DEVELOPMENT OF A NOVEL FERMENTATION PROCESS FOR THE ANAEROBIC CONVERSION OF GLYCEROL AND CO2 INTO SUCCINIC ACID USING ESCHERICHIA COLI

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: NRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0205996
• Grant No.: 2005-35504-16698
• Cumulative Award Amt.: $341,922.00
• Proposal No.: 2006-00675
• Project Start Date: Aug 1, 2005
• Project End Date: Jul 31, 2009
• Grant Year: 2006
• Program Code: [71.2]- Biobased Products & Bioenergy Production Research

Recipient Organization
RICE UNIVERSITY
PO BOX 1892
HOUSTON,TX 77251

Performing Department
(N/A)

Non Technical Summary
The value of the project is the conversion of two byproducts in the production of biofuels (glycerol and CO2) into a value added chemical (succinic acid). The objectives of the proposed project is to develop a novel fermentation process for the conversion of glycerol and CO2 into succinic acid, a higher-value feedstock used to produce industrially important chemicals having a domestic market of more than $1.3 billion per year.

Animal Health Component

20%

Research Effort Categories

Basic

80%

Applied

20%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
511	4010	1020	20%
511	4010	1060	30%
511	4010	2020	50%

Knowledge Area
511 - New and Improved Non-Food Products and Processes;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
2020 - Engineering; 1060 - Biology (whole systems); 1020 - Physiology;

Keywords

fermentation

metabolic engineering

escherichia coli

raw glycerol

carbon dioxide

Goals / Objectives
Specific Aims The specific aims of this project are: 1. Metabolically engineer E. coli for the anaerobic conversion of glycerol into succinic acid using genetic engineering tools. 1.1 Introduce specific genetic modifications into E. coli to improve the conversion of glycerol into succinic acid and block the synthesis of other fermentation products. 1.2 Characterize the conversion of glycerol into succinic acid by wild-type and recombinant strains through intensive fermentation profiling. 2 Evaluate wild-type and engineered strains using functional genomics and metabolic engineering tools. 2.1 Characterize wild-type and recombinant strains via metabolic flux analysis (MFA) using conventional and NMR-based MFA. 2.2 Evaluate changes in global gene expression resulting from introduced genetic perturbations using DNA Microarrays. 2.3 Integrate transcriptional and metabolic data, and introduce further genetic modifications informed by this analysis. 3. Formulate an optimum cultivation medium and identify optimum culture conditions using industrial-grade medium components. 3.1 Formulate an optimum culture medium using raw glycerol streams and cost-effective nutritional supplements. 3.2 Identify optimum pH, temperature, and dissolved CO2 concentrations for producing succinic acid using the culture medium formulated in 3.1.

Project Methods
APPROACH AND RESEARCH PLAN Overview The proposed project will encompass the two steps involved in developing a microbial fermentation process: (1) the development of a biocatalyst that efficiently catalyzes the desired biochemical reaction(s), and (2) the identification of the optimum culture conditions and the appropriate fermentation system. Two general approaches are envisioned for obtaining E. coli strains with improved capacity to convert glycerol and CO2 into succinic acid: (1) the empirical strain improvement via mutagenesis and selection, and (2) a more rational approach as proposed by ME. The empirical strain improvement approach, which has traditionally yielded mutant strains with improved properties, has three major drawbacks. First, mutagenesis and selection are laborious, time-consuming, and have unpredictable duration times and results. Second, the random mutations responsible for high titers cannot readily be identified, preventing the future use of these mutations for engineering other strains. Third, the obtained strains often acquire detrimental mutations, such that their general metabolism is impaired. Due to these limitations, we will use the more rational ME approach, including all steps ME has been defined to consist of. Optimization of culture conditions and development of an appropriate fermentation system. Once an efficient E. coli strain is obtained, it will be tested for its capacity to produce succinic acid using industrial-grade media. Optimum composition of the media and culture conditions will be determined. Using the engineered biocatalyst and optimized medium and culture conditions, we will identify the appropriate fermentation system. Two valuable statistical tools will be used in this part of the project: (1) statistically-based experimental design for the designing and planning of the experiments and (2) response surface methodology to analyze the results and identified optimum conditions.

Progress 08/01/07 to 07/31/08

Outputs
OUTPUTS: In the reporting period we created several knockout mutants and constructed several expression vectors that are central to our strategy for improving succinate production. Each expression vector was characterized through activity assays. Overexpressed genes are: (1) E. coli gldA and dhaKLM, which encodes glycerol dehydrogenase and PEP-dependent dihydroxyacetone kinase, respectively; C. freundii dhaKL, which encodes an ATP-dependent dihydroxyacetone kinase; (2) A. succinogenes and E. coli pckA, which encode phosphoenolpyruvate carboxykinases; (3) pyc from L. lactis, which encodes an ATP-dependent pyruvate carboxylase, and (4) E. coli maeB, which encodes the malic enzymes. Both low- and high-copy vectors were used: derivatives of plasmid pZSKLM reported in and derivatives of plasmid pTrc99A originally from Pharmacia Biotech, respectively. We also constructed strains with reduced production of competing byproducts ethanol, acetate and lactate by introducing mutations in the following genes: adhE (encoding aldehyde/alcohol dehydrogenase), pta (encoding phosphate acetyltransferase), poxB (encoding pyruvate oxidase), pflB (encoding pyruvate formate-lyase), and ldhA (encoding lactate dehydrogenase). The outcome of this work is reported in the section below. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The main outcomes of the reported period relate to the improvement of succinate yield and concentration by eliminating competing pathways and overexpressing those that divert carbon from the phosphoenolpyruvate and pyruvate nodes toward the synthesis of succinate, as described in the section "outputs". A succinate yield of 1.1 mole of succinate per mole of glycerol was realized, with concentrations exceeding 10 g/L in 72-hour cultures. The values of these fermentation parameters represent more than 10-fold increase over those observed in wild-type strains.

Publications

• -Gonzalez, R. Murarka. A., Dharmadi, Y., and Yazdani, S. S. 2008. A new model for the anaerobic fermentation of glycerol in enteric bacteria: trunk and auxiliary pathways in Escherichia coli. Metab. Eng. 10 (5): 234-245.
• -Murarka. A., Dharmadi, Y., Yazdani, S. S., and Gonzalez, R. 2008. Fermentative utilization of glycerol in Escherichia coli and its implications for the production of fuels and chemicals. App. Environ. Microbiol. 74 (4): 1124-1135.
• -Gonzalez, R., Clomburg, J., Yazdani, S.S., Murarka, A., and Dharmadi, Y. 2008. Understanding and harnessing the microbial fermentation of glycerol: a new path for the production of biochemicals. Metabolic Engineering VII: Health and Sustainability, Puerto Vallarta, Mexico, September 14-19, 2008.
• -Gonzalez, R. 2008. Understanding and harnessing the microbial fermentation of glycerol. A new path for the production of biochemicals. 2008 Annual AIChE meeting, Philadelphia, PA, November 16-21, 2008.
• -Gonzalez, R. 2008. Understanding and harnessing microbial fermentation of glycerol: A new path to biofuels and biochemicals. 236th ACS National Meeting & Exposition, Philadelphia, PA, August 17-21, 2008.

Progress 08/01/06 to 07/31/07

Outputs
In the reporting period we cloned several genes that are central to our strategy for improving succinate production. Their expression was also and characterized through activity assays. The genes included: (1) C. freundii dhaKL, which encodes an ATP-dependent dihydroxyacetone kinase and (2) pyc from L. lactis, which encodes an ATP-dependent pyruvate carboxylase. Both dhaKL and pyc were cloned in two vectors, one low copy (pZSdhaKL and pZSpyc, derivatives of plasmid pZSKLM reported in ) and one high copy (pTrcKL and pTrcpyc, derivatives of plasmid pTrc99A originally from Pharmacia Biotech). We also constructed strains with reduced production of competing byproducts ethanol and acetate by introducing mutations in the following genes: adhE (encoding aldehyde/alcohol dehydrogenase), pta (encoding phosphate acetyltransferase), poxB (encoding pyruvate oxidase), and pflB (encoding pyruvate formate-lyase). We also evaluated the use of carbon dioxide and carbonate salts as source of CO2. The outcome of this work is reported in the section below.

Impacts
The main outcomes of the reported period relate to the improvement of succinate yield and concentration by eliminating competing pathways and overexpressing those that favor the synthesis of succinate, as described in the section "outputs". A succinate yield of 86% (g succinate/g glycerol) was realized, with concentrations exceeding 10 g/L in 96-hour cultures. The values of these fermentation parameters represent more than 10-fold increase over those observed in wild-type strains.

Publications

• −Murarka. A., Dharmadi, Y., Yazdani, S. S., and Gonzalez, R. (2008). Fermentative Utilization of Glycerol in Escherichia coli and its Implications for the Production of Reduced Chemicals and Fuels. App. Environ. Microbiol. 74 (4).
• −Yazdani. S. S. and Gonzalez, R. (2007). Anaerobic Fermentation of Glycerol: A Path to Economic Viability for the Biofuels Industry. Curr. Opin. Biotechnol. 18 (3): 213-219.
• −Gonzalez, R., Murarka, A., Dharmadi, Y., and Yazdani, S. Anaerobic Fermentation of Glycerol in Escherichia coli: A New Path to BioFuels and BioChemicals. 2007 Annual AIChE Meeting Program and Abstract Book, Salt Lake City, UT, November 4-9, 2007.
• −Gonzalez, R., Murarka, A., Dharmadi, Y., and Yazdani, S. Anaerobic Fermentation of Glycerol in Escherichia coli: A New Path to BioFuels and BioChemicals. 2007 Annual Meeting Program and Abstract Book, Society for Industrial Microbiology, July 29-August 2, 2007, Denver, CO.
• −Gonzalez, R., Murarka, A., Dharmadi, Y., and Yazdani, S. A new paradigm for glycerol fermentation in Escherichia coli and other enteric bacteria: Implications for the production of biofuels and biochemicals. Program and Abstract Book of Biochemical Engineering XV: Engineering Biology from Biomolecules to Complex Systems. Quebec City, Canada, July 15-19, 2007.
• −Gonzalez, R., Murarka, A., Dharmadi, Y., and Yazdani, S. (2007) Escherichia coli Ferments Glycerol in the Absence of External Electron Acceptors: A New Platform for Metabolic Engineering. 29th Symposium on Biotechnology for Fuels and Chemicals Program and Abstract Book. April 29- May 2, 2007, Denver, CO.
• −Gonzalez, R. (2007) Anaerobic Fermentation of Glycerol. PCT Appl No.: PCT/US2007/065726. Also: US Serial No.: 60/788,512 and US Serial No.: 60/867,58.

Progress 08/01/05 to 08/01/06

Outputs
In the reporting period we investigated the anaerobic fermentation of glycerol by E. coli, thus creating a significant knowledge base that enables the implementation of metabolic engineering strategies proposed in the project. We discovered that Escherichia coli can ferment glycerol in a pH-dependent manner. We hypothesized that glycerol fermentation is linked to the availability of CO2, which under acidic conditions is produced by the oxidation of formate by the enzyme formate hydrogen lyase (FHL). In agreement with this hypothesis, glycerol fermentation was severely impaired by blocking the activity of FHL. We demonstrated that, unlike CO2, hydrogen (the other product of FHL-mediated formate oxidation) had a negative impact on cell growth and glycerol fermentation. In addition, supplementation of the medium with CO2 partially restored the ability of an FHL-deficient strain to ferment glycerol. High pH resulted in low CO2 generation (low activity of FHL) and availability (most CO2 is converted to bicarbonate), and consequently very inefficient fermentation of glycerol. Most of the fermented glycerol was recovered in the reduced compounds succinate and ethanol (93% of the product mixture), which reflects the highly reduced state of glycerol and confirms the fermentative nature of this process.

Impacts
The worldwide surplus of glycerol generated as inevitable by-product of biodiesel fuel and oleochemical production is resulting in the shutdown of traditional glycerol-producing/refining plants and new applications are needed for this now abundant carbon source. Our findings should enable the development of an E. coli-based platform for the anaerobic production of reduced chemicals from glycerol at yields higher than those obtained from common sugars such as glucose (including succinate, the product of interest in this project)

Publications

• Dharmadi, Y., Murarka, A., and Gonzalez, R., (2006). Anaerobic Fermentation of Glycerol by Escherichia coli: A New Platform for Metabolic Engineering. Biotechnol. Bioeng. 94 (5): 821-829 (Accelerated Publication).