Enhanced Expression Levels of Cellulase Enzymes Using Multiple Transcription Units
- 320 Downloads
Transgenic cereals are an attractive option for the accumulation of foreign proteins when large volumes and low cost are required. Previous work has shown maize germ to be a particularly good location for accumulating enzymes that target cellulose for degradation. In this study, recently identified embryo-preferred promoters were used to investigate their ability to increase the accumulation of the enzymes endoglucanase E1 and cellobiohydrolase CBHI. The effect of increasing copy numbers of identical transcription units, as well as multiple copies of the enzyme driven by different promoters, was explored. Results show that accumulation of the E1 or CBHI enzymes can be significantly increased, particularly when using constructs with multiple copies of the transcription units. These findings demonstrate the highest levels of these enzymes obtained in a commercially relevant plant species observed thus far. The methodology described here may provide a low-cost plant-based source of enzymes enabling an economically viable solution for the conversion of cellulose to ethanol.
KeywordsCellulase Biofuels Maize Transcription units Embryo promoters Ethanol
This work was supported by grant #DOE DE FG36 GO88025, Foundation, Walton Family Foundation, and Arkansas State University Biosciences Institute. We would also like to acknowledge the technical assistance of Aaron Harry, Mackenzie Tageson, and Raghavendra Rayadurg in this project.
- 8.Hood EE, Love R, Bray J, Lane J, Clough RC, Pappu K, Drees C, Hood KR, Yoon S, Ahmad A, Howard JA (2007) Subcellular targeting is a key condition for high-level accumulation of cellulase protein in transgenic maize seed. Plant Biotechnol J 5:709–719Google Scholar
- 9.Ziegelhoffer T, Will J, Austin-Phillips S (1999) Expression of bacterial cellulase genes in transgenic alfalfa (Medicago sativa L.), potato (Solanum tuberosum L.) and tobacco (Nicotiana tabacum L.). Molecular Breeding New Strategies in Plant Improvement 5(4):309–318Google Scholar
- 10.Tsai GJ, Wu ZY, Su WH (2000) Antibacterial activity of a chitooligosaccharide mixture prepared by cellulase digestion of shrimp chitosan and its application to milk preservation. J Food Protect 63(6):747–752Google Scholar
- 15.Hood EE, Horn ME, Howard JA (2003) Production and Application of Proteins from Transgenic Plants. In: Vasil I (ed) Plant Biotechnology 2002 and Beyond, Proceedings of the 10th IAPTC&B Congress, Orlando, FL, Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 377–382Google Scholar
- 27.Armstrong C, Green C, Phillips R (1991) Development and availability of germplasm with high Type II culture formation response. Maize Genet Coop Newsletter 65:92–93Google Scholar
- 31.Streatfield SJ, Love R, Bray J (2007) Globulin-1 promoter from maize and method of using same USA Patent 7,169,967, January 30, 2007Google Scholar
- 35.Gray BN, Bougri O, Carlson AR, Meissner J, Pan S, Parker MH, Zhang D, Samoylov V, Ekborg NA, Michael Raab R (2011) Global and grain specific accumulation of glycoside hydrolase family 10 xylanases in transgenic maize (Zea mays). Plant Biotechnol J 9(9):1100–1108. doi: 10.1111/j.1467-7652.2011.00632.x PubMedCrossRefGoogle Scholar
- 37.Gale SE, Westover EJ, Dudley N, Krishnan K, Merlin S, Scherrer DE, Han X, Zhai X, Brockman HL, Brown RE, Covey DF, Schaffer JE, Schlesinger P, Ory DS (2009) Side chain oxygenated cholesterol regulates cellular cholesterol homeostasis through direct sterol-membrane interactions. J Biol Chem 284(3):1755–1764. doi: 10.1074/jbc.M807210200 PubMedCrossRefGoogle Scholar
- 40.Naqvi S, Zhu C, Farre G, Ramessar K, Bassie L, Breitenbach J, Perez Conesa D, Ros G, Sandmann G, Capell T, Christou P (2009) Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways. Proc Natl Acad Sci USA 106(19):7762–7767. doi: 10.1073/pnas.090141210 PubMedCrossRefGoogle Scholar
- 41.Naqvi S, Farre G, Zhu C, Sandmann G, Capell T, Christou P (2010) Simultaneous expression of Arabidopsis r-hydroxyphenylpyruvate dioxygenase and MPBQ methyltransferase in transgenic corn kernels triples the tocopherol content. Transgenic Res 20(1):177–181. doi: 10.1007/s11248-010-9393-6 PubMedCrossRefGoogle Scholar
- 42.Hennegan K, Yang DC, Nguyen D, Wu LY, Goding J, Huang JM, Guo FL, Huang N, Watkins S (2005) Improvement of human lysozyme expression in transgenic rice grain by combining wheat (Triticum aestivum) puroindoline b and rice (Oryza sativa) Gt1 promoters and signal peptides. Transgenic Res 14(5):583–592. doi: 10.1007/s11248-004-6702-y PubMedCrossRefGoogle Scholar