Source: AGRENEW, INC. submitted to NRP
INNOVATIVE AND LOW COST PROCESS TO PRODUCE ISOAMYL ACETATE USING METABOLICALLY ENGINEERED E. COLI
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
SMALL BUSINESS GRANT
Reporting Frequency
Annual
Accession No.
0199575
Grant No.
2004-33610-14308
Cumulative Award Amt.
(N/A)
Proposal No.
2004-00503
Multistate No.
(N/A)
Project Start Date
May 15, 2004
Project End Date
Jun 30, 2005
Grant Year
2004
Program Code
[8.8]- (N/A)
Recipient Organization
AGRENEW, INC.
1500 HAYES DR.
MANHATTAN,KS 66502
Performing Department
(N/A)
Non Technical Summary
Natural flavor compounds find wide applications in the food, feed, cosmetic, chemical and pharmaceutical industry, and isoamyl acetate is an important flavor ester. In this research program, a low-cost process is proposed to convert glucose from renewable sources to isoamyl acetate using a high-yielding metabolically engineered E coli. Isoamyl acetate produced via chemical synthesis results in an undesirable racemic mixture. The fermentation route followed in this program will result in high yields of "natural" isoamyl acetate with additional usage in food industry along with other uses The proposed research will lay the foundation for isoamyl acetate biosynthesis at high yield from renewable sugars. Successful outcome of the project will provide a platform for the low-cost production of additional high-valued natural esters.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5011599100040%
5014010104060%
Knowledge Area
501 - New and Improved Food Processing Technologies;

Subject Of Investigation
1599 - Grain crops, general/other; 4010 - Bacteria;

Field Of Science
1040 - Molecular biology; 1000 - Biochemistry and biophysics;
Goals / Objectives
The first objective of this research will construct and characterize an improved metabolically engineered E.coli for high isoamyl acetate yields by overexpressing a more efficient alcohol transferase gene, ATF1 from yeast. The second objective is to develop high-cell-density fermentation technology that will utilize a low-cost source of glucose derived from corn and grain sorghum and other inexpensive nutrient sources. The third objective is to identify an effective separation process to recover the volatile isoamyl acetate
Project Methods
The proposed research focuses on developing a process to produce high yields of isoamyl acetate using a proven and then an improved, genetically-stable metabolically engineered E coli. Renewable, inexpensive glucose derived from corn and grain sorghum will be used as fermentation feedstock. Operational parameters for efficient aerobic and anaerobic fermentation using both engineered E coli strains will be optimized. Fed-batch mode with cell mass recycle to obtain high-cell-density fermentation conditions will be developed. An efficient separation strategy to economically separate isoamyl acetate from the final fermentation broth will be determined and preliminary economic analysis will be performed. The proposed research methodology will result in a low-cost fermentation process for producing isoamyl acetate.

Progress 05/15/04 to 06/30/05

Outputs
A new engineered E.coli production strain for improved isoamyl acetate by overexpressing a more efficient alcohol tranferase gene ATF1 in pBAD-TOPO plasmid has been developed. The improved metabolically engineered strain, E.coli YBS121, has been characterized and evaluated at the shake flask and at the 2 L fermenter level. Shake flask experiments were performed to evaluate the different media components; inexpensive fusel oil, sorghum glucose and CSL were found to effectively replace synthetic isoamyl alcohol, glucose and yeast extract, respectively. Batch fermentation experiments at 2 L fermenter level with different isoamyl alcohol addition patterns were performed and the conditions optimized. The target yield was successfully attained with fusel oil addition, and high-cell-density fermentation for increased isoamyl acetate yield was demonstrated.

Impacts
In this research program, a low-cost bio-based fermentation process to produce high yield of isoamyl acetate using engineered E coli and renewable sugars has been determined. Increased yield of 'natural'isoamyl acetate will lead to better production economics and wider application in food, feed, chemical and pharmaceutical industry.

Publications

  • No publications reported this period


Progress 10/01/03 to 09/30/04

Outputs
Good progress has been made in the Phase I project in the development of the improved engineered E.coli strain and in the experimental procedures to produce isoamyl acetate. A new engineered E.coli production strain for improved isoamyl acetate by overexpressing a more efficient alcohol tranferase gene ATF1 in pBAD-TOPO plasmid has been developed and characterized at the shake flask level. Issues relating to plasmid stability are being addressed. Experiments using glucose derived from corn and sorghum for isoamyl acetate production have been undertaken. Operational parameters for process optimization work of isoamyl acetate production under fed-batch and efficient dual-phase fermentation conditions will be determined in a 2 L fermenter using the improved E.coli YBS 121. An effective product recovery strategy will be identified and economic analysis of Phase I work will be executed.

Impacts
Production of a natural ester, such as isoamyl acetate, from renewable sugars and engineered E.coli will lead to a low-cost biobased process. The feasibility of producing high yields of isoamyl acetate will provide the framework for the bulk production of this high-valued specialty chemical.

Publications

  • No publications reported this period