Team:Tec-Monterrey/projectoverview

Cell surface display is a technique to display proteins on the surface of bacteria, fungi, or mammalian cells by fusing them to surface anchoring motifs. This technique has a wide range of biotechnological and industrial applications, including development of vaccines, peptide and antibody libraries, bioremediation, whole-cell-biosensors, and whole-cell-biocatalysis. When protein is expressed in the outer membrane of E. coli the cell envelope acts as a matrix. It is achievable thanks to several systems as outer membrane porins, lipoproteins, GPI-anchored-proteins, fimbriae, and autotransporters. (Jana S & Deb JK, 2005; Lee SH et al., 2004) Displaying proteins on the cell surface also makes preparing or purifying the protein unnecessary in many instances. Whole cells displaying the molecule of interest can be used in reactions or analytical assays and then can be simply removed by centrifugation. (Joachim J & Meyer TF, 2007)

The type V autotransporters are natural transportation system to import/export substrates through the periplasm/membrane, and are composed of an N-terminal sec-dependent signal peptide, a passenger domain, and a translocator domain that are predicted to form a β-barrel. (Rutherford et al., 2006) In this project, the natural passenger domain of the autotransporter estA from Pseudomonas sp was replaced by a cellulase (CelD) and an invertase (SacC) to display them at the bacterial surface by the translocator domain of the estA protein. The estA protein is inserted into the cytoplasmic membrane of E. coli by the Sec machinery, which translocates unfolded substrates across the membrane while the Tat (twin-arginine translocation) system functions to translocate folded proteins. (Yuan J et al ., 2010) The Sec translocase is comprised of the SecYEG translocation channel and the accessory components SecA, SecDFYajC, and YidC. (Yuan J et al., 2010) Using signal peptide of a protein which is naturally transported to the cytoplasma (signal peptide of PhoA), we expect successful localization of the cellulase and the invertase at the external surface of E. coli. In the other hand, a fragment of an integral outer membrane ompA with signal peptide of a lipoprotein lpp (BBa_K103006) was used to express the same enzymes by the type II Sec-secretion system.

C. thermocellum endoglucanase CelD is an enzyme that belongs to family E cellulases. Family E includes, beside C.thermocellum CelD, a number of cellulases such as Butyrivibrio fibrisolvens cellodextrinase Cedl, C. thermocellum endoglucanase CelF, Cellulomonas fimi endoglucanase CenB, Clostridium stercorarium Avicelase I, Persea americana endoglucanase, Dictyostelium discoideum endoglucanase, Cellulomonas fimi endoglucanase CenC, and Pseudomonas fluorescens var. cellulosa endoglucanase A. The sequence of CelD used to construct genetic frame was modified according to Chauvaux et al., substituing Asp523 by Ala, since that mutation increases specific activity of CelD to 224% . (Chauvaux S et al., 1992)

The extracellular sucrase SacC was obtained from Zymomonas mobilis by using PCR tecniques with the help of PhD. José Manuel Aguilar Yáñez. Zymomonas mobilis is a gram negative bacterium that has been shown to produce ethanol at a rate three to four fold, and at a higher final yield compared to the traditionally used yeast strains (Rogers et al., 1982). However, this organism uses only a narrow range of substrates, which is limited to glucose, fructose and sucrose (Swings & DeLey, 1977) An extracellular sucrase (SacC) has a high specific activity for sucrose hydrolysis. This enzyme contributes to nearly 60% of the extracellular sucrase activity of Zymomonas mobilis. The purified active enzyme from Zymomonas mobilis is a monomer with a molecular weight of 46 kDa and the sacC gene has already been cloned and expressed in E. coli. (Gurunathan S & Gunasekaran P, 2004)

Inverted sugar contains fructose and glucose in equal proportions. This product is greater in demand than pure glucose as a food and drink sweetener due to many useful physical and functional attributes of fructose including sweetness, flavor enhancement, humectancy, color and flavor development, freezing-point depression, and osmotic stability. (Hanover LM & White JS, 1993) The conventional method of manufacturing inverted sugar involves acid hydrolysis of sucrose. However, such reaction has a low conversion efficiency and high-energy consumption.

Our new genetic construct will be able to immobilize invertase by a cell surface display technique, fusing the enzyme to fragments of ompA and estA. This system will be capable of transforming sucrose into fructose by an enzymatic method without destroying the bacteria; thus reducing the amount of unit operations required to purify solely the enzyme and cutting production costs. At the same time, we will immobilize cellulase with the same strategy to take advantage of cellulose residues from the sugarcane process, making the device sustainable.

• Chauvaux S, Beguing P, & Aubert JP (1992) Site-directed Mutagenesis of Essential Carboxylic Residues in Clostridium thermocellum Endoglucanase CelD. The Journal of Biological Chemistry Vol 267(7) 4472-4478.

• Gurunathan S & Gunasekaran P (2004) Differential Expression of Zymomonas mobilis Sucrase Genes (sacB and sacC) in Escherichia coli and Sucrase Mutants of Zymomonas mobilis. Brazilian Archives of Biology and Technology Vol 47(3):329-338.

• Hanover LM & White JS (1993) Manufacturing, composition, and applications of fructose. Am J Clin Nutr. Vol. 58:724S-32S.

• Jana S & Deb JK (2005) Strategies for efficient production of heterologous proteins in Escherichia coli. Appl Microbiol Biotechnol 67: 289–298.

• Joachim J & Meyer TF (2007) The Autodisplay Story, from Discovery to Biotechnical and Biomedical Applications. Microbiology and Molecular Biology Reviews. Vol. 71, No. 4. p. 600–619

• Lee SH, Choi JI, Park SJ, Lee SY & Park BC. (2004) Display of Bacterial Lipase on the Escherichia coli Cell Surface by Using FadL as an Anchoring Motif and Use of the Enzyme in Enantioselective Biocatalysis. Applied and Environmental Microbiolgy. Vol. 70, No. 9 p. 5074–5080

• Rogers PL, Lee KJ, Skotnicki ML & Tribe DE (1982), Ethanol production by Zymomonas mobilis. Adv. Biochem. Engg. Biotechnol Vol 23:37-84.

• Rutherford N & Mourez M.(2006) Review Surface display of proteins by Gram-negative bacterial autotransporters. Microbial Cell Factories 5:22

• Swings J & De Ley J (1977) The biology of Zymomonas. Bacteriol Rev. Vol.41:1-46.

• Yuan J, Zweers JC, Maarten van Dijl J, Dalbey RE. (2010) Protein transport across and into cell membranes in bacteria and archaea. Cell. Mol. Life Sci. 67:179–199