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DESCIPTION

L-tert-leucine, an unnatural amino acid, plays an important role in the pharmaceutical, agrochemical, food additives and cosmetic industry. With its special importance, many methodologies, including chemical and biological resolutions, were developed for its preparation in the past decades. Chemical resolution could be easily carried out on a large scale, however, the tedious process in low yield and the difficulties in the racemization of chiral separation were also observed [1]. Biocatalytic protocols, which can be conducted under mild conditions with high chiral selectivity, usually offer greater benefits than chemical procedures and thus gain more and more attention from organic chemists. However, most of these biological resolution procedures are tedious and possess an inherent 50% yield limit [2].

Figure 1. Comparison of biological method and chemical method of L-tert-leucine synthesis

In order to solve the problem, scientists developed enzymatic reductive amination to produce L-tert-leucine by using leucine dehydrogenase and formate dehydrogenase [3]. This technology greatly improve the yield and excellent enantiomeric excess of L-tert-leucine. It is regarded to be one of the most efficient routes.

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 an pretty expensive material, which will make the mass production of L-tert-leucine not cost-effective.

The circuits with gene LeuDH and with gene FDH were inserted into two E.coli cells separately. Then they add different wet biomass of two E.coli cells [4]. They hope to keep the activity of two enzyme equal through this method. Researchers using isolated enzymes find it disadvantageous because enzymes are easily destabilized in the isolation and purification process.

Figure 2. Circuits inserted into two E.coli cells separately

Then researchers plan to use whole-cell biocatalyst to stabilize enzymes and reduce the need of cofactor NADH. They envisaged that a promising strategy for a successful co-expression could be based on the same inducible promoter for both genes but located on one E.coli plasmids with different copy numbers, producing LeuDH and FDH on different level. FDH was inserted in the plasmid with the higher copy number plasmid, while LeuDH was inserted in the medium copy number one. They hope to regulate the copy number of plasmid to ensure the continuous recycling of the cofactor NADH. Presumably, this was achieved by a higher production of FDH compared to LeuDH due to the higher copy number vector for the FDH gene. Furthermore, this LeuDH/FDH-strain is suitable for high-cell density fermentation. Compared with isolated enzymes，whole cell-catalyzed asymmetric process has many advantages, such as simple, efficient, environmentally and economically attractive. However, researchers also find that using substrate concentrations of 500mM or higher, revealed a non-satisfactory reaction course, indicating significant inhibitions effects. The activity of two enzymes are both inhibited [1]. Obviously, successful coexpression of two genes is still a challenge for scientists.

Owing to the low activity of FDH, scientists have tried a variety of methods recently. Some scientists used mutational FDH or FDH from other bacterial strains to take the place of FDH from Candida boidinii [5,6]. And some other scientists chose glucose dehydrogenase (GDH) to regenerate NADH [7,8]. However, in our opinion, none of these methods made a difference. Cofactor regeneration with the FDH from Candida boidinii is a stable system which has been used for many years. Mutational FDH or FDH from other bacterial strains may be unstable and difficult to be introducted into industrial production. And as for the GDH, the substrate is glucose and the product is gluconate. If we apply LeuDH and GDH into industrial production, the cost must be raised owing to the excess substate and purification process.

So when we started to do our project though biological methods, the problems are clear. One is researchers find it disadvantageous using isolated enzymes because enzymes are easily destabilized in the isolation and purification process. What's more, owing to different activities of leucine dehydrogenase and formate dehydrogenase, the NADH consumption rate does not equal to its regeneration. Therefore, it is necessary to add excess NADH. Whereas the cofactor-NADH is a pretty expensive raw material, which will make the mass production of L-tert-leucine not cost-effective. Given all these problems we plan to insert leudh and fdh to one circuit and use whole-cell biocatalyst to stabilize enzymes.

Figure 3. Different activities of LeuDH and FDH

But why do we choose one circuit containing two genes and use whole-cell catalysts? Firstly, 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. Secondly, transforming two plasmids with different resistance to one bacterium will make the E.coli hard to grow. Thirdly, the biocatalyst is suitable for high-cell density fermentations. No isolation and purification of the enzymes is required. Fourthly, whole-cell catalysts can achieve the effect of premix, which help to make the two enzymes cooperate well. Fifthly, it’s good for us to control variable in one cell. Last but not least, the most attractive thing is none or only very little external cofactor is required, because it is already contained in the whole-cell biocatalyst. In a word, the method we adopt not only can save a lot of cost but also can make it easy for us to adjust the expression level of 2 genes [8,9,10].

Here comes our project. The whole plan is to regulate the efficiency of ribosome binding site (RBS) to control the activity of leucine dehydrogenase and formate dehydrogenase. With experimental results, the most suitable efficiency of RBS of leucine dehydrogenase will be obtained. Consequently, the cofctor NADH can be self-sufficient as shown in this cycle.

To achieve our goal, we got two gene circuits, one contains LeuDH gene and the other one contains FDH gene. We combined them together and transformed the whole circuit into E.coli so that two genes can express independently according to the RBS strength we arranged. Then, the E.coli will be broken and two enzymes are realeasd. At that moment we add the substrate trimethylpyruvic acid (TMP) and ammonium formate to the system, so the cyclic catalysis begins and the L-tert-leucine will be produced.

Figure 4. Project flowchart view

We have totally constructed 3 circuits which contain different RBS pairs. Because the catalytic efficiency of FDH is much more weaker, we use the most strong RBS B0034 for FDH gene, and we change the RBS for LeuDH gene B0032, B0030 and B0034. As we expect, the data of characterization showed the circuit with B0030 and B0034 has the highest expression efficiency.

Figure 5. Gene circuits we have constructed

Reference:

[1] Gröger, H., May, O., Werner, H., Menzel, A., & Altenbuchner, J. A “Second-Generation Process” for the Synthesis of L-Neopentylglycine: Asymmetric Reductive Amination Using a Recombinant Whole Cell Catalys. Org. Process Res. & Dev.10, 666-669 (2006)
[2] Hong, E., Y., Cha, M., Yun, H. & Kim, B., G. Asymmetric synthesis of L-tert-leucine and L-3-hydroxyadamantyglycine using branched chain aminotransferase. J. Mol. Catal. B-Enzym. 66, 228-233 (2010)
[3] Gröger, H., May, O., Werner, H., Menzel, A., & Altenbuchner, J. A “Second-Generation Process” for the Synthesis ofL-Neopentylglycine:Asymmetric Reductive Amination Using a Recombinant Whole Cell Catalys. Org. Process Res. & Dev.. 10,666-669 (2006)
[4] Menzel, A., Werner, H., Altenbuchner, J., Gröger, H. From enzymes to "designer bugs" in reductive amination: A new process for the synthesis of L-tert-leucine using a whole cell-catalyst. Eng. Life Sci. 4, 573-576, (2004)
[5] Liu,W., Ma, H., Luo, J., Shen, W., Xu, X., Li, S., Hu,Y. & Huang, H. Efficient synthesis of l-tert-leucine through reductive amination using leucine dehydrogenase and formate dehydrogenase coexpressed in recombinant E. coli. Biochem. Eng. J. 91, 204–209 (2014)
[6] Bai, J., Song, Y., Luo, X., Yang, H., Du, W., & Zhang, T. Construction of L-tert-Leucine Producing Strain by Expressing Heterologous Leucine Dehydrogenase and Formate Dehydrogenase in Escherichia coli. Advances in Applied Biotechnology. 21-30 (2015)
[7] Shioiri, T., Izawa, K. & Konoike, T. Application of Whole‐Cell Biocatalysts in the Manufacture of Fine Chemicals. Pharmaceutical Process Chemistry.184-205 (2011)
[8] 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)
[9] Zhong, J., J., Chang, D. & L., Zhang, J. Discovery and application of new bacterial strains for asymmetric synthesis of L-tert-butyl leucine in high enantioselectivity. Appl. Biochem. Biotechnol. 164, 376–385 (2011)
[10] Liu, W., Luo, J., Zhuang, X., Shen, W., Zhang, Y., Li,SH., Hu, Y. & Huang, H. Efficient preparation of enantiopure L-tert-leucine through immobilized penicillin G acylase catalyzed kinetic resolution in aqueous medium. Biochem. Eng. J. 83, 116-120 (2014)