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 methods were developed for its preparation in the past decades. Some of them have low conversion rate 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 is not perfect(Figuer 1) [1]. The cofactor regeneration may be broken in reaction processes.

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 method 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) [2].

Figure 2 Method 1: changing ribosome binding site to regulate the activity of leucine dehydrogenase

Optimizing L-tert-leucine synthesis by changing the promoter is an alternative method. Promoter is also an important factor of translation process. So the second method is changing different promoters and finding out the optimal promoter.

However, the parts of different ribosome binding sites and promoters which we got from the iGEM authority cannot meet our demands, because these parts are not continuously adjustable. That means we may not be able to get optimal RBS or promoter for leudh. However, we were illuminated by the project did by Peking University in 2011. We get the idea that we could use genetic rheostats as RBS regulators. Then, we could get the excellent RBS efficiency and use RBS calculators to edit the RBSs' sequence to a suitable efficiency(Figure 3) [3,4].

Figure 3 Method 3: Using genetic rheostat to regulate the activity of leucine dehydrogenase

Reference:

[1] Li, J., Pan, J., Zhang, J. & Xu, H., J. Stereoselective synthesis of L-tert-leucine by a newly cloned leucine dehydrogenase from Exiguobacterium sibiricum. J. Mol. Catal. B-Enzym. 105, 11-17 (2014)
[2] http://https://2010.igem.org/Team:Warsaw/Stage1
[3] https://2011.igem.org/Team:Peking_R/Project/RNAToolkit
[4] Reeve, B., Hargest, T., Gilbert, C. & Ellis T. Predicting translation initiation rates for designing synthetic biology. Mini Review Article. 2, 1-6 (2014)