Enzymatic synthesis of cyclic imino acids

Optically active cyclic imino acids are widely used as important pharmaceutical intermediates and it is necessary to develop cost effective synthetic method for their production. We have already established one-pot synthesis of L-cyclic imino acids from diamino acids by using N-methyl-L-amino acid dehydrogenase (NMAADH) from Pseudomonas putida. In order to make this process more efficient, we established a recombinant Escherichia coli which expresses NMAADH, lysine racemase from P. putida and D-lysine dehydrogenase from Selenomonas ruminantium in a single cell. The recombinant E. coli makes it possible to recycle NADPH by combination of reductive reaction using NMAADH and oxidative reaction using D-lysine dehydrogenase (Fig.1). Please click Additional Files below to see the full abstract

Driving Forces Enable High-Titer Anaerobic 1-Butanol Synthesis in Escherichia coli▿†

1-Butanol, an important chemical feedstock and advanced biofuel, is produced by Clostridium species. Various efforts have been made to transfer the clostridial 1-butanol pathway into other microorganisms. However, in contrast to similar compounds, only limited titers of 1-butanol were attained. In this work, we constructed a modified clostridial 1-butanol pathway in Escherichia coli to provide an irreversible reaction catalyzed by trans-enoyl-coenzyme A (CoA) reductase (Ter) and created NADH and acetyl-CoA driving forces to direct the flux. We achieved high-titer (30 g/liter) and high-yield (70 to 88% of the theoretical) production of 1-butanol anaerobically, comparable to or exceeding the levels demonstrated by native producers. Without the NADH and acetyl-CoA driving forces, the Ter reaction alone only achieved about 1/10 the level of production. The engineered host platform also enables the selection of essential enzymes with better catalytic efficiency or expression by anaerobic growth rescue. These results demonstrate the importance of driving forces in the efficient production of nonnative products