Results

NK is toxic to yeast but NA is not.


       By establishing a gradient plate, we found that NK is toxic for yeast, with little yeast growth already in the region of about 3 g/L. However, the product NA was not toxic to yeast and colonies were still growing even in the area up to 10 g/L. This predicts that high-throughput screening is feasible for the experiment.（Fig. 1）

Fig. 1. Toxicity analysis of substrates and products

Target plasmid and strain construction


       We constructed the target plasmids by homologous recombination. And we transferred into yeast cells in the order of r-p426/r-p415/r-YEGAP.
       We designed a total of five WT mutants, named TBR1/TBR2/TBR3(R-type) and TBS1/TBS2, where the genes of TBR2/TBR3/TBS2 were obtained by purchase and the genes of TBR1 and TBS1 were completed by targeted mutation.

Fig. 2. Target plasmid construction

       We also successfully reconstituted the glucose dehydrogenase from Bacillus subtilis and transformed into E. coli BL21 for induction treatment. For practical application reasons, the GDH used in the experiments were all cell lysis supernatants.

Fig. 3. GDH Recombination.

Protein Expression


       We successfully expressed the rationally designed and wild-type proteins, which were isolated and purified by nickel columns.

Fig. 4. SDS-PAGE

Influence of temperature on enzyme activity


       The conversion of NADPH to NADP+ was catalyzed using ethanol dehydrogenase, with the former having an absorption peak at 340 nm and the latter not. The change in absorbance at 340 nm within 7 min of the reaction system was detected. The protein concentration was determined by the Komas Brilliant Blue colorimetric method. The final conversion was made to the enzyme activity per unit concentration. We can see that TBR1 has a significant increase in activity and heat resistance compared to WT.

Fig. 5 Influence of temperature on enzyme activity

GC-MS Analysis


       The results of GCMS analysis of WT and TBR1 is shown below.(Fig. 6)

Fig. 6 GCMS analysis of WT and TBR1.

Discussion


     According to N-Boc-3-pyrrolidinol global industry analysis to 2021, the global (R)-(-)-N-Boc-3-pyrrolidinol market size reached USD 4 million in 2020 and is expected to reach USD 9 million by 2027, at a compound annual growth rate (CAGR) of 8.4% (2021-2027). Therefore, the use of greener and more efficient enzymatic production has a very promising application.

     The EvolvR-based continuous targeting system is very promising because we have demonstrated through pre-experiments that NK to NA transformation can be used in this way to achieve high-throughput automated screening. However, due to time constraints, we did not achieve goals. However, during the design process, our proposed continuous directed evolution strategy for substrate and product toxicity is feasible and has implications for the development of other directed evolution topics.

     In constructing the reaction system, we identified several important reasons why this reaction is difficult to produce enzymatically.

     On the one hand, it is a matter of phase transfer. NK is insoluble in water and soluble in organic solvents, while NA is soluble in water. During the reaction we need to find a suitable phase transfer catalyst, otherwise the rate and the extent of the reaction will be greatly reduced. Another aspect is the problem of reaction cycle and temperature. TbsADH is a heat-resistant enzyme and its Tm value is around 65 °C. In order to achieve the recycling of NADPH industry often uses glucose dehydrogenase (GDH) to reduce NADP+ to NADPH again. But the optimum temperature of traditional GDH is around 40°C. The difference between the working temperature of these two enzymes is too large to the correct combination. To solve this problem, we also designed a thermal modification for GDH to better match TbsADH, while the higher temperature also facilitates the increase of chemical reaction rate. This part is not shown in the text. We will continue our work on directed evolution.

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