Search

The use of self-inducible promoters is a promising strategy to address metabolic imbalances caused by overexpression. However, the low activity of natural self-inducible promoters hinders their widespread application. To overcome this limitation, we selected the fic promoter as a model promoter to create an enhanced self-inducible promoter library using saturation mutations and high-throughput screening. Sequence analysis revealed that these promoters share certain characteristics, including semi-conservation in the −35 hexamer, highly conserved cytosine in the −17 motif (compared to −13 for other promoters), and moderate A+T content between positions −33 and −18 in the spacer region. Additionally, the discriminator region of these promotors features high A+T content in the first five bases. We identified PficI-17, PficII-33, and PficIII-14 promoters as the optional promoters in the −35 hexamer, spacer region, and discriminator mutation libraries, respectively. These promotors were used as representatives to measure the specific fluorescence and OD600 nm dynamics in different media and to confirm their effect on the expression of different proteins, including egfp (enhanced green fluorescence protein) and rfp (red fluorescence protein). Overall, our findings provide valuable guidance for modifying promoters and developing a promoter library suitable for regulating target genes. [ABSTRACT FROM AUTHOR]

The thermal stability of enzymes dramatically limits their application in the industrial field. Based on the crystal structure, we conducted a semi-rational design according to the B-factor and free energy values to improve the stability of dextranase from Streptococcus mutans (SmdexTM). The B-factor values of Asn102, Asn503, Asp501 and Asp500 were the highest predicted by B-FITTER. Then Rosetta was used to simulate the saturation mutations of Asn102, Asn503, Asp501 and Asp500. The mutated amino acid was designed according to the change of acG. The results showed that the thermal stability of N102P, N102C, D500G, and D500T was improved, and the half-lives of N102P/D500G and N102P/D500T at 45 °C were increased to 3.14 times and 2.44 times, respectively. Analyzing the interaction of amino acids by using Discovery Studio 4.5, it was observed that the thermal stability of dextranase was improved due to the increase in hydrophobicity and the number of hydrogen bonds of the mutant enzyme. The catalytic efficiency of N102P/D500T was increased. Compared with the hydrolyzed products of SmdexTM, the mutant enzymes do not change the specificity of hydrolysates. • The study provides a useful method to improve the thermal stability of dextranase according to the B-factor and ΔΔG. • The thermal stability and hydrolysis efficiency of the mutant dextranase were successfully improved. • The thermal stability of mutant enzyme is improved due to the increase of hydrogen bond, hydrophobic force and the decrease of swing amplitude of N-terminal peptide chain. [ABSTRACT FROM AUTHOR]

The use of self-inducible promoters is a promising strategy to address metabolic imbalances caused by overexpression. However, the low activity of natural self-inducible promoters hinders their widespread application. To overcome this limitation, we selected the fic promoter as a model promoter to create an enhanced self-inducible promoter library using saturation mutations and high-throughput screening. Sequence analysis revealed that these promoters share certain characteristics, including semi-conservation in the −35 hexamer, highly conserved cytosine in the −17 motif (compared to −13 for other promoters), and moderate A+T content between positions −33 and −18 in the spacer region. Additionally, the discriminator region of these promotors features high A+T content in the first five bases. We identified PficI-17, PficII-33, and PficIII-14 promoters as the optional promoters in the −35 hexamer, spacer region, and discriminator mutation libraries, respectively. These promotors were used as representatives to measure the specific fluorescence and OD600 nm dynamics in different media and to confirm their effect on the expression of different proteins, including egfp (enhanced green fluorescence protein) and rfp (red fluorescence protein). Overall, our findings provide valuable guidance for modifying promoters and developing a promoter library suitable for regulating target genes.

It is not clear how the amino acid composition of the X sites in GXSXG domain affects the properties of the lipase. To address this, saturation mutation at these sites of Penicillium expansum lipase (PEL) was carried out. After screening, a mutant L133M with significantly different activity was obtained. The activity of L133M was lower than that of wild-type PEL (WT) when the reaction time was 3–10 min. However, it was 1.61 times that of WT under long-term (48 h) catalysis. WT exhibited interfacial activation when the concentration of p-nitrophenylbutyrate was higher than 3 mM, whereas L133M could only show interfacial activation at higher micelle concentration (p-nitrophenylbutyrate concentration > 5 mM), which seemed to be slower. Further analysis showed that a new hydrophobic interaction was generated between the methionine introduced at position 133 and the lid, which made the interface activation process more difficult. However, the binding energy between the activated mutant and the substrate was smaller, showing stronger activity. This work provides some reliable clues for further understanding the complex role of GXSXG domain in enzyme catalysis. [ABSTRACT FROM AUTHOR]

Alcohol dehydrogenase (ADH) has attracted much attention due to its ability to catalyze the synthesis of important chiral alcohol pharmaceutical intermediates with high stereoselectivity. ADH protein engineering efforts have generally focused on reshaping the substrate-binding pocket. However, distant sites outside the pocket may also affect its activity, although the underlying molecular mechanism remains unclear. The current study aimed to apply evolutionary coupling-inspired engineering to the ADH CpRCR and to identify potential mutation sites. Through conservative analysis, phylogenic analysis and residues distribution analysis, the co-evolution hotspots Leu34 and Leu137 were confirmed to be highly evolved under the pressure of natural selection and to be possibly related to the catalytic function of the protein. Hence, Leu34 and Leu137, far away from the active center, were selected for mutation. The generated CpRCR-L34A and CpRCR-L137V variants showed high stereoselectivity and 1.24–7.81 fold increase in kcat/Km value compared with that of the wild type, when reacted with 8 aromatic ketones or β-ketoesters. Corresponding computational study implied that L34 and L137 may extend allosteric fluctuation in the protein structure from the distal mutational site to the active site. Moreover, the L34 and L137 mutations modified the pre-reaction state in multiple ways, in terms of position of the hydride with respect to the target carbonyl. These findings provide insights into the catalytic mechanism of the enzyme and facilitate its regulation from the perspective of the site interaction network.

Abstract Background Cellulases of glycosyl hydrolase (GH) family 5 share a (β/α)8 TIM-barrel fold structure with eight βα loops surrounding the catalytic pocket. These loops exposed on the surface play a vital role in protein functions, primarily due to the interactions of some key amino acids with solvent and ligand molecules. It has been reported that motions of these loops facilitate substrate access and product release, and loops 6 and 7 located at the substrate entrance of the binding pocket promote proton transfer reaction at the catalytic site motions. However, the role of these flexible loops in catalysis of GH5 cellulase remains to be explored. Results In the present study, an acidic, mesophilic GH5 cellulase (with optimal activity at pH 4.0 and 70 °C), GtCel5, was identified in Gloeophyllum trabeum CBS 900.73. The specific activities of GtCel5 toward CMC-Na, barley β-glucan, and lichenan were 1117 ± 43, 6257 ± 26 and 5318 ± 54 U/mg, respectively. Multiple sequence alignment indicates that one amino acid residue at position 233 on the loop 6 shows semi-conservativeness and might contribute to the great catalytic performance. Saturation mutagenesis at position 233 was then conducted to reveal the vital roles of this position in enzyme properties. In comparison to the wild type, variants N233A and N233G showed decreased optimal temperature (− 10 °C) but increased activities (27 and 70%) and catalytic efficiencies (k cat/K m; 45 and 52%), respectively. The similar roles of position 233 in catalytic performance were also verified in the other two GH5 homologs, TeEgl5A and PoCel5, by reverse mutation. Further molecular dynamics simulations suggested that the substitution of asparagine with alanine or glycine may introduce more hydrogen bonds, increase the flexibility of loop 6, enhance the interactions between enzyme and substrate, and thus improve the substrate affinity and catalytic efficiency. Conclusion This study proposed a novel cellulase with potentials for industrial application. A specific position was identified to play key roles in cellulase–substrate interactions and enzyme catalysis. It is of great importance for understanding the binding mechanism of GH5 cellulases, and provides an effective strategy to improve the catalytic performance of cellulases.

Raw starch glucoamylase (RSGA) can degrade the raw starch below the starch gelatinization temperature. In this study, to improve the catalytic activity of raw corn starch, N-glycosylation was introduced into the RSGA from Aspergillus fumigatus through site-directed mutation and the recombinant expression in Komagataella phaffii . Among them, the mutants G101S (N99-L100-S101) and Q113T (N111-S112-T113) increased the specific activity of raw corn starch by 1.19- and 1.21-fold, respectively. The optimal temperature of Q113T decreased from 70 to 60 °C. Notably, the combined mutant G101S/Q113T increased the specific activity toward raw starch by 1.22-fold and reduced the optimal temperature from 70 to 60 °C. Moreover, the mutant Q113M with a 1.5-fold increase in the catalytic activity was obtained via saturation mutation at site 113. Thus, the N-glycosylation site engineering is an efficient method to improve the activity of RSGA toward raw starch.

Owing to its unique fragrance, 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF) is widely used as a food flavoring agent and has high demand. Enone oxidoreductase is a vital enzyme involved in HEMF production. In this study, an enone oxidoreductase from Naumovozyma dairenensis CBS 421 (NDEO) was used for HEMF production for the first time. The mutant NDEO T183W,K290W was obtained through semirational protein engineering, which increased the HEMF yield by 75.2%. Finally, the engineered strain BM4 produced the highest HEMF yield, 194.42 mg L -1 in 132 h. Our study revealed that HEMF production can be improved in Saccharomyces cerevisiae and that this is an efficient method to improve the activity of enone oxidoreductase, which is important for the industrial synthesis of furanone.

As a highly efficient 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicide, topramezone is an ideal target for herbicide-resistant genetic engineering. In this study, two mutants, K-19 (N321Y) and K-63 (Q166R/E322V), with topramezone resistance increased by 205.3 and 58.5%, respectively, were screened from the random mutation library of Sp HPPDm, a topramezone-resistant HPPD mutant that we previously obtained. Sites N321 and E322 were identified as key sites for increased topramezone resistance by single-site mutation analysis. A mutant KB-145 (N321Y/E322K) was further obtained by saturation mutation at sites N321 and E322. The topramezone resistance of KB-145 increased by 955.3% compared to mutant Sp HPPDm. In conclusion, this study identifies two new sites that significantly affect the topramezone resistance of Sp HPPDm, which provides new insights into the molecular mechanism of herbicide resistance of HPPD, and the acquired mutants have great application potential in the construction of herbicide-resistant crops.

In order to explore the effect of the C-terminal loop domain of Penicillium expansum lipase (PEL) on its regioselectivity and activity, homologous substitution and site-directed saturation mutagenesis were used. Two mutants PEL-loopAO and M234F were selected to catalyze the hydrolysis of tricaprylin and the molar ratios of 1,2-dicaprylin to 1,3-dicaprylin in the products were 25.57 and 8.57 respectively, showing obvious sn -1,3 regioselectivity of the mutants. Bioinformatics analysis showed that the structures of the substrate binding pocket of the mutants changed significantly. Moreover, due to the different positions of sn -1(3) and sn -2 ester bonds in the substrate molecule, it is easier for the substrate molecule to adapt to the significant changes in the substrate binding pocket and form the correct sn -1(3) catalytic conformation instead of sn -2 catalytic conformation, which makes the mutants exhibit apparent sn -1,3 position selectivity. In addition, the decrease of hydrophobicity of C-terminal loop domain will cause the decrease of lipase activity, whereas maintaining or improving the hydrophobicity can maintain or improve the activity. These findings are of great value in elucidating the relationship between the C-terminal loop domain of the lipase and its regioselectivity and activity, and also provide some reliable clues for obtaining lipases with better regioselectivity. [Display omitted] • Engineering PEL to obtain 1,3-positional selectivity. • C-terminal loop domain significantly affects enzyme activity. • Relationship between the loop domain and regioselectivity was researched. [ABSTRACT FROM AUTHOR]

Abstract: The catalytic efficiency and pH optimum of Trichoderma reesei endo-β-1,4-glucanase II were improved by protein engineering. We subjected residue 342 to saturation mutagenesis, and further changed the enzyme by random mutagenesis and two rounds of DNA shuffling. Enzyme variants were purified and characterized. Variant N342V exhibited an optimal activity at pH 5.8, corresponding to a basic shift of 1pH unit compared with the wild-type enzyme, and had improved catalytic efficiency (1.5-fold of k cat/K m) for the main substrates at pH 6.2. Variants N342R and N39R/L218H/W276R/N342T both had a pH optimum of 6.2 and the latter had improved catalytic efficiency (1.4-fold of k cat/K m) at pH 6.2. Variants L218H, Q139R/N342T and Q139R/L218H/W276R/N342T all had more than 4.5-fold higher activity in reactions compared with the wild-type at pH 7.0. The relationship between the structures and the activities of the variants were analyzed by modeling the structures of the endoglucanase II variants. More stable helixes and changed electrostatic interactions between the catalytic residues and substrates may explain the higher activities and higher pH optima of the variants. [Copyright &y& Elsevier]

Alcohol dehydrogenase (ADH) has attracted much attention due to its ability to catalyze the synthesis of important chiral alcohol pharmaceutical intermediates with high stereoselectivity. ADH protein engineering efforts have generally focused on reshaping the substrate-binding pocket. However, distant sites outside the pocket may also affect its activity, although the underlying molecular mechanism remains unclear. The current study aimed to apply evolutionary coupling-inspired engineering to the ADH CpRCR and to identify potential mutation sites. Through conservative analysis, phylogenic analysis and residues distribution analysis, the co-evolution hotspots Leu34 and Leu137 were confirmed to be highly evolved under the pressure of natural selection and to be possibly related to the catalytic function of the protein. Hence, Leu34 and Leu137, far away from the active center, were selected for mutation. The generated CpRCR-L34A and CpRCR-L137V variants showed high stereoselectivity and 1.24-7.81 fold increase in k cat /K m value compared with that of the wild type, when reacted with 8 aromatic ketones or β -ketoesters. Corresponding computational study implied that L34 and L137 may extend allosteric fluctuation in the protein structure from the distal mutational site to the active site. Moreover, the L34 and L137 mutations modified the pre-reaction state in multiple ways, in terms of position of the hydride with respect to the target carbonyl. These findings provide insights into the catalytic mechanism of the enzyme and facilitate its regulation from the perspective of the site interaction network., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)