1,4-Butanediol (BDO) Metabolic Engineering Service

1,4-Butanediol (BDO) is a high-volume industrial chemical used as a bio-monomer for elastomers (e.g., polyurethanes) and engineering plastics (PBT). Industrial production via petrochemical routes faces environmental and cost pressures as chemical synthesis uses toxic/high-pressure reagents (e.g., maleic anhydride, butadiene), requiring severe operating conditions. Bio-based production is hampered by low flux from the central carbon metabolism (TCA cycle) to the BDO pathway intermediates (e.g., succinyl-CoA).

CD Biosynsis offers a complete metabolic pathway engineering solution to overcome these challenges. Our strategy includes: Pathway Design: Introduce a synthetic 4-Hydroxybutyryl-CoA (4-HB-CoA) pathway, requiring four heterologous enzymes, into E. coli . This bypasses TCA cycle bottlenecks by using Succinyl-CoA as an intermediate and directing flux to 4-HB (and then BDO). We further focus on Cofactor Balance: Optimize NADH/NADPH availability, which is critical for the final reduction steps . The final two BDO synthesis steps are reduction reactions highly dependent on NAD(P)H supply. Finally, we employ Enzyme Engineering: Directed evolution of the 4-HB-CoA reductase to improve efficiency and specificity . This key enzyme often has low activity or poor substrate specificity in heterologous hosts, limiting the overall flux to BDO.

Pain Points

Industrial BDO production faces these key challenges in bio-routes:

• Low Carbon Flux to Intermediate: The native TCA cycle is highly regulated, making it difficult to draw sufficient carbon flux to the initial BDO pathway intermediate, succinyl-CoA , limiting overall production.
• Cofactor Imbalance for Reduction: The final steps of BDO synthesis involve two successive NADPH or NADH-dependent reduction reactions . A lack of sufficient cofactor supply or the wrong ratio causes a bottleneck.
• Poor Enzyme Efficiency (4-HB-CoA reductase): The heterologous enzymes introduced into the host (specifically the 4-HB-CoA reductase and alcohol dehydrogenase) often have low specific activity or substrate promiscuity in the new cellular environment.
• Competing Native Pathways: Native metabolic routes (e.g., succinate fermentation pathways or acetate production) divert carbon away from BDO precursors, decreasing the theoretical yield .

A successful bio-BDO solution requires optimizing flux towards succinyl-CoA and maximizing the efficiency of the synthetic pathway steps.

Solutions

CD Biosynsis utilizes advanced metabolic and enzyme engineering to optimize BDO production:

Synthetic 4-HB-CoA Pathway Design

           

We engineer a high-flux synthetic pathway (e.g., succinyl-CoA to succinate semialdehyde to 4-hydroxybutyrate to BDO) using up to four optimized heterologous genes .

Cofactor Engineering (NADH/\text{NADPH)

We employ cofactor-switching or overexpress transhydrogenase (e.g., PntAB) or NAD-dependent enzymes to ensure adequate supply of NAD(P)H for final BDO reduction.

Directed Evolution of Key Enzymes

We use directed evolution to improve the catalytic efficiency and specificity of bottleneck enzymes, such as 4-HB-CoA reductase (4HB CoA DH) , for the host (E. coli or yeast).

Block Competing Pathways

We delete genes (e.g., adhE, ackA) that divert carbon flux to by-products (e.g., ethanol, acetate, succinate), and we engineer the TCA cycle to feed succinyl-CoA. [Image of High Conversion Efficiency Icon]