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DESCIPTION

L-tert-leucine is an unnatural amino acid which plays an important role in various industrial products, especially in pharmaceuticals.
With its importance, many methodologies were developed for its preparation in the past decades. Scientists have 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].

Figure 1 The enzymatic reductive amination for synthesizing L-tert-leucine and the bug of this method

To optimize this circle, we have tried many methods. Finally we constructed one genetic circuit containing two target genes and use whole-cell catalysts. There are several advantages using this method. Firstly, none or only very little external cofactor is required, because it is already contained in the whole-cell biocatalyst with the help of cofactor regeneration. Secondly, only one fermentation is required to produce the biocatalyst compared to two separate fermentations to clone cells containing leudh and fdh respectively, which is much more convenient. What’s more, the biocatalyst is suitable for high-cell density fermentations. No isolation or purification of enzymes are required so that the cost become lower. Last but not least, whole-cell catalysts can achieve the effect of premix, which help to make the two enzymes cooperate well. In a word, the method we adopt not only can lower the cost but also makes it easy for us to adjust the expression level of the two target genes.

Figure 2 The circuit constructed

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.