This is my project description.
L-tert-leucine is an important and attractive chiral building block. Owning to its bulky and hydrophobic tert-butyl side chain, this unnatural amino acid is widely used as chiral auxiliaries and catalysts in asymmetric synthesis in developing chiral pharmaceutically active chemicals. Many methods, such as strecker synthesis, amidocarbonylation and acetamidomolonic ester synthesis, have been used in L-tert-leucine synthesis, but products are usually racemic. In order to solve the problem, scientists developed enzymatic reductive amination to product L-tert-leucine by using leucine dehydrogenase and formate dehydrogenase. This technology greatly improve the yield and excellent enantiomeric excess of L-tert-leucine.
Initially, they used isolated enzymes, which can be disadvantageous for the reason that enzymes are always destabilized in the isolation and purification process. What's more, the cofactor-NADH is rather an expensive raw material, which will enhance the cost of L-tert-leucine production. So scientists introduced whole-cell biocatalysts to L-tert-leucine production. Whole-cell biocatalysts could stabilize enzymes and reduce the addition level of cofactor NADH.
However, owing to different strength of leucine dehydrogenase and formate dehydrogenase, the NADH consumption rate does not equal to its regeneration. Therefore, it is necessary to add excess NADH. Many different methods have been used, but none of them made difference. Because the criminal is different strength of enzymes, we want to regulate the efficiency of ribosome binding site (RBS) to control the strength of leucine dehydrogenase. With the help of mathematical modeling, we will get the most suitable efficiency of RBS of leucine dehydrogenase. As a result, the addition of excess NADH could be decreased.