Team:Tec-Monterrey/projectoverview

A cellulase was considered for the new application of sugarcane bagasse. 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. (Chauvaux, Beguin & Aubert, 1992).
The second enzyme was an invertase. Zymomonas mobilis that 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. This sacC gene expressed in Escherichia coli BL21 exhibited sucrase activity of 1948 - 2672 U/mg while the un-induced strain expressed 12.8 – 24.6 U/mg . One unit of sucrase activity was defined as the amount of enzyme releasing 1umol of reducing sugar per minute. It is a monomer in its native state, with a molecular weight of 46 kDa. (Gurunathan S & Gunasekaran P, 2004)

Industrial production of invert sugar is usually based on the acid or enzymatic hydrolysis of sucrose. Acid hydrolysis is based on the application of appropriate strong mineral or weak organic acids. The disadvantage of acid hydrolysis is the possible presence of impurities in the product introduced by uncontrollable parameters during conversion. On the other hand this conversion can also be achieved by enzymatic action of invertase on sucrose with a conversion efficiency of almost 100% without the inherent disadvantages of acid hydrolysis. (Safarik et al., 2009) As an alternative method to the traditional chemical process to produce inverted sugar, cell surface display was suggested. The 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 E. 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 industrial process reactions or analytical assays and then can be simply recovered by centrifugation. (Joachim J & Meyer TF, 2007)

The Tat (twin-arginine translocation) system functions to translocate folded proteins across the membrane while the Sec secretory pathway translocates unfolded substrates. The Sec translocase is comprised of the SecYEG translocation channel and the accessory components SecA, SecDFYajC, and YidC. (Yuan J et al., 2010 & Yuan J et al ., 2010) Both the type II secretory system and the type V autotransporter system are natural translocation systems to import/export substrates through the periplasm and membrane. The type II secretory system can take both Tat and Sec system while the type V autotransporters use the Sec system. Sec secretory pathway is 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) Both lpp and phoA signal peptides are natural Sec-dependent E. coli signal peptides which permits the translocation of the outer membrane proteins by the type II and the type V system, respectively.

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. Also, these enzymes were fused with an integral outer membrane fragment of ompA protein (BBa_K103006) to display them at the outer membrane of E. coli. Using the signal peptide of a protein which is naturally transported to the cytoplasm (signal peptide of phoA and lpp), we intend to export CelD and SacC to the external surface of E. coli. The sequence of CelD used to construct our genetic frame was modified according to Chauvaux et al., substituing Asp523 by Ala, since that mutation increases the specific activity of CelD to 224% . (Chauvaux S et al., 1992) Our genetic construct will be able to immobilize an invertase (sacC) and a cellulase (celD) on the outer membrane of E. coli, fusing the enzymes with fragments of ompA and estA. This chassis will be capable of transforming sucrose into fructose or hydrolyzing a complex material of sugarcane bagasse into reducing sugars without further downstream primary recovery, purification, or immobilization steps to obtain enzymes; thus reducing the amount of unit operations and cutting production costs.

• 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

• Lavarack BP, Griffn GJ, Rodman D. (2002) The acid hydrolysis of sugarcane bagasse hemicellulose to produce xylose,arabinose,glucose and other products. Biomass and Bioenergy. Vol. 23: 367 – 380

• 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(9): 5074–5080

• Pandey A, Soccol CR, Nigam P, Soccol VT. (2000) Biotechnological potential of agro-industrial residues. I: sugarcane bagasse. Bioresource Technology. Vol 74(1):69-80

• 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

• Safarik I, Sabatkova Z & Safarikova M. (2009) Invert sugar formation with Saccharomyces cerevisiae cells encapsulated in magnetically responsive alginate microparticles. Journal of Magnetism and Magnetic Materials Vol.321:1478–1481

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

• Yanase H, Yamamoto K, Sato D, Okamoto K. (2005) Ethanol production from cellobiose by Zymobacter palmae carrying the Ruminocuccus albus �-glucosidase gene. Journal of Biotechnology Vol. 118:35–43

• 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