Computational design of stable, soluble and highly active alcohol dehydrogenase 2 for NADPH regeneration

: 1 Nicotinamide adenine dinucleotide phosphate (NADPH), as a well-known cofactor, is 2 widely used in the most of enzymatic redox reactions, playing an important role in 3 industrial catalysis. However, the absence of a comparable method for efficient NADP + 4 to NADPH cofactor regeneration radically impairs efficient green chemical synthesis. 5 Alcohol dehydrogenase (ADH) enzymes, allowing the in situ regeneration of the redox 6 cofactor NADPH with high specific activity and easy by-product separation process, 7 are provided with great industrial application potential and research attention. 8 Accordingly, herein a NADP + specific ADH from Clostridium beijerinckii was selected 9 to be engineered for cofactor recycle, using an automated algorithm named Protein 10 Repair One-stop Shop (PROSS). The mutant CbADH-6M exhibited a favorable soluble 11 and highly active expression with an activity of 46.3 U/mL, which was 16 times higher 12 than the wild type (2.9 U/mL), and a more stable protein conformation with an 13 enhanced thermal stability: Δ 𝑇 1/260𝑚𝑖𝑛 = +3.6°C (temperature of 50% inactivation after 14 incubation for 60 min). Furthermore, the activity of CbADH-6M was up-graded to 15 2401.8 U/mL by high cell density fermentation strategy, demonstrating its industrial 16 potential. Finally, the superb efficiency for NADPH regeneration of the mutant enzyme 17 was testified in the synthesis of some fine chiral aromatic alcohols coupling with anther 18 ADH from Lactobacillus kefir (LkADH). 19


INTRODUCTION
The ability of enzymes to operate simply in aqueous systems in a highly efficient 2 manner makes them attractive environmentally benign synthetic reagents. However, 3 many classes of biocatalysts are not fully exploited, and their use in the large-scale 4 enzymatic synthesis of high added-value chemicals is often limited by the need for 5 expensive cofactors. Typical cofactor dependent enzymes are oxidoreductases, which 6 represent some 25% of all known enzymes (Liu & Wang, 2007), catalyzing about 30% 7 of the biotransformations in industry (Straathof et al., 2002), and the vast majority are 8 dependent on one of the two nicotinamide cofactors NADH or NADPH. Although this 9 two cofactors differ only by the 2'-phosphate group that is attached to the adenine ribose 10 in NADPH, they play a completely different function in nature. NADH is used almost 11 exclusively for oxidative degradations that eventually lead to production of ATP, 12 whereas NADPH is confined with few exceptions to the biosynthetic reactions (Carugo 13 & Argos, 1997), involving a spectrum of over 300 known, repeatedly-used reaction  U/mg at 25-40°C) (Peretz et al., 1997;Widdel & Wolfe, 1989) and easy by-product 20 separation process when using isopropanol as cheap H-donor ( Fig. 1), is therefore of 21 considerable commercial interest as a catalyst for NADPH regeneration in the synthesis 22 and/or biotransformation of valuable compounds. 23 However, the absence of industrializable ADH as an excellent NADPH regenerator  Although some ADHs can catalyze the synthesis of product and the regeneration of 28 cofactor simultaneously in a substrate-coupled system, the activities of the two 29 reactions hardly coordinate, not to mention the competitive inhibition of substrates.
Moreover, advanced enzyme engineering technologies are used to improve ADH 1 performances that are optimized to the chemical manufacturing process, such as 2 substrate specificity, enantioselectivity and catalytic activity, no exclusively NADPH 3 regenerating ADH was explored.

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Here an ADH from Clostridium beijerinckii (CbADH) was identificated with excellent 5 thermostability and high specific activity for NADPH regeneration. Aiming to improve 6 the poor heterologous expression level of CbADH (Peretz et al., 1997), an automated 7 structure-and sequence-based computational protein redesign, namely Protein Repair CbADH to obtain better mutants that could satisfy laboratory and even industrial 10 application. Furthermore, a two-phase high cell density fermentation strategy was 11 explored to the large-scale production of the best mutant, to demonstrate its industrial 12 potentiality. Last but not least, we showed the enzyme's efficacy at in situ NADPH   Culture and induction conditions 9 The recombinant E. coli cells were first cultured for 6-8 hours at 37℃ in 5 mL Luria-   15 The CbADH related parameters and protein crystal structure were submitted online on   The induced cells were harvested by centrifugation and washed three times using 23 deionized water. Finally, the harvested cells were resuspended in 0.1 M phosphate 24 buffer (pH 7.5) and disrupted ultrasonically. The resultant slurry was used for enzyme 25 assay. 26 The standard assay mixture (1 mL) consisted of 0.1 M phosphate buffer (pH 7.5), 50 27 mM isopropyl alcohol, 1 mM NADP+, and enzyme. The substrate and coenzyme 28 solution were incubated in a metal bath at 35℃ and 650rpm for 10 min. Once the 1 enzyme solution was added, the reaction solution was scanned at 340nm by 2 spectrophotometer for 60 second, and the change in absorbance was recorded to 3 calculate the enzyme activity. One unit of enzyme activity was defined as the amount 4 of enzyme required to catalyze the formation of 1 μmol NADPH per minute.   Optimum temperature and temperature stability: The temperature dependence was 26 determined over the range 15-75°C. The mixture except for enzyme was preincubated 27 for 10 min at a serious of temperature, and the reaction was initiated by the addition of 28 enzyme liquor. The thermal stability was measured through determination of T50 60 , the temperature where the enzyme activity is reduced to 50% of its initial activity after 1 incubation for 60 min. 2 Optimum pH and pH stability: The optimum pH was determined at 35°C using different 3 buffer systems to cover the pH scale: Phosphate buffer (pH 6.0-7.5), Tris-HCl buffer 4 (pH 7.5-9.0), Glycine-NaOH buffer (pH 9.5-11.0), Na2HPO4 buffer (pH 11.0-13.0). The 5 pH stability was measured by incubation of purified enzyme in buffer with different pH 6 for 24 h at 35°C, and the residual activity was then measured by standard assay. High-density fermentation 9 Single colonies were selected from the plate and incubated in 50 mL fermentation

Results and discussion
1 Alcohol dehydrogenase library construction and activity identification 2 So far, NADPH-specific ADH (EC 1.1.1.2) from different sources has been reported a 3 lot according to the enzyme database BRENDA, but the high-activity ADH is not much.

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Thus, an ADH library was constructed by selecting the one with specific activity 5 exceeding about 10 U/mg protein (Table S1). However, some members of this group 6 (though highly-active) are not appropriate as NADPH regenerator , due to their function 7 of oxidization only primary alcohols that will be transformed to aldehydes to vitiate 8 enzyme proteins. The others that can act also on secondary alcohols were selected to 9 further investigate. Soluble expression levels and NADPH regenerating activities (per 10 volume of fermentation broth) of these secondary ADHs were then tested except for 11 MtADH (Fig. S1), which necessitates unavailable F420 as cofactor (Widdel & Wolfe,12 1989). All the left six enzymes were successfully expressed in E. coli (Fig. S2) and 13 considerable activities of TgADH, CbADH, TbADH and LkADH were obtained (Fig.   14   2A). Among them, TgADH exhibited a highest activity but a vulnerable stability in 15 atmosphere, and LkADH was also demonstrated to be unstable than CbADH (Fig. 2B). 16 CbADH showed the second highest activity (2.9 U/mL) with a soluble enzyme protein 17 ratio of 25.1%, which was the lowest and less than a quarter of TbADH (96.9%), 18 indicating that the specific activity of CbADH was supreme. Considering the activity 19 and stability, CbADH was testified to be the best choice for NADPH regeneration, 20 despite that the heterogenous expression of CbADH urgently needed to be improved. the total enzyme activity increased slightly, but the proportion of soluble enzyme 7 protein decreased ( Fig. 3A and Fig. S4). Additionally, the induction temperature had 8 little influence on the activity and soluble enzyme-protein ratio (Fig. 3B and Fig. S3). 9 The maximum enzyme activity was only 3.7 U/mL with the lowest soluble target protein ratio of about 59.6% (Fig. S5 and S6). Subsequently, the chaperone co-   First of all, functionally relevant key sites, including Zn 2+ and cofactor ligand binding 27 sites, and dimer interface sites were excluded from mutation ( Fig. S7 and Table S3). 28 After three rounds of online calculations, five random mutations were selected to test functionally. Synthetic genes encoding wild-type CbADH (CbADH-WT) and the five 1 designs were expressed in E. coli BL21 (DE3), and the results was showed that 2 mutations except CbADH-24M were solubly and actively expressed much better than 3 the wild-type CbADH, and the soluble enzyme protein ratio and activity dramatically 4 decreased with the number of mutation sites increased (Fig. 4A and Fig. S8), that was 5 different from Goldenzweig's work. What' more, thermal stability of three mutations,  (Fig. 4B). The terrific design CbADH-6M was tested to 9 possess an expressional activity of 46.3 U/mL, which was 15 fold higher than the nature 10 enzyme, and a soluble enzyme protein ratio of 82.4% (Fig. 4C). Although completely 11 soluble expression was not achieved by sequence redesign of CbADH, it is 12 demonstrated that PROSS was an simple and applicable method for computational 13 design of stable and soluble biocatalysts.      The activity of purified CbADH-6M was measured at various temperature ranging from 9 15 to 75℃. As the temperature increased, the enzyme activity increased constantly, reaching the highest at 65℃ (Figure. 7A). Thermostability of the purified CbADH-1 6M was investigated at temperatures of 50, 60 and 70℃. It can be seen from the 2 deactivation curve ( Figure. 7B), the enzyme had half-lives ( 1/2 60 ) of 62.4, 4.9 and 3 0.4h at 50, 60 and 70℃ (Table 1), respectively. The optimum pH was determined by 4 measuring the enzyme activities at different pH from 6.0-13.0 ( Figure. 7C). The 5 maximum activity was observed at pH 9.5(Glycine-NaOH). In the case of pH stability,    employed in the synthesis of some fine chiral aromatic alcohols ( Figure. 8A). As can 4 be seen in the reduction reaction for the synthesis of (S)-1-phenylethanol, if only 5 LKADH was used, the reaction rate was slow, and the conversion rate ( Figure. 8B hours. Not only in the production of (S)-1-phenylethanol, but also in the synthesis of 10 other important chiral aromatic alcohols ( Table 2), LkADH&CbADH exhibited 11 excellent stereoselectivity with a ee value over 99% and the conversion rate of reaction 12 was mostly above 99% much higher than LkADH only, indicating the great potential 13 for industrial application.   CbADH with better stability and higher specific activity was screened for NADPH 2 regeneration, In this work, a computational protein design named PROSS was applied 3 for the soluble modification of target enzyme. The multipoint mutant CbADH-6M 4 displayed a superb solubility, which was 18 times that of the wild type. Meanwhile the 5 enzyme activity of CbADH-6M reached 46.3 U/mL (wild type 2.9 U/mL). In addition, 6 the multipoint superimposing effect improves the conformational stability of the protein, 7 as well as the thermal stability and pH stability. When coupling CbADH with LkADH, 8 the catalytic rate of the reduction reaction was greatly improved and the conversion rate 9 was significantly higher than that of the LkADH reaction with 99% conversion. The ee 10 value of the product in the final reaction mixture was ＞ 99%, showing strict 11 stereoselectivity. This ADH mutant appears to an attractive biocatalyst for the 12 asymmetric synthesis of chiral alcohols.

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Availability of data and materials 15 The data and materials in this work are available from the corresponding author on 16 reasonable request.   Availability of data and materials 11 The data and materials in this work are available from the corresponding author on 12 reasonable request.

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Ethics approval and consent to participate 15 Not applicable.

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Consent for publication 18