Research*and*Innova3on*ac3ons*H2020ZLEIT*BIOZ2014Z1*and*from*the*Austrian* BMWFJ,*BMVIT,*SFG,*Standortagentur*Tirol*and*ZIT*through*the*Austrian*FFGZCOMETZ*Funding* Program.* Disclaimer:*This*publica3on*reﬂects*the*author's*view*and*the*Agency+is*not*responsible*for*any*use* that*may*be*made*of*the*informa3on*it*contains.*

IMIZCHEM21* The*ac3vity*leading*to*the*present*poster*has*received*funding*from*the*European*Community´s*Seventh*Framework* Programme*(FP7/2007Z2013)*and*EFPIA*companies’*in*kind*contribu3on*for*the*Innova3ve*Medicine*Ini3a3ve*under* Grant*Agreement*No.*115360*(Chemical*manufacturing*methods*for*the*21st*century*pharmaceu3cal*industries,* CHEM21).*In*addi3on,*the*work*has*been*supported*by*the*Austrian*BMWFW,*BMVIT,*SFG,*Standortagentur*Tirol,* Government*of*Lower*Austria*and*Business*Agency*Vienna*through*the*Austrian*FFGZCOMETZ*Funding*Program.**

The*research*leading*to*these*results*has*received*funding*from*the*European*Union's*Seventh*Framework* Programme*FP7/2007Z2013*under*grant*agreement*n°*266025*(BIONEXGEN).**

Hands on Pichia Feb/2018 Graz

Acknowledgements Acknowledgements The activity leading to the present poster has received funding from the European Community´s Seventh Framework The activity leading to the present poster has received funding from the European Community´s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in kind contribution for the Innovative Medicine Initiative under Grant Programme (FP7/2007-2013) and EFPIA companies’ in kind contribution for the Innovative Medicine Initiative under Grant Agreement No. 115360 (Chemical manufacturing methods for the 21st century pharmaceutical industries, CHEM21). In Agreement No. 115360 (Chemical manufacturing methods for the 21st century pharmaceutical industries, CHEM21). In addition, the work has been supported by the Austrian BMWFW, BMVIT, SFG, Standortagentur Tirol, Government of Lower addition, the work has been supported by the Austrian BMWFW, BMVIT, SFG, Standortagentur Tirol, Government of Lower Austria and Business Agency Vienna through the Austrian FFG-COMET- Funding Program. Austria and Business Agency Vienna through the Austrian FFG-COMET- Funding Program.