CH391L/S12/MetabolicEngineering

Metabolic Engineering

Introduction

Metabolic engineering deals with modifying the metabolic and chemical pathways of a cell in order to alter the amount of a certain product that the cell can produce. This entails computation and bioinformatics to predict the effect of gene deletions on a cell’s metabolic pathways. Research on the metabolic engineering of Escherichia coli has been successful because there is an abundance of information and experiments on the genetic sequence and metabolic pathways of E. coli.

Flux Balance Analysis

Edwards and Palsson focused their work on E. coli metabolism by the flux balance analysis (FBA) method, a mathematical approach to predict metabolic capabilities. When a gene is deleted, then the flux, or output, of the corresponding pathway is constrained to zero. Genes encoding products of central metabolic pathways were deleted in E. coli in silico, and the resulting cell’s ability to grow were measured through FBA. These results matched 86% with existing mutants production. The goal of these studies is to utilize this information to predict the best a cell can produce under certain conditions, and what it cannot produce under those conditions. [1] [2]

Biofuel Applications

Biofuel Pathways

E. coli has become a primary source in the microbial production of biofuels because of the ease of metabolic engineering on E. coli. There are many carbon sources that can be used by E. coli to produce intermediate metabolites, and there is great potential in the engineering of effective pathways of these carbon sources; the difficulty comes in engineering pathways to turn these intermediates into useful biofuels as described in Clomburg and Gonzalez. These secondary pathways can be either fermentative or nonfermentative. The issue with fermentative pathways is that they use simple sugars to not only produce ethanol, but also other acids such as acetic acid, lactic acid, and others. So the goal of metabolic engineering in relation to the fermentative pathways is to refine the pathway to simply produce ethanol. Engineering nonfermentative pathways have led to biofuels closer to gasoline, however, these pathways are synthetic and do not naturally occur in E. coli. Through additional research, there is hope that microorganisms, especially E. coli can be used to synthesize biofuels vital to society today from renewable carbon sources. [3]

Industrial Applications

Industrial applications.

The latest experiments have engineered microorganism like E. coli to produce fuels compatible with modern engines. E. coli is ideal also because it has been engineered to produce a wider range of chemicals than any other known organism. However, there are many issues involved with the attempt to make E. coli use lignocellulose as a carbon source:

• E. coli needs more glycoside hydrolase to break down cellulose to sugars
• Techniques to overcome this need engineered organisms that convert biomass into biofuels in one step are not practical for mass production
• E. coli does not have the capacity for protein export in order to produce enzymes to digest lignocellulose in amounts needed for industrial use
• Integrating the needed concentration of enzymes to break down cellulose and the pathways to create biofuels into a microbe would create a great burden on the cell and would need to be highly regulated

Bokinsky et al engineered E. coli to overcome these obstacles by regulating the expressions of enzymes capable of being exported by E. coli that allow the cell to digest cellulose that has been pretreated with ionic liquids (IL), but as of now these IL are too expensive for commercial use. IL eliminates crystallinity and decreases lignin concentration decreasing the amount of enzymes needed to break down cellulose. This is a major advancement because the products from digestion of IL pretreated cellulose can be used in E. coli biosynthetic pathways to create gasoline, diesel, and jet fuel without the need for extra enzymes. At the same time major improvements in breaking down biomass and producing biofuels are necessary before these processes can be applied to industrial uses. [4]

References

1. Edwards JS and Palsson BO. The Escherichia coli MG1655 in silico metabolic genotype: its definition, characteristics, and capabilities. Proc Natl Acad Sci U S A. 2000 May 9;97(10):5528-33. DOI:10.1073/pnas.97.10.5528 |
2. Feist AM and Palsson BO. The biomass objective function. Curr Opin Microbiol. 2010 Jun;13(3):344-9. DOI:10.1016/j.mib.2010.03.003 |
3. Clomburg JM and Gonzalez R. Biofuel production in Escherichia coli: the role of metabolic engineering and synthetic biology. Appl Microbiol Biotechnol. 2010 Mar;86(2):419-34. DOI:10.1007/s00253-010-2446-1 |
4. Bokinsky G, Peralta-Yahya PP, George A, Holmes BM, Steen EJ, Dietrich J, Lee TS, Tullman-Ercek D, Voigt CA, Simmons BA, and Keasling JD. Synthesis of three advanced biofuels from ionic liquid-pretreated switchgrass using engineered Escherichia coli. Proc Natl Acad Sci U S A. 2011 Dec 13;108(50):19949-54. DOI:10.1073/pnas.1106958108 |