Team:Tec-Monterrey/projectdescription

Production of sugarcane used to be a high profit activity in the Mexican industry. Nonetheless, the increasing demand of high fructose syrup has become a rising threat to most sugar companies. Our project expects to apply synthetic biology to use sugar, obtained from sugarcane, in an industrial sugar-fructose process intending to make it easier and more profitable. The new genetic construct will be able to immobilize invertase by fusing it to bacterial natural membrane protein fragments using a technique for cell surface display. This system will catalyze the transformation of sucrose into fructose directly, without the need of any chemical or mechanical purification process to obtain the enzyme, reducing the amount of unit operations, and cutting production costs. Also, we will use the same principle to immobilize cellulase, converting cellulose from bagasse into something useful to produce biofuels.

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-biocatalysis, and whole-cell-biosensors. 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 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 and an invertase to display them at the bacterial surface by the translocator domain of the estA protein. The Sec machinery operates to insert proteins the cytoplasmic membrane of bacteria, but it cannot translocate folded substrates across the membrane, while the Tat (twin-arginine translocation) system functions to translocate folded proteins across the cytoplasmic membrane in bacteria. (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, we expect successful localization of the cellulase and the invertase at the external surface of E. coli. In the other hand, we used a fragment of an integral outer membrane ompA with signal peptide of a lipoprotein lpp (BBa_K103006) to express the same enzymes by the type II sec secretion system.

Inverted sugar contains fructose and glucose in roughly equal proportions. This product is greater in demand than pure glucose as a food and drink sweetener. The conventional method of manufacturing inverted Sugar involves acid hydrolysis of sucrose. However, such reaction has a low conversion efficiency and high-energy consumption, so the cost of production is high. Our new genetic construct will be able to immobilize invertase by cell surface display technique fusing them to fragments of ompA and estA. This system will be capable to transform sucrose into fructose by an enzymatic method without destroying the bacteria; thus reducing the amount of unit operations required to purify only 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.
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

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

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

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