Journal of Zhejiang University SCIENCE B

, Volume 12, Issue 7, pp 545–551 | Cite as

Construction of the yeast whole-cell Rhizopus oryzae lipase biocatalyst with high activity

  • Mei-ling Chen
  • Qin Guo
  • Rui-zhi Wang
  • Juan Xu
  • Chen-wei Zhou
  • Hui Ruan
  • Guo-qing He
Article

Abstract

Surface display is effectively utilized to construct a whole-cell biocatalyst. Codon optimization has been proven to be effective in maximizing production of heterologous proteins in yeast. Here, the cDNA sequence of Rhizopus oryzae lipase (ROL) was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae, and based on the Saccharomyces cerevisiae cell surface display system with α-agglutinin as an anchor, recombinant yeast displaying fully codon-optimized ROL with high activity was successfully constructed. Compared with the wild-type ROL-displaying yeast, the activity of the codon-optimized ROL yeast whole-cell biocatalyst (25 U/g dried cells) was 12.8-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate (pNPP) as the substrate. To our knowledge, this was the first attempt to combine the techniques of yeast surface display and codon optimization for whole-cell biocatalyst construction. Consequently, the yeast whole-cell ROL biocatalyst was constructed with high activity. The optimum pH and temperature for the yeast whole-cell ROL biocatalyst were pH 7.0 and 40 °C. Furthermore, this whole-cell biocatalyst was applied to the hydrolysis of tributyrin and the resulted conversion of butyric acid reached 96.91% after 144 h.

Key words

Rhizopus oryzae lipase (ROL) Yeast surface display Codon optimization Whole-cell biocatalyst 

CLC number

Q26 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Andersson, S.G.E., Kurland, C.G., 1990. Codon preferences in free-living microorganisms. Microbiol. Rev., 54(2): 198–210.PubMedGoogle Scholar
  2. Bucarey, S.A., Noriega, J., Reyes, P., Tapia, C., Saenz, L., Zuniga, A., Tobar, J.A., 2009. The optimized capsid gene of porcine circovirus type 2 expressed in yeast forms virus-like particles and elicits antibody responses in mice fed with recombinant yeast extracts. Vaccine, 27(42): 5781–5790. [doi:10.1016/j.vaccine.2009.07.061]PubMedCrossRefGoogle Scholar
  3. Das, S., Roymondal, U., Sahoo, S., 2009. Analyzing gene expression from relative codon usage bias in yeast genome: a statistical significance and biological relevance. Gene, 443(1–2):121–131. [doi:10.1016/j.gene.2009.04.022]PubMedCrossRefGoogle Scholar
  4. Esteban, L., Munio, M.D., Robles, A., Hita, E., Jimenez, M.J., Gonzalez, P.A., Camacho, B., Molina, E., 2009. Synthesis of 2-monoacylglycerols (2-MAG) by enzymatic alcoholysis of fish oils using different reactor types. Biochem. Eng. J., 44(2–3):271–279. [doi:10.1016/j.bej.2009.01.004]CrossRefGoogle Scholar
  5. Guldener, U., Heck, S., Fiedler, T., Beinhauer, J., Hegemann, J.H., 1996. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res., 24(13): 2519–2524. [doi:10.1093/nar/24.13.2519]PubMedCrossRefGoogle Scholar
  6. Guo, Q., Zhang, W., Ma, L.L., Chen, Q.H., Chen, J.C., Zhang, H.B., Ruan, H., He, G.Q., 2010. A food-grade industrial arming yeast expressing β-1,3-1,4-glucanase with enhanced thermal stability. J. Zhejiang Univ.-Sci. B (Biomed. & Biotechnol.), 111 (1):41–51. [doi:10.1631/jzus.B0900185]CrossRefGoogle Scholar
  7. Hama, S., Yamaji, H., Kaieda, M., Oda, M., Kondo, A., Fukuda, H., 2004. Effect of fatty acid membrane composition on whole-cell biocatalysts for biodiesel-fuel production. Biochem. Eng. J., 21(2):155–160. [doi:10.1016/j.bej.2004.05.009]CrossRefGoogle Scholar
  8. Hasan, F., Shah, A.A., Hameed, A., 2006. Industrial applications of microbial lipases. Enzyme Microb. Technol., 39(2):235–251. [doi:10.1016/j.enzmictec.2005.10.016]CrossRefGoogle Scholar
  9. Ikemura, T., 1985. Codon usage and transfer-RNA content in unicellular and multicellular organisms. Mol. Biol. Evol., 2(1):13–34.PubMedGoogle Scholar
  10. Kaewthong, W., Sirisansaneeyakul, S., Prasertsan, P., H-Kittikun., A., 2005. Continuous production of monoacylglycerols by glycerolysis of palm olein with immobilized lipase. Process Biochem., 40(5):1525–1530. [doi:10.1016/j.procbio.2003.12.002]CrossRefGoogle Scholar
  11. Kato, M., Fuchimoto, J., Tanino, T., Kondo, A., Fukuda, H., Ueda, M., 2007. Preparation of a whole-cell biocatalyst of mutated Candida antaretica lipase B (mCALB) by a yeast molecular display system and its practical properties. Appl. Microbiol. Biotechnol., 75(3):549–555. [doi:10.1007/s00253-006-0835-2]PubMedCrossRefGoogle Scholar
  12. Klibanov, A.M., 2001. Improving enzymes by using them in organic solvents. Nature, 409(6817):241–246. [doi:10.1038/35051719]PubMedCrossRefGoogle Scholar
  13. Kondo, A., Ueda, M., 2004. Yeast cell-surface display-applications of molecular display. Appl. Microbiol. Biotechnol., 64(1):28–40. [doi:10.1007/s00253-003-1492-3]PubMedCrossRefGoogle Scholar
  14. Kristensen, J.B., Xu, X.B., Mu, H.L., 2005. Diacylglycerol synthesis by enzymatic glycerolysis: screening of commercially available lipases. J. Am. Oil Chem. Soc., 82(5): 329–334. [doi:10.1007/s11746-005-1074-5]CrossRefGoogle Scholar
  15. Kurland, C.G., 1991. Codon bias and gene-expression. FEBS Lett., 285(2):165–169. [doi:10.1016/0014-5793(91)80797-7]PubMedCrossRefGoogle Scholar
  16. Lithwick, G., Margalit, H., 2003. Hierarchy of sequence-dependent features associated with prokaryotic translation. Genome Res., 13(12):2665–2673. [doi:10.1101/gr.1485203]PubMedCrossRefGoogle Scholar
  17. Lo, S.K., Baharin, B.S., Tan, C.P., Lai, O.M., 2004. Lipase-catalysed production and chemical composition of diacylglycerols from soybean oil deodoriser distillate. Eur. J. Lipid Sci. Technol., 106(4):218–224. [doi:10.1002/ejlt.200300888]CrossRefGoogle Scholar
  18. Matsumoto, T., Takahashi, S., Kaieda, M., Ueda, M., Tanaka, A., Fukuda, H., Kondo, A., 2001. Yeast whole-cell biocatalyst constructed by intracellular overproduction of Rhizopus oryzae lipase is applicable to biodiesel fuel production. Appl. Microbiol. Biotechnol., 57(4):515–520.PubMedCrossRefGoogle Scholar
  19. Murai, T., Ueda, M., Shibasaki, Y., Kamasawa, N., Osumi, M., Imanaka, T., Tanaka, A., 1999. Development of an arming yeast strain for efficient utilization of starch by co-display of sequential amylolytic enzymes on the cell surface. Appl. Microbiol. Biotechnol., 51(1):65–70. [doi: 10.1007/s002530051364]PubMedCrossRefGoogle Scholar
  20. Prim, N., Blanco, A., Martinez, J., Pastor, F.I.J., Diaz, P., 2000. estA, a gene coding for a cell-bound esterase from Paenibacillus sp. BP-23, is a new member of the bacterial subclass of type B carboxylesterases. Res. Microbiol., 151(4):303–312. [doi:10.1016/S0923-2508(00)00150-9]PubMedCrossRefGoogle Scholar
  21. Proshkin, S., Rahmouni, A.R., Mironov, A., Nudler, E., 2010. Cooperation between translating ribosomes and RNA polymerase in transcription elongation. Science, 328(5977): 504–508. [doi:10.1126/science.1184939]PubMedCrossRefGoogle Scholar
  22. Shibasaki, S., Maema, H., Ueda, M., 2009. Molecular display technology using yeast-arming technology. Anal. Sci., 25(1):41–49.PubMedCrossRefGoogle Scholar
  23. Sinclair, G., Choy, F.Y.M., 2002. Synonymous codon usage bias and the expression of human glucocerebrosidase in the methylotrophic yeast, Pichia pastoris. Protein Expr. Purif., 26(1):96–105. [doi:10.1016/S1046-5928(02)00526-0]PubMedCrossRefGoogle Scholar
  24. Tamalampudi, S., Talukder, M.R., Hama, S., Numata, T., Kondo, A., Fukuda, H., 2008. Enzymatic production of biodiesel from Jatropha oil: a comparative study of immobilized-whole cell and commercial lipases as a biocatalyst. Biochem. Eng. J., 39(1):185–189. [doi:10.1016/j.bej.2007.09.002]CrossRefGoogle Scholar
  25. Thompson, J.R., Register, E., Curotto, J., Kurtz, M., Kelly, R., 1998. An improved protocol for the preparation of yeast cells for transformation by electroporation. Yeast, 14(6): 565–571. [doi:10.1002/(SICI)1097-0061(19980430)14:6<565::AID-YEA251>3.0.CO;2-B]PubMedCrossRefGoogle Scholar
  26. Washida, M., Takahashi, S., Ueda, M., Tanaka, A., 2001. Spacer-mediated display of active lipase on the yeast cell surface. Appl. Microbiol. Biotechnol., 56(5–6):681–686. [doi:10.1007/s002530100718]PubMedCrossRefGoogle Scholar
  27. Zaks, A., Klibanov, A.M., 1988. Enzymatic catalysis in nonaqueous solvents. J. Biol. Chem., 263(7):3194–3201.PubMedGoogle Scholar

Copyright information

© Zhejiang University and Springer-Verlag Berlin Heidelberg 2011

Authors and Affiliations

  • Mei-ling Chen
    • 1
  • Qin Guo
    • 2
  • Rui-zhi Wang
    • 1
  • Juan Xu
    • 1
  • Chen-wei Zhou
    • 1
  • Hui Ruan
    • 1
  • Guo-qing He
    • 1
  1. 1.Department of Food Science and NutritionZhejiang UniversityHangzhouChina
  2. 2.School of Food and Biological EngineeringJiangsu UniversityZhenjiangChina

Personalised recommendations