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Specific activities and thermostability of UbP4H and its variants. Thermostability was determined by measuring the residual activity after incubation of the enzymes at 37 °C for 1 h. UbP4H: wild-type, (uncultured bacterium esnapd13, trans-P4H). UbP4H-Da and UbP4H-Sc were loop variants of UbP4H constructed by replacing the predicted lid loop (A162-K178) of UbP4H with the corresponding sequence of DaP4H and ScP4H, respectively. DaP4H (Dactylosporangium sp. RH1, trans-P4H); ScP4H (Sorangium cellulosum, trans-P4H). Each value in the panel was presented as mean ± standard deviation (SD) (n = 3)
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trans-Proline 4-hydroxylases (trans-P4Hs) hydroxylate free L-proline to trans-4-hydroxy-L-proline (trans-4-Hyp) is a valuable chiral synthon for important pharmaceuticals such as carbapenem antibiotics. However, merely few microbial trans-P4Hs have been identified, and trans-4-Hyp fermentations using engineered Escherichia coli strains expressing t...
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Understanding the molecular determinants for recognition, binding and transport of antibiotics by multidrug efflux systems is important for basic research and useful for the design of more effective antimicrobial compounds. Imipenem and meropenem are two carbapenems whose antibacterial activity is known to be poorly and strongly affected by MexAB-O...
Citations
... [23][24][25][26] The advantages offered by this enzyme family include a high degree of chemical flexibility in the iron-containing active site due to multiple open coordination sites, the utilization of benign molecular oxygen as an oxidant, and use of the inexpensive and readily available cofactor αKG. However, Fe(II)/αKGs can be relatively unstable, 27,28 which may limit their practical applications in organic synthesis. ...
... 40 In another case, the Fe(II)/αKG UbP4H was successfully screened for improved stability after initial screening rounds destabilized the enzyme. 27 However, the benefits of starting with a highly stable enzyme for directed evolution are well-established. [12][13][14] A stabilized protein scaffold could potentially increase the population of active, properly-folded protein or provide access to other mutants that otherwise do not fold. ...
Directed evolution has emerged as a powerful tool for engineering new biocatalysts. However, introducing new catalytic residues can be destabilizing, and it is generally beneficial to start with a stable enzyme parent. Here we show that the deep learning tool ProteinMPNN can be used to redesign an Fe(II)/alphaKG superfamily enzyme for greater stability, solubility, and expression while retaining both native activity and an industrially-relevant non-native function. We performed site-saturation mutagenesis with both the wild type and stabilized design variant and screened for activity increases in a non-native C-H hydroxylation reaction. We observed substantially larger increases in non-native activity for variants obtained from the stabilized scaffold compared to those from the wild-type enzyme. Deep learning tools like ProteinMPNN are user-friendly and widely-accessible, and relatively straightforward structural criteria were sufficient to obtain stabilized variants while preserving catalytic function. Our work suggests that stabilization by computational sequence redesign could be routinely implemented as a first step in directed evolution campaigns for novel biocatalysts.
... Research [18,20] has revealed a strong correlation between the optimal growth temperature of the host organisms and the T m value of proteins. Furthermore, the utilization of information from homologous protein families allows for a more robust characterization of protein thermal stability [17,18,[25][26][27][28]. Simultaneously, protein contact maps [29] can capture the underlying relationships between residue-residue pairs in the spatial dimension of proteins [30]. ...
Thermally stable proteins find extensive applications in industrial production, pharmaceutical development, and serve as a highly evolved starting point in protein engineering. The thermal stability of proteins is commonly characterized by their melting temperature (Tm). However, due to the limited availability of experimentally determined Tm data and the insufficient accuracy of existing computational methods in predicting Tm, there is an urgent need for a computational approach to accurately forecast the Tm values of thermophilic proteins. Here, we present a deep learning-based model, called DeepTM, which exclusively utilizes protein sequences as input and accurately predicts the Tm values of target thermophilic proteins on a dataset consisting of 7790 thermophilic protein entries. On a test set of 1550 samples, DeepTM demonstrates excellent performance with a coefficient of determination (R²) of 0.75, Pearson correlation coefficient (P) of 0.87, and root mean square error (RMSE) of 6.24 ℃. We further analyzed the sequence features that determine the thermal stability of thermophilic proteins and found that dipeptide frequency, optimal growth temperature (OGT) of the host organisms, and the evolutionary information of the protein significantly affect its melting temperature. We compared the performance of DeepTM with recently reported methods, ProTstab2 and DeepSTABp, in predicting the Tm values on two blind test datasets. One dataset comprised 22 PET plastic-degrading enzymes, while the other included 29 thermally stable proteins of broader classification. In the PET plastic-degrading enzyme dataset, DeepTM achieved RMSE of 8.25 ℃. Compared to ProTstab2 (20.05 ℃) and DeepSTABp (20.97 ℃), DeepTM demonstrated a reduction in RMSE of 58.85% and 60.66%, respectively. In the dataset of thermally stable proteins, DeepTM (RMSE=7.66 ℃) demonstrated a 51.73% reduction in RMSE compared to ProTstab2 (RMSE=15.87 ℃). DeepTM, with the sole requirement of protein sequence information, accurately predicts the melting temperature and achieves a fully end-to-end prediction process, thus providing enhanced convenience and expediency for further protein engineering.
... In order to enhance the activity and thermos-stability of P4H, a new P4H from the uncultured bacterium esnapd13 putative "lid" loop in combination with site-directed mutagenesis was performed. Finally, 12.9 g/L t4Hyp was obtained in a fed-batch fermentation [13]. Recently, Long et al. significantly enhancing production of t4Hyp through rare codon selected evolution, dynamic precursor modulation, and metabolic engineering. ...
Background
In recent years, there has been a growing demand for microbial production of trans -4-hydroxy-L-proline ( t 4Hyp), which is a value-added amino acid and has been widely used in the fields of medicine, food, and cosmetics. In this study, a multivariate modular metabolic engineering approach was used to remove the bottleneck in the synthesis pathway of t 4Hyp.
Results
Escherichia coli t 4Hyp synthesis was performed using two modules: a α-ketoglutarate (α-KG) synthesis module (K module) and L-proline synthesis with hydroxylation module (H module). First, α-KG attrition was reduced, and then, L-proline consumption was inhibited. Subsequently, to improve the contribution to proline synthesis with hydroxylation, optimization of gene overexpression, promotor, copy number, and the fusion system was performed. Finally, optimization of the H and K modules was performed in combination to balance metabolic flow. Using the final module H1K4 in a shaking flask culture, 8.80 g/L t 4Hyp was produced, which was threefold higher than that produced by the W0 strain.
Conclusions
These strategies demonstrate that a microbial cell factory can be systematically optimized by modular engineering for efficient production of t 4Hyp.
... Compared with the structures of leucine hydroxylase and transproline-4-hydroxylases, replacing the lid loop may alter its enzymatic properties. Liu et al. replaced a new transproline-4-hydroxylase from the uncultured bacterium esnapd13 (UbP4H) putative "lid" loop in combination with site-directed mutagenesis to enhance hydroxylase activity and thermos-stability [57]. ...
... After introducing the evolutive proline 4-hydroxylase, a fed-batch fermentation in a 5-L bioreactor at 37 °C using glucose as the sole carbon source without proline addition was performed. Although only 12.9 g/L of t4Hyp was obtained, this concentration was 3.3-fold higher than that produced by the wild-type proline 4-hydroxylase control and simultaneously improved the activity and thermostability [57]. The highest t4Hyp production to date was 54.8 g/L at 60 h using integrated system engineering in E. coli without L-proline addition in a 7.5-L bioreactor, in which the deletion of putA, proP, putP, and aceA, and the mutation of proB contributed to l-proline and relieved its feedback inhibition, and a tunable circuit based on quorum sensing for the dynamic regulation of ODHC activity and rationally designed l-proline hydroxylase [69]. ...
Trans- 4-hydroxy- l -proline is an important amino acid that is widely used in medicinal and industrial applications, particularly as a valuable chiral building block for the organic synthesis of pharmaceuticals. Traditionally, trans- 4-hydroxy- l -proline is produced by the acidic hydrolysis of collagen, but this process has serious drawbacks, such as low productivity, a complex process and heavy environmental pollution. Presently, trans- 4-hydroxy- l -proline is mainly produced via fermentative production by microorganisms. Some recently published advances in metabolic engineering have been used to effectively construct microbial cell factories that have improved the trans- 4-hydroxy- l -proline biosynthetic pathway. To probe the potential of microorganisms for trans -4-hydroxy- l -proline production, new strategies and tools must be proposed. In this review, we provide a comprehensive understanding of trans -4-hydroxy- l -proline, including its biosynthetic pathway, proline hydroxylases and production by metabolic engineering, with a focus on improving its production.
... Residues involved in the lid region bind to the product via its peptide backbone (Fig. 2b), and the loop shifts towards the active site that appears after substrate binding. Liu et al. [63] engineered l-proline 4-hydroxylase (UbP4H) to enhance the activity and thermostability of UbP4H by replacing the lid region, in combination with site-directed mutagenesis, with putative lid loops identified based on multiple sequence alignment. ...
The asymmetric hydroxylation of inactive carbon atoms in organic compounds remains an important reaction in the industrial synthesis of valuable chiral compounds. Fe(II) and 2-ketoglutarate-dependent dioxygenases (Fe/2-kg DOs) are the largest known subgroups of mononuclear nonheme-Fe(II)-dependent oxygenases, catalyzing various oxidation reactions of C–H bonds. Recent developments in Fe/2-kg DO-related researches have coupled concepts from bioinformatics, synthetic biology, and computational biology to establish effective biotransformation systems. The most well-studied and characterized activity of the Fe/2-kg DOs is substrate hydroxylation, with regard to which mechanistic studies involving the Fe center assist in engineering the protein frameworks of these enzymes to obtain the desired catalytic enhancements. Amino acids are typical substrates of Fe/2-kg DOs and are usually converted into hydroxyl amino acids, which are widely used as intermediates in pharmaceutical and fine chemical industries. Herein, we have reviewed prior structural and mechanistic studies on Fe/2-kg DOs, as well as studies on the Fe/2-kg DO-mediated selective C–H oxidation process for selective hydroxyl amino acid synthesis, which will further our journey along the promising path of building complexity via C–H bond oxidation. Further, new bioinformatics techniques should be adopted with structure-based protein rational design to mine sequence databases and shrink mutant libraries to produce a diverse panel of functional Fe/2-kg DOs capable of catalyzing targeted reactions.
... Higher t4HYP production will be anticipated when P4H can act on at high temperature, i.e., 37 • C for E. coli growth. Recently, UbP4H, a P4H from uncultured bacterium esnapd13, was reported to produce t4HYP at 37 • C (Liu et al., 2019). Loop grafting and site-directed mutagenesis were employed to enhance UbP4H's catalytic activity and thermostability (Liu et al., 2019), which led to UbP4H-Da-E112 P that produce a 3.3-fold increase of t4HYP (Liu et al., 2019). ...
... Recently, UbP4H, a P4H from uncultured bacterium esnapd13, was reported to produce t4HYP at 37 • C (Liu et al., 2019). Loop grafting and site-directed mutagenesis were employed to enhance UbP4H's catalytic activity and thermostability (Liu et al., 2019), which led to UbP4H-Da-E112 P that produce a 3.3-fold increase of t4HYP (Liu et al., 2019). ...
... Recently, UbP4H, a P4H from uncultured bacterium esnapd13, was reported to produce t4HYP at 37 • C (Liu et al., 2019). Loop grafting and site-directed mutagenesis were employed to enhance UbP4H's catalytic activity and thermostability (Liu et al., 2019), which led to UbP4H-Da-E112 P that produce a 3.3-fold increase of t4HYP (Liu et al., 2019). ...
Non-proteinogenic trans-4-hydroxy-L-proline (t4HYP), a crucial naturally occurred amino acid, is present in most organisms. t4HYP is a regio- and stereo-selectively hydroxylated product of L-proline and a valuable building block for pharmaceutically important intermediates/ingredients synthesis. Microbial production of t4HYP has aroused extensive investigations because of its low-cost and environmentally benign features. Herein, we reported metabolic engineering of endogenous L-proline biosynthetic pathway to enhance t4HYP production in trace L-proline-producing Escherichia coli BL21(DE3) (21-S0). The genes responsible for by-product formation from L-proline, pyruvate, acetyl-CoA, and isocitrate in the biosynthetic network of 21-S0 were knocked out to channel the metabolic flux towards L-proline biosynthesis. PdhR was knocked out to remove its negative regulation and aceK was deleted to ensure isocitrate dehydrogenase’s activity and to increase NADPH/NADP⁺ level. The other genes for L-proline biosynthesis were enhanced by integration of strong promoters and 5′-untranslated regions. The resulting engineered E. coli strains 21-S1 ∼ 21-S9 harboring a codon-optimized proline 4-hydroxylase-encoding gene (P4H) were grown and fermented. A titer of 4.82 g/L of t4HYP production in 21-S6 overexpressing P4H was obtained at conical flask level, comparing with the starting 21-S0 (26 mg/L). The present work paves an efficient metabolic engineering way for higher t4HYP production in E. coli.
... For instance, substituting a putative "lid" loop of trans-proline 4-hydroxylases from uncultured bacterium esnapd13 with the corresponding fragment from other trans-proline 4-hydroxylases has resulted in a significant improvement of enzymatic activity. 15 Dong et al. replaced a loop of 5β-mannanase from Aspergillus usamii with other loops, and discovered a significant improvement of half-life and Tm. 16 Site-directed mutagenesis is another alternative strategy for changing the features of enzymes. ...
The cell free system has been paid more attention due to its potential of facilitating efficient catalysis of multistep reactions. In this study, an efficient enzymatic cascade of glutathione (GSH) production was developed through the evolution of bifunctional glutathione synthetase (GshF), coupled with polyphosphate kinase (PPK). First, the stability and activity of GshF were enhanced by loop interchange and site‐directed mutagenesis. As a result, the GshF half‐value period increased 163.32‐fold, and its activity raised 18.16%. PPK from Jhaorihella thermophile (PPKJT) was characterized and used to regenerate ATP in the GSH synthesis, with hexametaphosphate (PolyP (6)) as the phosphate donor. After the process optimization, 99.92 mM GSH and 7.61 mM oxidized glutathione (GSSG) were produced within 2 hr. The molar yield was 0.96 mol/mol based on the amino acid added, while the productivities of GSH achieved 49.95 mM/hr, which were the highest yield and productivity ever reported about GSH synthesis.
... Strategies of protein engineering to boost enzyme stability included rational design [28,29] and non-rational engineering employing multiple cycles of error-prone PCR [30], directed evolution with random mutagenesis [31][32][33], and site-directed mutagenesis [34,35] including saturation mutagenesis (SM) [36] on the basis of homologous structures and sequences. Recently, B-factor analysis has been extensively developed and applied in protein engineering [37][38][39]. ...
D-allulose has received increasing attention due to its excellent physiological properties and commercial potential. The D-allulose 3-epimerase from Rhodopirellula baltica (RbDAEase) catalyzes the conversion of D-fructose to D-allulose. However, its poor thermostability has hampered its industrial application. Site-directed mutagenesis based on homologous structures in which the residuals on high flexible regions were substituted according to B-factors analysis, is an effective way to improve the thermostability and robustness of an enzyme. RbDAEase showed substrate specificity toward D-allulose with a Km of 58.57 mM and kcat of 1849.43 min-1. It showed a melting temperature (Tm) of 45.7 °C and half-life (t1/2) of 52.3 min at pH 8.0, 60 °C with 1 mM Mn2+. The Site-directed mutation L144 F strengthened the thermostability to a Δt1/2 of 50.4 min, ΔTm of 12.6 °C, and ΔT5060 of 22 °C. It also improved the conversion rate to 28.6%. Structural analysis reveals that a new hydrophobic interaction was formed by the mutation. Thus, site-directed mutagenesis based on B-factors analysis would be an efficient strategy to enhance the thermostability of designed ketose 3-epimerases.
... Already today, 2-oxoglutarate-dependent oxygenases are employed for a variety of industrially and academically relevant transformations, ranging from the production of amino acid derivatives to the chemo-enzymatic total synthesis of complex natural products (vide supra). As attested by recent enzyme engineering studies on 2-oxoglutarate-dependent oxygenases [25,99,140], the progress made in the field of protein design and evolution over the last few decades [147,148] facilitates the development of robust and active C-H functionalization biocatalysts [149]. As the integration of automation and the use of advanced computer algorithms continue to facilitate enzyme engineering, the industrial implementation of 2OGX-driven processes will be further accelerated. ...
C–H functionalization is a chemically challenging but highly desirable transformation. 2-oxoglutarate-dependent oxygenases (2OGXs) are remarkably versatile biocatalysts for the activation of C–H bonds. In nature, they have been shown to accept both small and large molecules carrying out a plethora of reactions, including hydroxylations, demethylations, ring formations, rearrangements, desaturations, and halogenations, making them promising candidates for industrial manufacture. In this review, we describe the current status of 2OGX use in biocatalytic applications concentrating on 2OGX-catalyzed oxyfunctionalization of amino acids and synthesis of antibiotics. Looking forward, continued bioinformatic sourcing will help identify additional, practical useful members of this intriguing enzyme family, while enzyme engineering will pave the way to enhance 2OGX reactivity for non-native substrates.
Biocatalytic oxidations are an emerging technology for selective C‐H bond activation. While promising for a range of selective oxidations, practical use of enzymes catalyzing aerobic hydroxylation is presently limited by their substrate scope and stability under industrially relevant conditions. Here, we report the engineering and practical application of a non‐heme iron and α‐ketoglutarate‐dependent dioxygenase for the direct stereo‐ and regio‐selective hydroxylation of a non‐native fluoroindanone en route to the oncology treatment belzutifan, replacing a five‐step chemical synthesis with a direct enantioselective hydroxylation. Mechanistic studies indicated that formation of the desired product was limited by enzyme stability and product overoxidation, with these properties subsequently improved by directed evolution, yielding a biocatalyst capable of >15,000 total turnovers. Highlighting the industrial utility of this biocatalyst, the high‐yielding, green, and efficient oxidation was demonstrated at kilogram scale for the synthesis of belzutifan.
