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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 pepti- dyl-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 pro- duction 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 heterolo- gous 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 modula- tors 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.
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