Jonathan W. Chin
Olubolaji Akinterinwa
Caroline Monroe
Engineering E. coli to Maximize the Flux of Reducing Equivalents Available for Cofactor-Dependent Transformations
This research project is aimed at utilizing biomass-derived sugars as sources of energy and carbon backbone for the production of "renewable" chemicals and fuels through metabolic engineering. In collaboration with Costas Maranas, we are studying knowledge-based and model-predicted genetic manipulations in E. coli to develop robust, efficient whole-cell biocatalysts. One goal is to design bacteria which divert reduced cofactors (generated through central carbon metabolism) away from native electron transport or fermentation pathways and toward desired, heterologous reaction pathways. That is, we aim to maximize the efficiency with which sugars are utilized for driving NAD(P)H-dependent transformations by engineering strains which essentially "respire" on the substrates of these reactions. Transformations of primary interest are those catalyzed by reductases and oxygenases. Important considerations in this research include the cofactor requirement or specificity for the enzyme of interest, ensuring efficient (low energy) substrate transport and appropriate genetic modifications.
An experimental system has been developed for studying the influence of growth conditions and genetic modifications on production of non-fermentative, reduced compounds in E. coli. Our representative electron-accepting reaction is the reduction of xylose to xylitol (a low-calorie, non-cariogenic polyol sweetener) via NADPH-dependent xylose reductase. Expression of a cAMP-independent CRP variant allows for the simultaneous uptake of glucose and xylose, while a deletion in xylB prevented further metabolism of xylulose-5-phosphate. A variety of gene deletions introduced into our production strain are being studied to gain insights into which NADH- and NADPH-producing and consuming reactions are most prevalent in the context of NADPH-dependent xylitol production under different conditions (e.g., growing versus resting cells, aerobic versus anaerobic versus microaerobic, choice and availability of carbon/energy source, etc.). Gene targets include pgi, zwf, pfl, ldhA, adhE, nuo, ndh, sthA, pntAB (refer to the figure below). This experimental platform is being used to implement and further develop computational frameworks for metabolic optimization and strain improvement.
- Some Primary Experimental Goals:
- Understand the role of transhydrogenase during NADPH-dependent xylose reduction
- Maximize yield on xylitol produced per glucose consumed under aerobic conditions using non-growing "resting" cells with appropriate gene knockouts and/or inhibitors (minimize growth and aerobic respiration)
- Achieve complete NADH oxidation via xylose reduction under anaerobic conditions (minimize fermentation products)
- Maximize xylitol titer in controlled fermentations
The energy requirements associated with xylose transport critically effect cell growth and xylitol yield, particularly under anaerobic conditions. This aspect is being studied in detail in a related project.
Click on the diagram to view the enlargement
Overview of E. coli central metabolism highlighting NAD(P)H generation sites and other reactions likely to play an important role in our studies. Genes listed denote knockout mutations (or in some cases overexpressions) that are being studied.
This material is based upon work supported by the
National Science Foundation under Grant No. 0519516.