PROJECT B

Metabolic Engineering

Keywords: xylose metabolism, fermentation, pentose, lignocellulosic bioconversion, ethanol, metabolism, E. coli, Z. mobilis.

Overview: For the biotechnological production of low-cost high-volume commodities, such as ethanol, it is economically important that all sugars, hexoses and pentoses obtained from biomass, are used as substrates. Microorganisms that ferment both monosaccharides at acceptable yields have not been found in nature and ethanol produced from xylose is an inefficient pathway in most natural organisms. To bypass these natural barriers it is necessary to genetically alter the metabolism of natural living cells to reshape the fermentation pathway towards the production of ethanol. E. coli is unable to produce ethanol because it lacks two key enzymes. However, if the missing activities are provided through genetic engineering recombinant E. coli cells are able to convert glucose and other sugars into ethanol (see Figure 1). Successfully engineering metabolic pathways to adjust microorganisms to the conditions required by industrial operations is the key to the introduction of new fermentation technologies.

 

Goal: The objective of this laboratory assignment is to construct an E. coli recombinant strain that uses glucose and xylose as a carbon source to produce ethanol. Plasmids encoding the required metabolic activities will be provided. Strain construction, fermentation protocols and analytical methods to determine the consumption of substrates and production of metabolites is to be decided by each group.

Recommended Reading

(***** Easy Reading (very useful); *** Classic Paper; * Important Contribution)

 

Xylose fermentation, Ethanologenic bacteria and Metabolic Engineering

 

*** 1 - Alterthum, F. and L. O. Ingram (1989). Efficient ethanol production from glucose, lactose, and xylose by recombinant Escherichia coli. Appl Environ Microbiol 55: 1943-1948.

 

***** 2 - Glick, B. R. and J. J. Pasternak (1994). Chapter 10: Bioremediation and Biomass Utilization. Molecular Biotechnology: Principles and applications of recombinant DNA . Washington D.C., ASM Press. 235-265.

 

***** 3 - Glick, B. R. and J. J. Pasternak (1994). Chapter 13: Large-scale production of proteins from recombinant microorganisms. Molecular Biotechnology: Principles and applications of recombinant DNA . Washington D.C., ASM Press. 305-327.

 

*** 4 - Hahn-Hägerdal, B., T. Linden, T. Senac and K. Skoog (1991). Ethanolic fermentation of pentoses in lignocellulose hydrolysates. Appl Biochem Biotechnol 29: 131-144.

 

*** 5 - Ingram, L. O., T. Conway, D. P. Clark, G. W. Sewell and J. F. Preston (1987). Genetic engineering of ethanol production in Escherichia coli. Appl. Environ. Microbiol. 53: 2420-2425.

 

* 6 - Jeffries, T. W. and C. P. Kurtzman (1994). Strain selection, taxonomy, and genetics of xylose-fermenting yeasts. Enz Microb Technol. 16: 922-932.

 

***** 7 - Lodish, H., D. Baltimore, A. Berk, L. Zipursky, P. Matsudaira and J. Darnell (1995). Chapter 17: Cellular energetics: Formation of ATP by glycolysis and oxidative phosphorilation. Molecular Cell Biology Ed. t. edition. New York, W.H. Freeman and Company. 739-777.

 

* 8 - Ohta, K., D. S. Beall, J. P. Mejia, K. T. Shanmugam and L. O. Ingram (1991a). Genetic improvement of Escherichia coli for ethanol production: chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II. Appl Environ Microbiol. 57: 893-900.

 

* 9 - Ohta, K., D. S. Beall, J. P. Mejia, K. T. Shanmugam and L. O. Ingram (1991b). Metabolic engineering of Klebsiella oxytoca M5A1 for ethanol production from xylose and glucose. Appl Environ Microbiol. 57: 2810-2815.

 

***** 10 - Primrose, S. B. (1991). Part III: The exploitation of microbes. Molecular Biotechnology . Oxford, UK, Blackwell Scientific Publications. second edition, ed. 61-108.

 

*** 11 - Schneider, H. (1989). Conversion of pentoses to ethanol by yeasts and fungi. Crit Rev Biotechnol. 9: 1-40.

 

* 12 - Takahashi, D. F., M. L. Carvalho and F. Alterthum (1994). Ethanol production from pentose and hexoses by recombinant Escherichia coli. Biotechnol Lett. 16: 747-750.

 

*** 13 - Zhang, M., C. Eddy, K. Deanda, M. Finkelstein and S. Picataggio (1995). Metabolic engineering of a pentose metabolism pathway in ethanologenic Zymomonas mobilis. Science 267: 240-243.

 

Reference to Methods and Molecular Biology Handbooks

 

14 - Ausubel, F. M. (1987). Current Protocols in Molecular Biology.

 

15 - Glick, B. R. and J. J. Pasternak (1994). Chapter 1: The molecular biology revolution. Molecular Biotechnology: Principles and applications of recombinant DNA . Washington D.C., ASM Press. 5-16.

 

16 - Glick, B. R. and J. J. Pasternak (1994). Chapter 2: Recombinant DNA technology. Molecular Biotechnology: Principles and applications of recombinant DNA . Washington D.C., ASM Press. 17-54.

 

17 - Glick, B. R. and J. J. Pasternak (1994). Chapter 3: Molecular research procedures. Molecular Biotechnology: Principles and applications of recombinant DNA . Washington D.C., ASM Press. 55-82.

 

18 - Glick, B. R. and J. J. Pasternak (1994). Chapter 4: Manipulation of gene expression in prokaryotes. Molecular Biotechnology: Principles and applications of recombinant DNA . Washington D.C., ASM Press. 83-112.

 

19 - Lodish, H., D. Baltimore, A. Berk, L. Zipursky, P. Matsudaira and J. Darnell (1995). Chapter 7: Recombinant DNA technology. Molecular Cell Biology Ed. t. edition. New York, W.H. Freeman and Company. 221-260.

 

20 - Primrose, S. B. (1991). Part II: Recombinant DNA technology. Molecular Biotechnology . Oxford, UK, Blackwell Scientific Publications. second edition, ed. 13-57.

 

21 - Primrose, S. B. (1991). Part III: The exploitation of microbes. Molecular Biotechnology . Oxford, UK, Blackwell Scientific Publications. second edition, ed. 61-108.

 

22 - Sambrook, J., E. F. Fritsch and T. Maniatis (1989). Molecular Cloning. New York, CSHL Press.

Ethics and Regulatory Issues in Biotechnology

 

23 - Glick, B. R. and J. J. Pasternak (1994). Chapter 18: Regulating the use of biotechnology. Molecular Biotechnology: Principles and applications of recombinant DNA . Washington D.C., ASM Press. 425-441.

 

24 - Glick, B. R. and J. J. Pasternak (1994). Chapter 19: Patenting biotechnology inventions. Molecular Biotechnology: Principles and applications of recombinant DNA . Washington D.C., ASM Press. 443-450.