acryloyl-coenzyme-a and acrylic-acid

Topics: Acrylates; Acyl Coenzyme A; Amino Acid Sequence; Carbon-Sulfur Lyases; Inactivation, Metabolic; Oxidoreductases; Rhodobacteraceae; Roseobacter; Sulfides; Sulfonium Compounds; Sulfur

Acrylic acid is an important industrial feedstock. In this study, a de novo acrylate biosynthetic pathway from inexpensive carbon source glycerol was constructed in Escherichia coli. The acrylic acid was produced from glycerol via 3-hydroxypropionaldehyde, 3-hydroxypropionyl-CoA, and acrylyl-CoA. The acrylate production was improved by screening and site-directed mutagenesis of key enzyme enoyl-CoA hydratase and chromosomal integration of some exogenous genes. Finally, our recombinant strain produced 37.7 mg/L acrylic acid under shaking flask conditions. Although the acrylate production is low, our study shows feasibility of engineering an acrylate biosynthetic pathway from inexpensive carbon source. Furthermore, the reasons for limited acrylate production and further strain optimization that should be performed in the future were also discussed.

Topics: Acrylates; Acyl Coenzyme A; Amino Acid Sequence; Biosynthetic Pathways; Chromosomes, Bacterial; Culture Media; Enoyl-CoA Hydratase; Escherichia coli; Glyceraldehyde; Glycerol; Industrial Microbiology; Mutagenesis, Site-Directed; Propane; Recombinant Proteins

Clostridium propionicum converts lactate to propionate (Cardon, B.P., and Barker, H.A. (1947) Arch. Biochem. Biophys. 12, 165-171). We have obtained a soluble system that carries out this conversion as well as the hydration of acrylate to lactate and the reduction of acrylate to propionate. 3-Pentynyl-CoA inhibits reduction of acrylate and lactate to propionate, but not hydration of acrylate to lactate by cell extracts. The conversion probably involves CoA esters. When [beta-2H3] lactate is used as a substrate, the rate of propionate formation is reduced 1.8-fold, and the methyl group of the resulting propionate has lost 1.4 deuterium atoms. These results are consistent with the intermediate formation of acrylate (acrylyl-CoA) in the conversion of D-lactate to propionate. Two proteins, which we designate E I and E II, were purified to greater than 90% homogeneity. Together, they catalyze the hydration of acrylyl-CoA to lactyl-CoA. E I has an apparent molecular mass of 27,000 daltons and is rapidly and irreversibly inactivated by O2. E II consists of two subunits of molecular mass 41,000 and 48,000 daltons and contains equal amounts of riboflavin and flavin mononucleotide. Hydration of acrylyl-CoA to lactyl-CoA requires Mg2+ and catalytic quantities of ATP. GTP can replace ATP, but ADP and adenylyl imidodiphosphate cannot. We were unable to detect any stable intermediate during acrylyl-CoA hydration. Finally, we proposed a mechanism for this reaction.

Topics: Acrylates; Acyl Coenzyme A; Adenosine Triphosphate; Clostridium; Deuterium; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Hydro-Lyases; Kinetics; Lactates; Lactic Acid; Magnesium; Magnetic Resonance Spectroscopy; Molecular Weight; Oxidation-Reduction; Oxygen; Propionates; Spectrophotometry; Structure-Activity Relationship