Protein Quality Control Acts on Folding Intermediates to Shape the Effects of Mutations on Organismal Fitness

What are the molecular properties of proteins that fall on the radar of protein quality control (PQC)? Here we mutate the E. coli’s gene encoding dihydrofolate reductase (DHFR) and replace it with bacterial orthologous genes to determine how components of PQC modulate fitness effects of these genetic changes. We find that chaperonins GroEL/ES and protease Lon compete for binding to molten globule intermediate of DHFR, resulting in a peculiar symmetry in their action: overexpression of GroEL/ES and deletion of Lon both restore growth of deleterious DHFR mutants and most of the slow-growing orthologous DHFR strains. Kinetic steady-state modeling predicts and experimentation verifies that mutations affect fitness by shifting the flux balance in cellular milieu between protein production, folding, and degradation orchestrated by PQC through the interaction with folding intermediates.Chemistry and Chemical Biolog

Synthesis of allitol from D-psicose using ribitol dehydrogenase and formate dehydrogenase

Purpose: To synthesize allitol from D-psicose by a combination of novel ribitol dehydrogenase (RDH) and formate dehydrogenase (FDH) under optimised production conditions.Methods: RDH and FDH genes were cloned and introduced into pET-22b(+) vectors for expression in Escherichia coli to produce the corresponding enzymes. The effects of temperature, pH, shaking velocity (75, 100, 125, and 150 rpm), and shaking type (horizontal and vortex) were optimised to maximise the production yield of allitol. The final product was purified and subjected to nuclear magnetic resonance (NMR) spectroscopy, infrared (IR) spectrometry, and liquid chromatography-mass spectrometry (LC-MS) to confirm its structure.Results: The optimal pH and temperature for the reaction were 7.5 and 40 °C, respectively. The results revealed that allitol yield significantly increased with increase in reaction shaking velocity and reached a maximum yield of 95.60 ± 0.54 % at 150 rpm shaking velocity after 6 h of reaction. When the reaction was run under horizontal shaking, allitol yield increased from 100.00 ± 6.05 (without shaking) to 124.20 ± 9.70 %. Twenty milligrams of D-psicose were successfully reduced to allitol under optimum conditions with a high production yield of 16.7 ± 0.62 mg after 6 h. No by-products were formed during or after the reaction. The produced allitol had a purity of 95 %, and its structure was confirmed by HPLC, IR, LCMS, and NMR spectral analyses.Conclusion: Using D-psicose as a substrate, allitol with 95 % purity was successfully produced by the combination of novel RDH and FDH.Keywords: Allitol, Ribitol dehydrogenase, Formate dehydrogenase, D-Psicose, Providencia alcalifacien

Crystal Structure of Levansucrase from the Gram-Negative Bacterium Brenneria Provides Insights into Its Product Size Specificity

Microbial levansucrases (LSs, EC 2.4.1.10) have been widely studied for the synthesis of β-(2,6)-fructans (levan) from sucrose. LSs synthesize levan-type fructo-oligosaccharides, high-molecular-mass levan polymer or combinations of both. Here, we report crystal structures of LS from the G-bacterium Brenneria sp. EniD 312 (Brs-LS) in its apo form, as well as of two mutants (A154S, H327A) targeting positions known to affect LS reaction specificity. In addition, we report a structure of Brs-LS complexed with sucrose, the first crystal structure of a G-LS with a bound substrate. The overall structure of Brs-LS is similar to that of G- and G+-LSs, with the nucleophile (D68), transition stabilizer (D225), and a general acid/base (E309) in its active site. The H327A mutant lacks an essential interaction with glucosyl moieties of bound substrates in subsite +1, explaining the observed smaller products synthesized by this mutant. The A154S mutation affects the hydrogen-bond network around the transition stabilizing residue (D225) and the nucleophile (D68), and may affect the affinity of the enzyme for sucrose such that it becomes less effective in transfructosylation. Taken together, this study provides novel insights into the roles of structural elements and residues in the product specificity of LSs

Enzymatic Preparation of Gentiooligosaccharides by a Thermophilic and Thermostable β-Glucosidase at a High Substrate Concentration

Gentiooligosaccharides (GnOS) are a kind of oligosaccharide formed by glucose with β-1-6 glycosidic bonds, which has become a new type of functional oligosaccharide for its unique refreshing bitter taste and valuable probiotic effects. However, the research on the enzymatic preparation of GnOS is not thorough enough. In this study, a GH1 thermophilic β-glucosidase from Thermotoga sp. KOL6 was used as a biocatalyst for the synthesis of GnOS. TsBgl1 exhibited excellent thermophilic and thermostable properties by possessing a melting temperature of 101.5 °C and reacting at 80–90 °C efficiently. Its half-life at 90 °C was approximately 5 h, suggesting its high heat resistance as well. TsBgl1 also showed excellent glucose tolerance with an inhibition constant (Ki) of 1720 mM and was stimulated in the presence of 0–900 mM glucose. TsBgl1 showed the highest hydrolytic activity on laminaribiose (Glc-β-1,3-Glc), but mainly synthetized gentiobiose (Glc-β-1,6-Glc) during transglycosylation. By optimizing the reaction conditions and substrate concentration, the highest yield of GnOS synthesized by TsBgl1 reached 144.3 g·L−1 when 1000 g·L−1 glucose was used as a substrate, which was higher than the highest yield ever reported. The thermophilic and thermostable properties of TsBgl1 were considered to be significant advantages in the industrial production of GnOS, where long periods of high-temperature reactions are required. This study was expected to provide an excellent candidate enzyme for industrial production of GnOS and also provide a reference for studying the transglycosylation of GH1 β-glucosidases

Recent advances on N-acetylneuraminic acid: Physiological roles, applications, and biosynthesis

N-Acetylneuraminic acid (Neu5Ac), the most common type of Sia, generally acts as the terminal sugar in cell surface glycans, glycoconjugates, oligosaccharides, lipo-oligosaccharides, and polysaccharides, thus exerting numerous physiological functions. The extensive applications of Neu5Ac in the food, cosmetic, and pharmaceutical industries make large-scale production of this chemical desirable. Biosynthesis which is associated with important application potential and environmental friendliness has become an indispensable approach for large-scale synthesis of Neu5Ac. In this review, the physiological roles of Neu5Ac was first summarized in detail. Second, the safety evaluation, regulatory status, and applications of Neu5Ac were discussed. Third, enzyme-catalyzed preparation, whole-cell biocatalysis, and microbial de novo synthesis of Neu5Ac were comprehensively reviewed. In addition, we discussed the main challenges of Neu5Ac de novo biosynthesis, such as screening and engineering of key enzymes, identifying exporters of intermediates and Neu5Ac, and balancing cell growth and biosynthesis. The corresponding strategies and systematic strategies were proposed to overcome these challenges and facilitate Neu5Ac industrial-scale production

Thermostability engineering of an inulin fructotransferase for the biosynthesis of difructose anhydride I

© 2022The thermostability of enzymes is an essential factor that performs a vital role during practical applications. Inulin fructotransferases can efficiently convert inulin into bio-functional difructose anhydrides (DFAs). The present study aimed to improve the thermostability of a previously reported inulin fructotransferase, SpIFTase, and apply it to the biosynthesis of DFA I. In silico rational design was used to predict mutation sites, based on sequential and structural information. Two triple-site mutants, Q69L/Q234L/K310G and E201I/Q234L/K310G, were characterized and exhibited enhanced thermostability with approximately 5 °C higher in melting temperature (Tm), respectively, and a 45-fold longer half-life (t1/2) at 70 °C, compared to that of SpIFTase. Molecular dynamic simulations and elaborate structural analysis suggested that the combinations of hydrophobic interaction, electrostatic potential distribution, and decreased flexibility via stabilization of loops and α-helix improved the thermostability of SpIFTase. Additionally, the promising mutants exhibited great potential to the industrial production of DFA I.N

Characterization of a novel metal-dependent D-psicose 3-epimerase from Clostridium scindens 35704.

The noncharacterized protein CLOSCI_02528 from Clostridium scindens ATCC 35704 was characterized as D-psicose 3-epimerase. The enzyme showed maximum activity at pH 7.5 and 60°C. The half-life of the enzyme at 50°C was 108 min, suggesting the enzyme was relatively thermostable. It was strictly metal-dependent and required Mn²⁺ as optimum cofactor for activity. In addition, Mn²⁺ improved the structural stability during both heat- and urea-induced unfolding. Using circular dichroism measurements, the apparent melting temperature (T m) and the urea midtransition concentration (C m) of metal-free enzyme were 64.4°C and 2.68 M. By comparison, the Mn²⁺-bound enzyme showed higher T m and C m with 67.3°C and 5.09 M. The Michaelis-Menten constant (K m), turnover number (k cat), and catalytic efficiency (k cat/K m) values for substrate D-psicose were estimated to be 28.3 mM, 1826.8 s⁻¹, and 64.5 mM⁻¹ s⁻¹, respectively. The enzyme could effectively produce D-psicose from D-fructose with the turnover ratio of 28%

Efficient Utilization of Fruit Peels for the Bioproduction of D-Allulose and D-Mannitol

Currently, the demand for low-calorie sweeteners has grown dramatically because consumers are more mindful of their health than they used to be. Therefore, bioproduction of low-calorie sweeteners from low-cost raw materials becomes a hot spot. In this study, a two-stage strategy was established to efficiently utilize D-fructose from fruit and vegetable wastes. Firstly, ketose 3-epimerase was used to produce D-allulose from D-fructose of pear peels. Secondly, the residual D-fructose was converted to D-mannitol by the engineered strain co-expression of D-mannitol 2-dehydrogenase and formate dehydrogenase. Approximately 29.4% D-fructose of pear peels was converted to D-allulose. Subsequently, under optimal conditions (35 °C, pH 6.5, 1 mM Mn2+, 2 g/L dry cells), almost all the residual D-fructose was transformed into D-mannitol with a 93.5% conversion rate. Eventually, from 1 kg fresh pear peel, it could produce 10.8 g of D-allulose and 24.6 g of D-mannitol. This bioprocess strategy provides a vital method to biosynthesize high-value functional sugars from low-cost biomass