New Efficient Recombinant Expression System To Engineer Candida antarctica Lipase B

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 2010, p. 2684–2687 0099-2240/10/$12.00 doi:10.1128/AEM.03057-09 Copyright © 2010, American Society for Microbiology. All Rights Reserved.

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New Efficient Recombinant Expression System To Engineer Candida antarctica Lipase B䌤† Ste´phane Emond,1‡ Ce´dric Montanier,1‡ Jean-Marc Nicaud,3 Alain Marty,1 Pierre Monsan,1,2 Isabelle Andre´,1 and Magali Remaud-Sime´on1* Inge´nierie des Syste`mes Biologiques et des Proce´de´s, Universite´ de Toulouse, INSA, UPS, INP, 135 Avenue de Rangueil, F-31077 Toulouse, France, and INRA, UMR792, F-31400 Toulouse, France1; Institut Universitaire de France, 103 Boulevard Saint-Michel, F-75005 Paris, France2; and Laboratoire de Microbiologie et Ge´ne´tique Mole´culaire, INRA, UMR1238, CNRS, UMR2585, AgroParisTech, F-78850 Thieverval-Grignon, France3 Received 18 December 2009/Accepted 8 February 2010

Here, we report the use of Yarrowia lipolytica as a versatile expression host for developing protein engineering approaches to modify the properties of Candida antarctica lipase B. A reliable screening protocol was defined and validated using a saturation mutagenesis library, yielding mutants displaying higher catalytic efficiencies than the wild-type enzyme. high transformation efficiency (8,000 transformants/␮g of DNA) (3). Here, as a prerequisite for future protein engineering experiments, we used this approach to develop a new secretion system for CalB allowing the reliable screening of libraries as well as the rapid purification and characterization of interesting mutants (see detailed descriptions of experimental procedures in the supplemental material). The DNA sequence encoding CalB without its natural Nterminal prepro region was PCR amplified from the pColdIII/ CalB plasmid (14). This product was then fused by overlap extension PCR to the DNA sequence encoding the prepro region of Y. lipolytica Lip2 (Preprolip2), which targets the protein to the secretory pathway. The final fusion product was cloned into the JMP62-His6Cter vector, yielding a plasmid with a cassette containing the Ura3 selection marker and the Preprolip2-CalB-His6 fusion gene under the control of the oleic acid-inducible POX2 promoter (Fig. 1A). Upon NotI digestion, the JMY1212 strain was transformed with this expression cassette for single-copy integration into the genome (3). Four Ura-positive colonies were randomly chosen for inoculation into 20-ml flask cultures with oleic acid as an inducer. After 48 h of growth, the culture media were assayed for lipase activity toward para-nitrophenyl butyrate (p-NPB), yielding on average 513 ⫾ 34 U/liter of culture (mean ⫾ standard deviation). SDS-PAGE (Fig. 1B, lane 1) confirmed that CalB-His6 was the major secreted protein. The production of the lipase was then scaled up in a 3-liter reactor by using an optimized culture procedure described previously (12). Under these conditions, CalB-His6 was produced in amounts 10-fold larger than those in flask cultures (lipase activity, 5,090 ⫾ 136 U/liter of culture; amount of protein, 190 ⫾ 5 mg/liter of culture). An easy one-step procedure was also set up to purify the recombinant protein (to ⬎95% purity) from the culture medium by using immobilized-metal affinity chromatography (Fig. 1B, lanes 8 and 9). The culture conditions enabling the secretion of CalB-His6 were miniaturized in the 96-well microplate format to set up a reliable high-throughput screening protocol (Fig. 2A; see also the detailed protocol in the supplemental material). Briefly,

Lipase B from Candida antarctica (CalB) is one of the most widely used enzymes in industrial biocatalysis (1, 4, 10). Modifying its properties by using protein engineering methods (including rational engineering, directed evolution, or the combination of both) (9, 20) is therefore a challenging goal for many research groups nowadays. Due to the facility of genetic manipulation, high transformation efficiency, and rapid growth rates, Escherichia coli remains to date the most popular expression host in such experimental strategies. However, expression of CalB as an active enzyme in E. coli is a challenge since most of the protein is insoluble and found in inclusion bodies (2, 11, 14, 16, 24). Eukaryotic expression using several host systems, such as Aspergillus oryzae (7, 28), Saccharomyces cerevisiae (27, 29), Pichia pastoris (11, 21) and Hansenula polymorpha (26), has also been described previously. However, to date, very few reports describe the engineering of CalB toward improved expression (26), increased thermostability (5, 27, 29), or modified enantioselectivity (18, 19, 25). Several problems, including production of insoluble proteins in E. coli, low secretion efficiency in S. cerevisiae, and poor transformation efficiency in P. pastoris, have limited the development of efficient screening protocols and thus the identification of CalB variants with interesting properties. The yeast Yarrowia lipolytica has proven to be an efficient system for recombinant protein expression (15). Recently, a high-throughput screening procedure with the endogenous lipase Lip2 as a model protein was optimized using a strain (JMY1212) enabling single-copy cassette integration by homologous recombination into the genome at a zeta docking platform (derived from the Ylt1 retrotransposon) (23) with * Corresponding author. Mailing address: Laboratoire d’Inge´nierie des Syste`mes Biologiques et des Proce ´de´s, Institut National des Sciences Applique´es de Toulouse, 135 Avenue de Rangueil, F-31400 Toulouse, France. Phone: 33-561-559-446. Fax: 33-561-559-400. E-mail: remaud @insa-toulouse.fr. ‡ These authors contributed equally to this work. † Supplemental material for this article may be found at http://aem .asm.org/. 䌤 Published ahead of print on 19 February 2010. 2684

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FIG. 1. Secretion of CalB-His6 by Y. lipolytica and subsequent protein purification. (A) Schematic diagram of the expression cassette for CalB-His6 released from the shuttle plasmid JMP62-CalB-His6 upon NotI digestion. The cassette is flanked by zeta regions, allowing single-copy insertion into the genome of the Y. lipolytica JMY1212 strain. (B) SDS-PAGE analysis of CalB-His6 secretion and affinity purification. Lanes: 1, Y. lipolytica culture supernatant; 2, flowthrough; 3 and 4, washes; 5, wash with 5 mM imidazole; 6 and 7, washes with 10 mM imidazole; 8 and 9, elution with 100 mM imidazole.

precultures in a 96-well microplate were grown for 24 h with glucose as the carbon source to allow homogeneous growth. The preculture microplate was then replicated to start a production culture in the presence of oleic acid. The protein production was optimized in a 96-deep-well format to obtain larger amounts of enzymes in one step for use in the second stage, assaying the activities of each variant on several substrates. After 30 h of growth, the supernatants containing the secreted recombinant enzymes were assayed for p-NPB hydrolysis. The procedure reproducibility was analyzed with experimental data collected using transformants expressing wild-type CalB-His6 or the inactive S105A mutant (columns 2 to 7 or 9 to 11 in Fig. 2B, respectively). No growth due to cross-contamination was observed in uninoculated wells (columns 1, 8, and 12 in Fig. 2B). The mean wild-type activity calculated from results of repeated experiments was 199 ⫾ 29 U/liter of culture (coefficient of variation [CV], 14.6%; n ⫽ 144). The enzyme production in deep-well microplates is thus 2- to 3-fold lower than that in flask cultures, which provide better aeration conditions. The probability of measuring wild-type activities at or higher than a defined threshold was calculated by assuming that the activity measurements follow a Gaussian distribution (22). This probability represents the expected frequency of false-positive transformants in screening for variants with improved activity (above the defined threshold). With a CV of 14.6%, selection of at least one false-positive variant would be a very rare event (approximately 1 in 1.5 ⫻ 1011 screened variants) if our objective was to isolate mutants displaying 2-fold-increased activity. The miniaturized growth conditions described here thus resulted in satisfactorily reproducible activity levels for transformants expressing wild-type CalB, thereby strongly limiting the occurrence of false-positive variants during screening experiments. To validate our screening system, we chose to create a small library of CalB variants by using saturation mutagenesis and

screen the variants for p-NPB hydrolysis. Residue A281 was chosen based on findings from molecular docking studies that aimed at predicting the conformation of the substrate in the binding pocket and estimating the strength of the enzymesubstrate interaction (see Fig. S1A in the supplemental material). Following the construction of the library, 94 Ura-positive transformants were chosen randomly to cover at least 95% of the diversity (6) and screened for p-NPB hydrolysis. This experiment yielded 13 variants (positive hit rate, ⬃15% of the library) with at least 3-fold-higher activity than the wild-type enzyme. The four best mutants were isolated for further characterization. Analysis of their sequences revealed that A281 was replaced by either D, N, F, or Y. Resulting kinetic constants for p-NPB hydrolysis are listed in Table 1 (see also Fig. S2 in the supplemental material). Overall, these variants displayed 4.2- to 5.8-fold-higher catalytic efficiency (kcat/Km ratios) than the parental lipase. A directed evolution experiment had previously led to the identification of double and triple mutants (A281E/V210I and A281E/V210I/V221D enzymes) displaying 4.4- and 3.2-fold-higher kcat/Km ratios for p-NPB hydrolysis than the wild-type enzyme, respectively (29). Here, we thus identified variants possessing comparable and greater enhancement through a single substitution at position 281. To gain some insight into the effects of the mutations, we analyzed the three-dimensional models of p-NPB covalently docked into the different isolated single mutants. Comparisons with the docking mode predicted for p-NPB with the parental enzyme (see Fig. S1A in the supplemental material) showed that the mutations introduced at position 281 (D, N, F, and Y) had clear effects on the orientation of p-NPB in the catalytic pocket and, more importantly, on the stabilization of the tetrahedral intermediate complex. Additional hydrogen bonding interactions between p-NPB and amino acid residues (I189 and V190) of the catalytic pocket of CalB variants that could assist in the departure of the leaving para-nitrophenyl group during hydro-

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FIG. 2. High-throughput procedure to screen libraries of CalB variants. (A) Schematic flowchart of the screening protocol. The use of the p-NPB substrate is shown as an example. YTD medium, 10 g 䡠 liter⫺1 yeast extract, 10 g 䡠 liter⫺1 Bacto tryptone, and 10 g 䡠 liter⫺1 glucose; YTO medium, 10 g 䡠 liter⫺1 yeast extract, 20 g 䡠 liter⫺1 Bacto tryptone, 20 g 䡠 liter⫺1 oleic acid, and 50 mM Na/K phosphate buffer, pH 6.8. (B) Typical microplate profile of activities toward p-NPB obtained for wild-type CalB (columns 2 to 7) and the S105A inactive mutant (columns 9 to 11). Data were normalized with respect to the mean calculated for wild-type CalB activities and were plotted in order of well position (columns) or in descending order (squares). Columns 1, 8, and 12 represent samples containing culture medium but not inoculated with yeast. ␮ and ␴ represent the mean activity toward p-NPB for wild-type CalB and the standard deviation (n ⫽ 144), respectively.

lysis (see, for example, data for A281Y in Fig. S1B in the supplemental material) were also observed. These interactions may result in a favorable enthalpic contribution to the free energy of the system and thus explain the enhancement of the catalytic efficiency of the variants compared to that of wild-type CalB. Our results show that CalB is secreted by Y. lipolytica in relatively large amounts (up to 200 mg/liter of culture) by using

TABLE 1. Kinetic constants of wild-type CalB and selected variants for activity toward p-NPBa Lipase

wt CalB A9 B5 F12 H3 a

Amino acid change

Km (␮M) (% of wt value)

kcat (s⫺1) (% of wt value)

kcat/Km ratio (min⫺1 䡠 ␮M⫺1) (% of wt value)

A281D A281F A281N A281Y

583 (100) 614 (105) 444 (76) 413 (71) 491 (84)

110 (100) 581 (528) 463 (421) 324 (295) 537 (488)

11.3 (100) 56.7 (502) 62.6 (554) 47.1 (416) 65.6 (580)

wt, wild-type.

a single-copy integration strategy. This approach was chosen to allow reproducible expression levels compatible for the screening of variant libraries. Note that the production rate can be easily increased using multicopy vectors (17). High-yield CalB production (up to 1.3 g/liter of culture) in P. pastoris was achieved previously by using multicopy gene integration into the genome (8, 21). However, low transformation efficiencies (⬃102 transformants/␮g of DNA) and high expression variability among transformants make this host generally unsuitable for screening experiments. Expression of a FLAG-tagged CalB recombinant form in S. cerevisiae (yielding ⬃80 mg/liter of culture) provides a more reliable screening system (29). A 7-day growth period, however, is needed to achieve enzyme production in microplates, whereas only 2 days were necessary in our protocol. Moreover, due to the expense of FLAG tag affinity chromatography (13), only small amounts of protein can be purified for characterization upon screening. In contrast to other prokaryotic and eukaryotic expression systems described previously (14, 21, 29), the use of Y. lipolytica enabled us to establish efficient procedures for both the construction

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and the screening of CalB variant libraries, as well as for the overproduction, secretion, and purification of the lipase necessary for the rapid characterization of interesting variants. The methods described here will now be used in-house to create and screen larger libraries of CalB mutants in order to identify new variants displaying original properties. This work was supported by the French National Research Agency (under the POLYMER-BIO-PATH project, contract no. ANR-07CP2D-15) and the European Union (under the EU FP6 RTD project “Synthesis and application of nanostructured tethered silicates,” contract no. 033254). We thank Florence Bordes for valuable advice throughout this study and Rolf D. Schmid for the generous gift of the pColdIII/CalB plasmid. REFERENCES 1. Anderson, E. M., K. M. Larsson, and O. Kirk. 1998. One biocatalyst—many applications: the use of Candida antarctica B-lipase in organic synthesis. Biocatal. Biotransformation 16:181–204. 2. Blank, K., J. Morfill, H. Gumpp, and H. E. Gaub. 2006. Functional expression of Candida antarctica lipase B in Escherichia coli. J. Biotechnol. 125: 474–483. 3. Bordes, F., F. Fudalej, V. Dossat, J. M. Nicaud, and A. Marty. 2007. A new recombinant protein expression system for high-throughput screening in the yeast Yarrowia lipolytica. J. Microbiol. Methods 70:493–502. 4. Bornscheuer, U. T., and R. J. Kazlauskas. 1999. Hydrolases in organic synthesis: regio- and stereoselective biotransformations. Wiley-VCH, Weinheim, Germany. 5. Chodorge, M., L. Fourage, C. Ullmann, V. Duvivier, J. M. Masson, and F. Lefevre. 2005. Rational strategies for directed evolution of biocatalysts— applications to Candida antarctica lipase B (CALB). Adv. Synth. Catal. 347:1022–1026. 6. Denault, M., and J. N. Pelletier. 2007. Protein library design and screening: working out the probabilities. Methods Mol. Biol. 352:127–154. 7. Høegh, I., S. Patkar, T. Halkier, and M. T. Hansen. 1995. Two lipases from Candida antarctica—cloning and expression in Aspergillus oryzae. Can. J. Bot. 73:S869–S875. 8. Jahic, M., M. Gustavsson, A. K. Jansen, M. Martinelle, and S. O. Enfors. 2003. Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes. J. Biotechnol. 102:45–53. 9. Kazlauskas, R. J., and U. T. Bornscheuer. 2009. Finding better protein engineering strategies. Nat. Chem. Biol. 5:526–529. 10. Kirk, O., and M. W. Christensen. 2002. Lipases from Candida antarctica: unique biocatalysts from a unique origin. Org. Process Res. Dev. 6:446–451. 11. Larsen, M. W., U. T. Bornscheuer, and K. Hult. 2008. Expression of Candida antarctica lipase B in Pichia pastoris and various Escherichia coli systems. Protein Expr. Purif. 62:90–97. 12. Leblond, Y., A. Marty, N. Mouz, and J. L. Uribelarrea. December 2007. Method for producing lipase, transformed Yarrowia lipolytica cell capable of producing said lipase and their uses. W.O. patent 144,475.

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13. Lichty, J. J., J. L. Malecki, H. D. Agnew, D. J. Michelson-Horowitz, and S. Tan. 2005. Comparison of affinity tags for protein purification. Protein Expr. Purif. 41:98–105. 14. Liu, D., R. D. Schmid, and M. Rusnak. 2006. Functional expression of Candida antarctica lipase B in the Escherichia coli cytoplasm—a screening system for a frequently used biocatalyst. Appl. Microbiol. Biotechnol. 72: 1024–1032. 15. Madzak, C., C. Gaillardin, and J. M. Beckerich. 2004. Heterologous protein expression and secretion in the non-conventional yeast Yarrowia lipolytica: a review. J. Biotechnol. 109:63–81. 16. Narayanan, N., and C. P. Chou. 2009. Alleviation of proteolytic sensitivity to enhance recombinant lipase production in Escherichia coli. Appl. Environ. Microbiol. 75:5424–5427. 17. Pignede, G., H. J. Wang, F. Fudalej, M. Seman, C. Gaillardin, and J. M. Nicaud. 2000. Autocloning and amplification of LIP2 in Yarrowia lipolytica. Appl. Environ. Microbiol. 66:3283–3289. 18. Qian, Z., C. J. Fields, and S. Lutz. 2007. Investigating the structural and functional consequences of circular permutation on lipase B from Candida antarctica. Chembiochem 8:1989–1996. 19. Reetz, M. T., D. Kahakeaw, and R. Lohmer. 2008. Addressing the numbers problem in directed evolution. Chembiochem 9:1797–1804. 20. Reetz, M. T., D. Kahakeaw, and J. Sanchis. 2009. Shedding light on the efficacy of laboratory evolution based on iterative saturation mutagenesis. Mol. Biosyst. 5:115–122. 21. Rotticci-Mulder, J. C., M. Gustavsson, M. Holmquist, K. Hult, and M. Martinelle. 2001. Expression in Pichia pastoris of Candida antarctica lipase B and lipase B fused to a cellulose-binding domain. Protein Expr. Purif. 21: 386–392. 22. Salazar, O., and L. Sun. 2003. Evaluating a screen and analysis of mutant libraries. Methods Mol. Biol. 230:85–97. 23. Schmid-Berger, N., B. Schmid, and G. Barth. 1994. Ylt1, a highly repetitive retrotransposon in the genome of the dimorphic fungus Yarrowia lipolytica. J. Bacteriol. 176:2477–2482. 24. Seo, H. S., S. E. Kim, K. Y. Han, J. S. Park, Y. H. Kim, S. J. Sim, and J. Lee. 2009. Functional fusion mutant of Candida antarctica lipase B (CalB) expressed in Escherichia coli. Biochim. Biophys. Acta 1794:519–525. 25. Skjot, M., L. De Maria, R. Chatterjee, A. Svendsen, S. A. Patkar, P. R. Ostergaard, and J. Brask. 2009. Understanding the plasticity of the alpha/ beta hydrolase fold: lid swapping on the Candida antarctica lipase B results in chimeras with interesting biocatalytic properties. Chembiochem 10:520– 527. 26. So-Young, K., J. H. Sohn, Y. R. Pyun, I. S. Yang, K. H. Kim, and E. S. Choi. 2007. In vitro evolution of lipase B from Candida antarctica using surface display in Hansenula polymorpha. J. Microbiol. Biotechnol. 17:1308–1315. 27. Suen, W. C., N. Y. Zhang, L. Xiao, V. Madison, and A. Zaks. 2004. Improved activity and thermostability of Candida antarctica lipase B by DNA family shuffling. Protein Eng. Des. Sel. 17:133–140. 28. Tamalampudi, S., M. Talukder, S. Hama, T. Tanino, Y. Suzuki, A. Kondo, and H. Fukuda. 2007. Development of recombinant Aspergillus oryzae wholecell biocatalyst expressing lipase-encoding gene from Candida antarctica. Appl. Microbiol. Biotechnol. 75:387–395. 29. Zhang, N., W. C. Suen, W. Windsor, L. Xiao, V. Madison, and A. Zaks. 2003. Improving tolerance of Candida antarctica lipase B towards irreversible thermal inactivation through directed evolution. Protein Eng. 16:599–605.