Enhance Soluble Heteroexpression of a NADPH-Dependent Alcohol Dehydrogenase Based on the Chaperone Strategy
DENG Tong,ZHOU Hai-sheng,WU Jian-ping,YANG Li-rong
China Biotechnology ›› 2020, Vol. 40 ›› Issue (8) : 24-32.
Enhance Soluble Heteroexpression of a NADPH-Dependent Alcohol Dehydrogenase Based on the Chaperone Strategy
Objectives: Many of biocatalytic redox reactions which are widely used in the production of chiral chemicals involve the regeneration of the coenzyme NADPH in situ. Alcohol dehydrogenases that regenerate NADPH with isopropanol as substrate have the advantages of high specific activity and easy separation of byproduct acetone, attracting more and more attention. Therefore, an alcohol dehydrogenase from Clostridium beijerinckii, namely CbADH, was chosen as the research object for its more considerable specific activity and the most applicable potentiality within present literatures. To solve the problem of poor soluble expression of CbADH in E. coli genetically engineered strains and the consequent enzyme activity as low as 2.31 U / mg DCW, the following studies were carried out. Methods: Firstly, different chaperone proteins were expressed by inducible plasmids to increase the soluble expression level of CbADH, and the results showed that molecular chaperone GroES-GroEL significantly improved the soluble expression of CbADH by 3.57 times more than the original strain, with enzyme activity of 11.18 U/mg DCW which is 4.83 times more than the original strain. Secondly, three other different GroES-GroEL expression strategies were examined: pET-28a(+) single plasmid co-expression, genomic enhancing expression of chaperone, and constitutive-pGro7/pET-28a(+) dual plasmid co-expression. Results: The results indicated that the constitutive-pGro7/pET-28a(+) dual plasmid co-expression strategy had the best effect which improved the soluble expression of CbADH by 8.07 times more than the oringinal strain, with a CbADH activity of 21.79 U/mg DCW, which was 9.43 times higher than the oringinal strain. Conclusions: This study not only lays the foundation for the industrial application of CbADH but also provides a reference for heterologous soluble protein expression.
Alcohol dehydrogenase / Soluble heteroexpression / Chaperone / NADPH {{custom_keyword}} /
Table 1 Information of molecular chaperone plasmid表1 分子伴侣质粒信息 |
分子伴侣质粒 | 伴侣蛋白 | 启动子 | 诱导剂 | 抗性 |
---|---|---|---|---|
pG-KJE8 | DnaK-DnaJ-GrpE-GroES-GroEL | araB Pzt-1 | 0.5g/ml L-Arabinose+5ng/ml Tetracyclin | Cm |
pGro7 | GroES-GroEL | araB | 0.5g/ml L-Arabinose | Cm |
pKJE7 | DnaK-DnaJ-GrpE | araB | 0.5g/ml L-Arabinose | Cm |
pG-Tf2 | GroES-GroEL-Tf | Pzt-1 | 5ng/ml Tetracyclin | Cm |
pTf16 | Tf | araB | 0.5g/ml L-Arabinose | Cm |
Table 2 Information of constituent promoters表2 组成型启动子信息①(① 组成型启动子具体信息来源于http://parts.igem.org/Promoters/Catalog/Anderson) |
启动子名称 | 序列 | 相对强度 |
---|---|---|
J23119 | ttgacagctagctcagtcctaggtataatgctagc | n/a |
J23100 | ttgacggctagctcagtcctaggtacagtgctagc | 1 |
J23102 | ttgacagctagctcagtcctaggtactgtgctagc | 0.86 |
J23104 | ttgacagctagctcagtcctaggtattgtgctagc | 0.72 |
J23108 | ctgacagctagctcagtcctaggtataatgctagc | 0.51 |
J23110 | tttacggctagctcagtcctaggtacaatgctagc | 0.33 |
J23114 | tttatggctagctcagtcctaggtacaatgctagc | 0.10 |
Fig.3 SDS-PAGE analysis of molecular chaperones co-expression strainsLane M:molecular weight marker;Lane W:whole cell protein; Lane S:supernatant;Lane P:precipitation 图3 分子伴侣共表达菌株的SDS-PAGE分析 |
Table 3 CbADH activity of different strategies表3 不同策略的CbADH发酵液酶活测定结果 |
采用策略 | 菌株名称 | OD600 | 酶活(U/mg DCW) |
---|---|---|---|
出发菌株 | CbADH-WT | 5.91±0.34 | 2.31±0.3 |
分子伴侣共表达 | A | 6.24±0.38 | 11.18±0.96 |
B | 5.27±0.2 | 3.61±0.21 | |
C | 5.17±0.31 | 3.98±0.14 | |
D | 7.19±0.24 | 4.17±0.35 | |
E | 5.97±0.23 | 2.88±0.09 | |
单质粒共表达 | F | 5.4±0.24 | 10±0.35 |
G | 5.23±0.32 | 7.89±0.46 | |
H | 4.88±0.28 | 8.02±0.43 | |
I | 5.09±0.19 | 12.99±0.89 | |
基因组强化表达 | J | 5.23±0.35 | 9.13±0.31 |
K | 5.31±0.37 | 7.26±0.18 | |
L | 5.28±0.25 | 2.3±0.24 | |
组成型双质粒共表达 | M | 5.08±0.41 | 21.79±1.1 |
N | 5.44±0.24 | 17.74±0.92 | |
O | 5.43±0.32 | 16.8±0.42 | |
P | 5.56±0.21 | 15.57±0.52 | |
Q | 5.79±0.13 | 15.08±0.46 | |
R | 5.55±0.32 | 14.82±0.32 | |
S | 5.32±0.26 | 12.35±0.67 |
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AbstractNAD(P)-dependent oxidoreductases are valuable tools for the synthesis of chiral compounds. Due to the high cost of the pyridine cofactors, in situ cofactor regeneration is required for preparative applications. In recent years, existing regeneration methodologies have been improved and new approaches have been devised. These include the use of newly discovered dehydrogenases that are stable in high contents of organic solvent and novel enzymes that can regenerate either the reduced or oxidized forms of the cofactor. The use of electrochemical methods has allowed cofactor regeneration for monooxygenases and natural or engineered whole-cell systems provide alternatives to approaches relying on purified enzymes. {{custom_citation.content}}
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The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (R)-4-chloro-3-hydroxybutanoate [(R)-CHBE] using Escherichia coli cells, which coexpress both the aldehyde reductase gene from Sporobolomyces salmonicolor and the glucose dehydrogenase (GDH) gene from Bacillus megaterium as a catalyst was investigated. In an organic solvent-water two-phase system, (R)-CHBE formed in the organic phase amounted to 1610 mM (268 mg/ml), with a molar yield of 94.1% and an optical purity of 91.7% enantiomeric excess. The calculated turnover number of NADP+ to CHBE formed was 13,500 mol/mol. Since the use of E. coli JM109 cells harboring pKAR and pACGD as a catalyst is simple, and does not require the addition of GDH or the isolation of the enzymes, it is highly advantageous for the practical synthesis of (R)-CHBE.
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An alcohol dehydrogenase (ADH) from hyperthermophilic archaeon Thermococcus guaymasensis was purified to homogeneity and was found to be a homotetramer with a subunit size of 40 +/- 1 kDa. The gene encoding the enzyme was cloned and sequenced; this gene had 1,095 bp, corresponding to 365 amino acids, and showed high sequence homology to zinc-containing ADHs and L-threonine dehydrogenases with binding motifs of catalytic zinc and NADP(+). Metal analyses revealed that this NADP(+) -dependent enzyme contained 0.9 +/- 0.03 g-atoms of zinc per subunit. It was a primary-secondary ADH and exhibited a substrate preference for secondary alcohols and corresponding ketones. Particularly, the enzyme with unusual stereoselectivity catalyzed an anti-Prelog reduction of racemic (R/S)-acetoin to (2R, 3R)-2,3-butanediol and meso-2,3-butanediol. The optimal pH values for the oxidation and formation of alcohols were 10.5 and 7.5, respectively. Besides being hyper-thermostable, the enzyme activity increased as the temperature was elevated up to 95 degrees C. The enzyme was active in the presence of methanol up to 40% (vol/vol) in the assay mixture. The reduction of ketones underwent high efficiency by coupling with excess isopropanol to regenerate NADPH. The kinetic parameters of the enzyme showed that the apparent K(m) values and catalytic efficiency for NADPH were 40 times lower and 5 times higher than those for NADP(+), respectively. The physiological roles of the enzyme were proposed to be in the formation of alcohols such as ethanol or acetoin concomitant to the NADPH oxidation.
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Formate dehydrogenases (FDHs) are frequently used for the regeneration of cofactors in biotransformations employing NAD(P)H-dependent oxidoreductases. Major drawbacks of most native FDHs are their strong preference for NAD(+) and their low operational stability in the presence of reactive organic compounds such as alpha-haloketones. In this study, the FDH from Mycobacterium vaccae N10 (MycFDH) was engineered in order to obtain an enzyme that is not only capable of regenerating NADPH but also stable toward the alpha-haloketone ethyl 4-chloroacetoacetate (ECAA). To change the cofactor specificity, amino acids in the conserved NAD(+) binding motif were mutated. Among these mutants, MycFDH A198G/D221Q had the highest catalytic efficiency (k cat/K m) with NADP(+). The additional replacement of two cysteines (C145S/C255V) not only conferred a high resistance to ECAA but also enhanced the catalytic efficiency 6-fold. The resulting quadruple mutant MycFDH C145S/A198G/D221Q/C255V had a specific activity of 4.00 +/- 0.13 U mg(-1) and a K m, NADP(+) of 0.147 +/- 0.020 mM at 30 degrees C, pH 7. The A198G replacement had a major impact on the kinetic constants of the enzyme. The corresponding triple mutant, MycFDH C145S/D221Q/C255V, showed the highest specific activity reported to date for a NADP(+)-accepting FDH (v max, 10.25 +/- 1.63 U mg(-1)). However, the half-saturation constant for NADP(+) (K m, NADP(+) , 0.92 +/- 0.10 mM) was about one order of magnitude higher than the one of the quadruple mutant. Depending on the reaction setup, both novel MycFDH variants could be useful for the production of the chiral synthon ethyl (S)-4-chloro-3-hydroxybutyrate [(S)-ECHB] by asymmetric reduction of ECAA with NADPH-dependent ketoreductases.
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Proteins play a pivotal role in thermophily. Comparing the molecular properties of homologous proteins from thermophilic and mesophilic bacteria is important for understanding the mechanisms of microbial adaptation to extreme environments. The thermophile Thermoanaerobacter (Thermoanaerobium) brockii and the mesophile Clostridium beijerinckii contain an NADP(H)-linked, zinc-containing secondary alcohol dehydrogenase (TBADH and CBADH) showing a similarly broad substrate range. The structural genes encoding the TBADH and the CBADH were cloned, sequenced, and highly expressed in Escherichia coli. The coding sequences of the TB adh and the CB adh genes are, respectively, 1056 and 1053 nucleotides long. The TB adh gene encoded an amino acid sequence identical to that of the purified TBADH. Alignment of the deduced amino acid sequences of the TB and CB adh genes showed a 76% identity and a 86% similarity, and the two genes had a similar preference for codons with A or T in the third position. Multiple sequence alignment of ADHs from different sources revealed that two (Cys-46 and His-67) of the three ligands for the catalytic Zn atom of the horse-liver ADH are preserved in TBADH and CBADH. Both the TBADH and CBADH were homotetramers. The substrate specificities and thermostabilities of the TBADH and CBADH expressed inE. coli were identical to those of the enzymes isolated from T. brockii and C. beijerinckii, respectively. A comparison of the amino acid composition of the two ADHs suggests that the presence of eight additional proline residues in TBADH than in CBADH and the exchange of hydrophilic and large hydrophobic residues in CBADH for the small hydrophobic amino acids Pro, Ala, and Val in TBADH might contribute to the higher thermostability of the T. brockii enzyme.
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We have determined the X-ray structures of the NADP(H)-dependent alcohol dehydrogenase of Clostridiim beijerinckii (CBADH) in the apo and holo-enzyme forms at 2.15 A and 2.05 A resolution, respectively, and of the holo-alcohol dehydrogenase of Thermoanaerobacter brockii (TBADH) at 2.5 A. These are the first structures of prokaryotic alcohol dehydrogenase to be determined as well as that of the first NADP(H)-dependent alcohol dehydrogenase. CBADH and TBADH 75% have sequence identity and very similar three-dimensional structures. Both are tetramers of 222 symmetry. The monomers are composed of two domains: a cofactor-binding domain and a catalytic domain. These are separated by a deep cleft at the bottom of which a single zinc atom is bound in the catalytic site. The tetramers are composed of two dimers, each structurally homologous to the dimer of alcohol dehydrogenases of vertebrates. The dimers form tetramers by means of contacts between surfaces opposite the interdomain cleft thus leaving it accessible from the surface of the tetramer. The tetramer encloses a large internal cavity with a positive surface potential. A molecule of NADP(H) binds in the interdomain cleft to the cofactor-binding domain of each monomer. The specificity of the two bacterial alcohol dehydrogenases toward NADP(H) is determined by residues Gly198, Ser199, Arg200 and Tyr218, with the latter three making hydrogen bonds with the 2'-phosphate oxygen atoms of the cofactor. Upon NADP(H) binding to CBADH, Tyr218 undergoes a rotation of approximately 120 degrees about chi1 which facilitates stacking interactions with the adenine moiety and hydrogen bonding with one of the phosphate oxygen atoms. In apo-CBADH the catalytic zinc is tetracoordinated by side-chains of residues Cys37, His59, Asp150 and Glu60; in holo-CBADH, Glu60 is retracted from zinc in three of the four monomers whereas in holo-TBADH, Glu60 does not participate in Zn coordination. In both holo-enzymes, but not in the apo-enzyme, residues Ser39 and Ser113 are in the second coordination sphere of the catalytic zinc. The carboxyl group of Asp150 is oriented with respect to the active carbon of NADP(H) so as to form hydrogen bonds with both pro-S and pro-R hydrogen atoms.
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Previous research in our laboratory comparing the three-dimensional structural elements of two highly homologous alcohol dehydrogenases, one from the mesophile Clostridium beijerinckii (CbADH) and the other from the extreme thermophile Thermoanaerobacter brockii (TbADH), suggested that in the thermophilic enzyme, an extra intrasubunit ion pair (Glu224-Lys254) and a short ion-pair network (Lys257-Asp237-Arg304-Glu165) at the intersubunit interface might contribute to the extreme thermal stability of TbADH. In the present study, we used site-directed mutagenesis to replace these structurally strategic residues in CbADH with the corresponding amino acids from TbADH, and we determined the effect of such replacements on the thermal stability of CbADH. Mutations in the intrasubunit ion pair region increased thermostability in the single mutant S254K- and in the double mutant V224E/S254K-CbADH, but not in the single mutant V224E-CbADH. Both single amino acid replacements, M304R- and Q165E-CbADH, in the region of the intersubunit ion pair network augmented thermal stability, with an additive effect in the double mutant M304R/Q165E-CbADH. To investigate the precise mechanism by which such mutations alter the molecular structure of CbADH to achieve enhanced thermostability, we constructed a quadruple mutant V224E/S254K/Q165E/M304R-CbADH and solved its three-dimensional structure. The overall results indicate that the amino acid substitutions in CbADH mutants with enhanced thermal stability reinforce the quaternary structure of the enzyme by formation of an extended network of intersubunit ion pairs and salt bridges, mediated by water molecules, and by forming a new intrasubunit salt bridge.
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Two primary alcohols (1-butanol and ethanol) are major fermentation products of several clostridial species. In addition to these two alcohols, the secondary alcohol 2-propanol is produced to a concentration of about 100 mM by some strains of Clostridium beijerinckii. An alcohol dehydrogenase (ADH) has been purified to homogeneity from two strains (NRRL B593 and NESTE 255) of 2-propanol-producing C. beijerinckii. When exposed to air, the purified ADH was stable, whereas the partially purified ADH was inactivated. The ADHs from the two strains had similar structural and kinetic properties. Each had a native M(r) of between 90,000 and 100,000 and a subunit M(r) of between 38,000 and 40,000. The ADHs were NADP(H) dependent, but a low level of NAD(+)-linked activity was detected. They were equally active in reducing aldehydes and 2-ketones, but a much lower oxidizing activity was obtained with primary alcohols than with secondary alcohols. The kcat/Km value for the alcohol-forming reaction appears to be a function of the size of the larger alkyl substituent on the carbonyl group. ADH activities measured in the presence of both acetone and butyraldehyde did not exceed activities measured with either substrate present alone, indicating a common active site for both substrates. There was no similarity in the N-terminal amino acid sequence between that of the ADH and those of fungi and several other bacteria. However, the N-terminal sequence had 67% identity with those of two other anaerobes, Thermoanaerobium brockii and Methanobacterium palustre. Furthermore, conserved glycine and tryptophan residues are present in ADHs of these three anaerobic bacteria and ADHs of mammals and green plants.
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An efficient genome-scale editing tool is required for construction of industrially useful microbes. We describe a targeted, continual multigene editing strategy that was applied to the Escherichia coli genome by using the Streptococcus pyogenes type II CRISPR-Cas9 system to realize a variety of precise genome modifications, including gene deletion and insertion, with a highest efficiency of 100%, which was able to achieve simultaneous multigene editing of up to three targets. The system also demonstrated successful targeted chromosomal deletions in Tatumella citrea, another species of the Enterobacteriaceae, with highest efficiency of 100%.
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The contribution of co-translational chaperone functions to protein folding is poorly understood. Ribosome-associated trigger factor (TF) is the first molecular chaperone encountered by nascent polypeptides in bacteria. Here we show, using fluorescence spectroscopy to monitor TF function and structural rearrangements in real time, that TF interacts with ribosomes and translating polypeptides in a dynamic reaction cycle. Ribosome binding stabilizes TF in an open, activated conformation. Activated TF departs from the ribosome after a mean residence time of approximately 10 s, but may remain associated with the elongating nascent chain for up to 35 s, allowing entry of a new TF molecule at the ribosome docking site. The duration of nascent-chain interaction correlates with the occurrence of hydrophobic motifs in translating polypeptides, reflecting a high aggregation propensity. These findings can explain how TF prevents misfolding events during translation and may provide a paradigm for the regulation of nucleotide-independent chaperones.
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Efficient folding of many newly synthesized proteins depends on assistance from molecular chaperones, which serve to prevent protein misfolding and aggregation in the crowded environment of the cell. Nascent chain--binding chaperones, including trigger factor, Hsp70, and prefoldin, stabilize elongating chains on ribosomes in a nonaggregated state. Folding in the cytosol is achieved either on controlled chain release from these factors or after transfer of newly synthesized proteins to downstream chaperones, such as the chaperonins. These are large, cylindrical complexes that provide a central compartment for a single protein chain to fold unimpaired by aggregation. Understanding how the thousands of different proteins synthesized in a cell use this chaperone machinery has profound implications for biotechnology and medicine.
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GroEL and GroES form a chaperonin nano-cage for proteins up to approximately 60 kDa to fold in isolation. Here we explored the structural features of the chaperonin cage critical for rapid folding of encapsulated substrates. Modulating the volume of the GroEL central cavity affected folding speed in accordance with confinement theory. Small proteins (approximately 30 kDa) folded more rapidly as the size of the cage was gradually reduced to a point where restriction in space slowed folding dramatically. For larger proteins (approximately 40-50 kDa), either expanding or reducing cage volume decelerated folding. Additionally, interactions with the C-terminal, mildly hydrophobic Gly-Gly-Met repeat sequences of GroEL protruding into the cavity, and repulsion effects from the negatively charged cavity wall were required for rapid folding of some proteins. We suggest that by combining these features, the chaperonin cage provides a physical environment optimized to catalyze the structural annealing of proteins with kinetically complex folding pathways.
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A subset of essential cellular proteins requires the assistance of chaperonins (in Escherichia coli, GroEL and GroES), double-ring complexes in which the two rings act alternately to bind, encapsulate and fold a wide range of nascent or stress-denatured proteins. This process starts by the trapping of a substrate protein on hydrophobic surfaces in the central cavity of a GroEL ring. Then, binding of ATP and co-chaperonin GroES to that ring ejects the non-native protein from its binding sites, through forced unfolding or other major conformational changes, and encloses it in a hydrophilic chamber for folding. ATP hydrolysis and subsequent ATP binding to the opposite ring trigger dissociation of the chamber and release of the substrate protein. The bacteriophage T4 requires its own version of GroES, gp31, which forms a taller folding chamber, to fold the major viral capsid protein gp23 (refs 16-20). Polypeptides are known to fold inside the chaperonin complex, but the conformation of an encapsulated protein has not previously been visualized. Here we present structures of gp23-chaperonin complexes, showing both the initial captured state and the final, close-to-native state with gp23 encapsulated in the folding chamber. Although the chamber is expanded, it is still barely large enough to contain the elongated gp23 monomer, explaining why the GroEL-GroES complex is not able to fold gp23 and showing how the chaperonin structure distorts to enclose a large, physiological substrate protein.
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