Team:Amoy/Description
DESCIPTION
L-tert-leucine is an unnatural amino acid which plays an important role in various industrial products, especially in pharmaceuticals. With its application, many methodologies were developed for its preparation in the past decades. Some of them show low conversion ratio and poor chiral selectivity.Thirty years ago, scientists developed enzymatic reductive amination to produce L-tert-leucine by using leucine dehydrogenase and formate dehydrogenase. This technology greatly improved the yield and excellent enantiomeric excess value of L-tert-leucine. It is regarded to be one of the most efficient routes. But due to the different activity of leucine dehydrogenase and format dehydrogenase, this method are not perfect (Figuer 1)[1]. The cofactor regeneration mei be broken in the reaction process.
Figure 1 The enzymatic reductive amination for synthesizing L-tert-leucine and the bug of this method
In order to solve this problem, we put forward many methods. The basic idea is variable control. We constructed plasmids which bear leucine dehydrogenase gene (leudh) and formate dehydrogenase gene (fdh). One plasmid which contains both leucine dehydrogenase gene and format dehydrogenase gene controls the copy numbers and the transformation efficiency. And the only variable is efficiency of ribosome binding sites or strength of promoters. So the first methord is changing different ribosome binding sites. because of the weak activity of formate dehydrogenase, we connected fdh with the strongest ribosome binding site from iGEM registry, which is numbered BBa_B0034. Then, we changed the ribosome binding site of leudh (Figure 2).
Figure 2 The series connected circuit
However, owing to different activity of leucine dehydrogenase and formate dehydrogenase, the NADH consumption rate does not equal to its regeneration. Therefore, it is necessary to add excess NADH. The cofactor NADH is a pretty expensive raw material, which will make the mass production of L-tert-leucine not cost-effective.
Figure 3 Different activities of leucine dehydrogenase and formate dehydrogenase
The whole plan is to regulate the efficiency of ribosome binding site. In other words, RBS, to control the strength of leudh and fdh. With the help of mathematical modeling, the most suitable efficiency of RBS of leucine dehydrogenase will be obtained. Consequently, the cofactor NADH can be self-sufficient as shown in this cycle. We have investigated lots of RBSs and tried to choose the most probable pair of RBS and we chose B0032， B0030 and B0034 for plasmid construction. Through this way, it is quite easier to control RBS strength in one circuit.
CONTACT US
Email: igemxmu@gmail.com
Website: 2015.igem.org/Team:Amoy
Address: Xiamen University, No. 422, Siming South Road, Xiamen, Fujian, P.R.China 361005
