Sustainable microbial production of high-value organic compounds is becoming an increasingly attractive alternative to organic syntheses that utilize petrochemical feedstocks. For example, the high cost of and the numerous applications for 3-hydroxypropanoate (3HP) make it a valuable target for biosynthesis. 3HP can be used directly as a monomer for the production of polyhydroxypropanoate or as a raw material for the production of acrylic acid, 1,3 propanediol, or acrylamide. In the past numerous biosynthetic pathways have been proposed for the production of 3HP from pyruvate, and one of these pathways (pyruvate --> á-alanine --> â-alanine --> 3-oxopropanoate --> 3HP) has also been implemented in a microorganism.

As a case study, we applied the Biochemical Network Integrated Computational Explorer (BNICE) framework(1,2) for the automated construction and evaluation of metabolic pathways to explore novel biosynthetic routes for the production of 3HP from pyruvate. Among the pathways to 3HP generated by the BNICE framework were all of the known pathways for the production of 3HP. In addition to the known pathways, many novel pathways were discovered, two of which involve fewer steps than any of the known pathways to 3HP. Thermodynamics-based Metabolic Flux Analysis (TMFA)(3) was utilized to determine the maximum yield for the production of 3HP in E. coli growing anaerobically on glucose for each of the pathways discovered, and ATP consumption by the pathway was determined to have a significant effect on the yield. The thermodynamic constraints of TMFA also allowed for the maximum achievable intracellular activity of 3HP to be determined for each of the pathways. Although pathways consuming ATP allow for the production of 3HP at higher concentration, the trade off in yield is undesirable. Many of the novel pathways discovered require no ATP for the production of 3HP including the novel two-step pathway, while only one of the known pathways to 3HP consumes no ATP. Overall, we demonstrate the capability of the BNICE framework to reproduce all of the known pathways to 3HP and generate numerous promising alternative pathways, and we also demonstrate the capability of our pathway evaluation techniques in identifying the promising pathways for implementation.

1. Hatzimanikatis, V., C. Li, I. J. A., C. S. Henry, M. D. Jankowski and L. J. Broadbelt. 2004. Exploring the diversity of complex metabolic networks. Bioinformatics 21(8):1603-1609.

2. Li, C., J. A. Ionita, C. S. Henry, M. D. Jankowski, H. V. and L. J. Broadbelt. 2004. Computational discovery of biochemical routes to specialty chemicals. Chemical Engineering Science 59(22-23):5051-5060.

3. Henry, C. S., L. J. Broadbelt and V. Hatzimanikatis. 2007. Thermodynamics-based metabolic flux analysis. Biophys J. 92(7):1792-1805.