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DISCUSSION

From March to September, nearly 200 days of learning and working, we made so much of mistakes in lab work but eventually obtained ideal data and results. From the results we had, we made following analysis of our system. See more data and results please click here.

Ⅰ. Whole-cell biocatalyst

Nowadays, there are mainly two methods applying to produce L-tert-leucine, isolated enzymes and whole-cell biocatalyst.

However, the use of isolated enzymes can be disadvantageous because of laborious isolation and purification of the enzymes and also often a reduced stability under process conditions. Then, because leucine dehydrogenase and formate dehydrogenase have different enzyme activities, we have to add different wet biomass of different E.coli [1]. If this method is applied into industrial production, it will cost more labors and machines. Furthermore, proteins expressions require more than twice raw materials by adopting this method. By choosing one circuit containing two genes, production becomes easier and cheaper because it simplify the production process and save costs. To begin with, there is not only no need to purify enzymes, but also can use host cells intensive mixing them and keep the enzyme activity. What`s more, only one fermentation required will save more time and raw materials [2].

As for the whole-cell biocatalyst, the coexpression was realized by location of the two related genes on two plasmids with different copy numbers, producing LeuDH and FDH at different levels. Obviously, compared with isolated enzymes, whole-cell biocatalysts could stabilize enzymes and reduce costs of production. However, whole-cell biocatalyst also has shortcomings. Firstly, it is inconvenient to transform two plasmids into one host cell. Secondly, we need to add two kinds of antibiotics into culture medium due to the different resistances of plasmids which will make host cells hard to grow. Thirdly, using substrate concentrations of 500mM or higher, revealed a non-satisfactory reaction course, indicating significant inhibitions effects. The activities of two enzymes are both inhibited [3,4]. By choosing one circuit containing two genes, we can obtain a more stable protein expression system and simplify the transformation process at the same time. More importantly, activities of two enzymes improve significantly.

In conclusion, our team optimize the above two methods through putting two genes into one plasmid. It only requires one fermentation run and simple cell separation as a concentration step. Meanwhile, activities of enzymes improve. In other words, with the help of our engineering bacteria, cost will be saved and output is expected to improve.

Ⅱ. Different strength of RBS

The main purpose of our project this year is to make cofactor NADH self-sufficient in our redox reactions by using different strength of RBS. The expected result is that we find the best ratio of the two enzymes working together, leading to the equivalence of consumption rate and regeneration rate. Ribosome binding site plays an important role in regulating the process of protein translation.Different RBSs bind ribosomes with different efficiencies.Clearly we can regulate the yield of the two enzymes needed-LeuDH and FDH by using various kind of RBSs.

Consequently,we constructed three different plasmids with three types of RBS, B0030,B0032 and B0034. B0034 is stronger than B0030 and B0032 is the weakest. The second enzyme FDH with lower enzyme activity is equipped with the strongest RBS-B0034, the regulation lies on the first enzyme-LeuDH.

Figure 1 Different Strength of RBS

As the result of HPLC shows,we made conclusions as follows.

Apart from taking all three whole circuits into analysis, we detected circuits with LeuDH or FDH separately. No matter which kind of RBS, its conversion rate does not show too much of change compared with each other, indicating that RBS control would not make an effect if the whole system is expressed separately.

Subsequently, let’s take a deep look at the difference of separated circuits and series connected circuits. Obviously, the conversion rate of series connected circuits is much higher than the other. As we mentioned on the whole-cell biocatalyst part, our project which make two genes into series connection would largely promote the productivity and conversion rate of L-tert-Leucine.

In the end, we are comfort to find rbs_B0030 make the best performance among these three kinds of RBS which indicate that the most suitable strength of rbs is located between RBS_B0034 and RBS_B0032.

However, the parts of different RBS we get from the iGEM registry cannot meet our demands,because these parts are not continuously adjustable.That means we may not be able to make the reaction cycle one hundred percent self-sufficient or in another way, no perfect effects of productivity.what should we do next will be discussed in the future work.

Ⅲ. Different Concentration of IPTG

IPTG(Isopropyl β-D-1-Thiogalactopyranoside)is known to be a highly stable molecule in solution at room temperature and in bacterial cultures under commonly used conditions for recombinant protein production [5].

After reading some relevant review articles, we found that IPTG (Isopropyl β-D-1-Thiogalactopyranoside) is an effective inducer of protein expression. For example, it can promote the expression of a thermostable amidase in recombinant E.coli the GST-GnRH/TRS gene [6,7]. So we design an experiment which using different concentration of IPTG to explore whether IPTG promotes the expression of our gene circuits or not. After experiments, we found that it can increase the expression of gene by determining the enzyme activity. And we found the optimal concentration of IPTG.

Figure 2 IPTG Induction of Isolated circuits

Figure 3 IPTG Induction of series connected circuits

Ⅳ. Adaptability of cofactor regeneration systems

Whole-cell biocatalysts based on cofactor regeneration system have showed board future throughout our project study [8]. Recently genetic engineering has made it possible to construct a new yeast strain to simplify the use of cofactor-requiring enzymes by introducing heterologous genes [9]. Thereby we demonstrate nearly all cofactor regeneration systems could be suitable under our framework.

For example, NADH Oxidase is a kind of oxidoreductase that catalyzes the oxidation of NADH by oxygen to yield H₂O and NAD⁺. NOX can be used to the regeneration of NADH. glycerol dehydrogenase is an enzyme in the oxidoreductase family that utilizes the NAD⁺ to catalyze the oxidation of glycerol to form glycerone (dihydroxyacetone). We will engineer fusion enzyme (GDH-NOX) of glycerol dehydrogenase and NADH oxidase. The fusion enzyme may successfully express in Escherichia coli and characterize.

A variety of potential useful enzymes require nicotinamide cofactors. And cofactor regeneration system played an important role in reducing the costs. Apart from the case of NADH Oxidase and glycerol dehydrogenase, our framework coupled with different enzymes could be perfectly optimized by different strengthen of ribosome binding site, which would be a huge progress in whole-cell biocatalyst.

Reference

[1] Shioiri, T., Izawa, K. & Konoike, T. Application of Whole‐Cell Biocatalysts in the Manufacture of Fine Chemicals. Pharmaceutical Process Chemistry.184-205 (2011)
[2] Kragl, U., Vasic-Racki, D.& Wandrey, C. Continuous production of L-tert-leucine in series of two enzyme membrane reactors. Bioprocess Eng. 14, 291-297 (1996)
[3] 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 catalysis. 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] Politi, N.,Pasotti, L., Zucca, S., Casanova, M., Micoli, G., M., Angelis, G., C., D. & Magni, P. Half-life measurements of chemical inducers for recombinant gene expression. J. Bio. Eng. 8, 1-22 (2014)
[6] Olaofea,O., A., Burtona, S., G., Cowanb, D. & Harrisona,A.,S., T., L. Improving the production of a thermostable amidase through optimising IPTG induction in a highly dense culture of recombinantEscherichia coli. Biochem. Eng. J.. 52. 19–24 (2010)
[7] Jin,Y., Liu, L., Su, X., et al.Effect of induce concentration, time and temperature of IPTG on the expression of the GST- GnRH/TRS gene. Heilongjiang Animal Science And Veterinary Medicine. 8,17-19 (2006)
[8]Stewart,JD.Organic transformations catalyzed by engineered yeast cells and related systems. Curr. Opin. Biotechnol. 11,363-368 (2000)
[9] Wichmann, R. & Vasic-Racki, D. Cofactor Regeneration at the Lab Scale. Adv. Biochem. Engin. Biotechnol. 92. 225–260 (2005)