Team:Tec-Monterrey/projectoverview

Justificación general del proyecto
Enzimas necesarias y en que procesos se utilizan
Producción tradicional
Sistemas alternativos (Cell surface display)

Inverted sugar contains fructose and glucose in equal proportions. This product has a 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) Inverted sugar is conventionaly synthetizing by the acid hydrolysis of sucrose. However, such reaction has a low conversion efficiency and high-energy consumption. (¿?)

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 proteins are expressed in the outer membrane of Escherichia coli the cell envelope acts as their matrix. This display is achievable thanks to several displaying 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 induatrial process reactions or analytical assays and then can be simply recovered by centrifugation. (Joachim J & Meyer TF, 2007)

The type V autotransporters are natural transportation system to import/export substrates through the periplasm/membrane. They are composed of an N-terminal sec-dependent signal peptide, a passenger domain, and a translocator domain that is predicted to form a β-barrel. (Rutherford et al., 2006)

The estA protein is inserted into the cytoplasmic membrane of E. coli by the Sec secretory pathway, 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)

Clostridium 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.

Zymomonas mobilis is a gram negative bacterium that produces ethanol from glucose, fructose and sucrose (Swings & DeLey, 1977) at a rate three to four fold, and at a higher final yield compared to the traditionally used yeast strains (Rogers et al., 1982). Almost 60% of the extracellular sucrase activity of Zymomonas mobilis is the result of the activity of the extracellular SacC. Theat particular enzyme has a specific activity to hydrolysis sucrose of ¿? that is a high standard for sucrose. (¿?) It is a monomer in its native state, with a molecular weight of 46 kDa and has already been expressed in E. coli. (Gurunathan S & Gunasekaran P, 2004)

In this project, the natural passenger domain of the autotransporter estA from Pseudomonas sp was replaced by a cellulase (CelD) and an invertase (SacC) as means to display them at the bacterial surface by the translocator domain of the estA protein.

Using the signal peptide of a protein which is naturally transported to the cytoplasma (signal peptide of PhoA), we intend to export CelD and SacC to the external surface of E. coli. On 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.
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)
Our new genetic construct will be able to immobilize invertase (sacC) on the outer membrane of E. coli, fusing the enzyme with fragments of ompA and estA. This chassis will be capable of transforming sucrose into fructose without further primary recovery, purification, and immobilization steps to obtain enzymes; thus reducing the amount of unit operations and cutting production costs. At the same time, we will immobilize cellulase (celD) with the same strategy to take advantage of cellulose residues from the sugarcane process, making the process more 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