Improvement of cellobiose 2-epimerase expression in Bacillus subtilis for efficient bioconversion of lactose to epilactose.

Epilactose, a lactose derivative known for its prebiotic properties and potential health benefits, has garnered significant interest. Cellulose 2-epimerase (CEase) is responsible for catalyzing the conversion of lactose to epilactose. In this study, the enhancement of food-grade CEase expression in Bacillus subtilis WB600 was systematically investigated. Among seven selected epilactose-producing CEases, Rhodothermus marinus CEase (RmCE) exhibited the highest epimerization activity when expressed in B. subtilis. Translational and transcriptional regulations were employed to enhance CEase expression by screening effective N-terminal coding sequences (NCSs) and promoters. The final strain demonstrated efficient production of CEase, with epimerization activity reaching 273.6 ±â€¯6.5 U/mL and 1255 ±â€¯26.4 U/mL in shake-flask and fed-batch cultivation, respectively. Utilizing only 0.25 % (V/V) of the fed-batch cultivation broth for lactose biotransformation, epilactose was efficiently produced from 300 g/L of lactose within 4 h, achieving a yield of 29.5 %. These findings provide significant support for the potential industrialization of enzymatic epilactose production.

Crystal structure and structure-guided tunnel engineering in a bacterial ß-1,4-galactosyltransferase.

Lacto-N-neotetraose (LNnT), a representative oligosaccharide found in human milk, has been previously examined for its beneficial traits. However, the LNnT titer is limited by the efficient glycosyltransferase pathway, particularly with respect to the catalysis of rate-limiting steps. As data on the crystal structure of the key enzyme required for synthesizing LNnT are lacking, the synthesis of LNnT remains an uncertainty. Here, for the first time we report the three-dimensional structure of a bacterial ß-1,4-galactosyltransferase, Aaß4GalT, and analyze the critical role played by residues in its catalytic efficacy. Guided by structural insights, we engineered this enzyme to enhance its catalytic efficiency using structure-guided tunnel engineering. The mutant enzyme L5 (K155M/H156D/F157W/K185M/Q216V) so produced, showed a 50-fold enhancement in catalytic activity. Crystal structure analysis revealed that the mechanism underlying the improvement in activity was of the swing door type. The closed conformation formed by dense hydrophobic packing with Q216V-K155M widened and permitted substrate entry. Our results show that altering the tunnel conformation helped appropriately accommodate the substrate for catalysis and provide a structural basis for the modification of other glycosyltransferases.

Rational modification of Neisseria meningitidis ß1,3-N-acetylglucosaminyltransferase for lacto-N-neotetraose synthesis with reduced long-chain derivatives.

Lacto-N-neotetraose (LNnT), as a neutral core structure within human milk oligosaccharides (HMOs), has garnered widespread attention due to its exceptional physiological functions. In the process of LNnT synthesis using cellular factory approaches, substrate promiscuity of glycosyltransferases leads to the production of longer oligosaccharide derivatives. Here, rational modification of ß1,3-N-acetylglucosaminyltransferase from Neisseria meningitidis (LgtA) effectively decreased the concentration of long-chain LNnT derivatives. Specifically, the optimal ß1,4-galactosyltransferase (ß1,4-GalT) was selected from seven known candidates, enabling the efficient synthesis of LNnT in Escherichia coli BL21(DE3). Furthermore, the influence of lactose concentration on the distribution patterns of LNnT and its longer derivatives was investigated. The modification of LgtA was conducted with computational assistance, involving alanine scanning based on molecular docking to identify the substrate binding pocket and implementing large steric hindrance on crucial amino acids to obstruct LNnT entry. The implementation of saturation mutagenesis at positions 223 and 228 of LgtA yielded advantageous mutant variants that did not affect LNnT synthesis while significantly reducing the production of longer oligosaccharide derivatives. The most effective mutant, N223I, reduced the molar ratio of long derivatives by nearly 70 %, showcasing promising prospects for LNnT production with diminished byproducts.

Multistrategy Engineering of an Inulosucrase to Enhance the Activity and Thermostability for Efficient Production of Microbial Inulin.

Inulin has found commercial applications in the pharmaceutical, nutraceutical, and food industries due to its beneficial health effects. The enzymatic biosynthesis of microbial inulin has garnered increasing attention. In this study, molecular modification was applied to Lactobacillus mulieris UMB7800 inulosucrase, an enzyme that specifically produces high-molecular weight inulin, to enhance its catalytic activity and thermostability. Among the 18 variable regions, R5 was identified as a crucial region significantly impacting enzymatic activity by replacing it with more conserved sequences. Site-directed mutagenesis combined with saturated mutagenesis revealed that the mutant A250 V increased activity by 68%. Additionally, after screening candidate mutants by rational design, four single-point mutants, S344D, H434P, E526D, and G531P, were shown to enhance thermostability. The final combinational mutant, M5, exhibited a 66% increase in activity and a 5-fold enhancement in half-life at 55 °C. These findings are significant for understanding the catalytic activity and thermostability of inulosucrase and are promising for the development of microbial inulin biosynthesis platforms.

Structural Insights into the Catalytic Cycle of Inulin Fructotransferase: From Substrate Anchoring to Product Releasing.

Carbohydrate degradation is crucial for living organisms due to their essential functions in providing energy and composing various metabolic pathways. Nevertheless, in the catalytic cycle of polysaccharide degradation, the details of how the substrates bind and how the products release need more case studies. Here, we choose an inulin fructotransferase (SpIFTase) as a model system, which can degrade inulin into functionally difructose anhydride I. At first, the crystal structures of SpIFTase in the absence of carbohydrates and complex with fructosyl-nystose (GF4), difructose anhydride I, and fructose are obtained, giving the substrate trajectory and product path of SpIFTase, which are further supported by steered molecular dynamics simulations (MDSs) along with mutagenesis. Furthermore, structural topology variations at the active centers of inulin fructotransferases are suggested as the structural base for product release, subsequently proven by substitution mutagenesis and MDSs. Therefore, this study provides a case in point for a deep understanding of the catalytic cycle with substrate trajectory and product path.

Combinatorial Optimization Strategies for 3-Fucosyllactose Hyperproduction in Escherichia coli.

3-Fucosyllactose (3-FL), an important fucosylated human milk oligosaccharide in breast milk, offers numerous health benefits to infants. Previously, we metabolically engineered Escherichia coli BL21(DE3) for the in vivo biosynthesis of 3-FL. In this study, we initially optimized culture conditions to double 3-FL production. Competing pathway genes involved in in vivo guanosine 5'-diphosphate-fucose biosynthesis were subsequently inactivated to redirect fluxes toward 3-FL biosynthesis. Next, three promising transporters were evaluated using plasmid-based or chromosomally integrated expression to maximize extracellular 3-FL production. Additionally, through analysis of α1,3-fucosyltransferase (FutM2) structure, we identified Q126 residues as a highly mutable residue in the active site. After site-saturation mutation, the best-performing mutant, FutM2-Q126A, was obtained. Structural analysis and molecular dynamics simulations revealed that small residue replacement positively influenced helical structure generation. Finally, the best strain BD3-A produced 6.91 and 52.1 g/L of 3-FL in a shake-flask and fed-batch cultivations, respectively, highlighting its potential for large-scale industrial applications.

Highly efficient biosynthesis of 3'-sialyllactose in engineered Escherichia coli.

3'-Sialyllactose (3'-SL), one of the abundant and important sialylated human milk oligosaccharides, is an emerging food ingredient used in infant formula milk. We previously developed an efficient route for 3'-SL biosynthesis in metabolically engineered Escherichia coli BL21(DE3). Here, several promising α2,3-sialyltransferases were re-evaluated from the byproduct synthesis perspective. The α2,3-sialyltransferase from Neisseria meningitidis MC58 (NST) with great potential and the least byproducts was selected for subsequent molecular modification. Computer-assisted mutation sites combined with a semi-rational modification were designed and performed. A combination of two mutation sites (P120H/N113D) of NST was finally confirmed as the best one, which significantly improved 3'-SL biosynthesis, with extracellular titers of 24.5 g/L at 5-L fed-batch cultivations. When NST-P120H/N113D was additionally integrated into the genome of host EZAK (E. coli BL21(DE3)ΔlacZΔnanAΔnanT), the final strain generated 32.1 g/L of extracellular 3'-SL in a 5-L fed-batch fermentation. Overall, we underscored the existence of by-products and improved 3'-SL production by engineering N. meningitidis α2,3-sialyltransferase.

Engineering Bacillus subtilis for highly efficient production of functional disaccharide lactulose from lactose.

Bioconversion of lactose to functional lactose derivatives attracts increasing attention. Lactulose is an important high-value lactose derivative, which has been widely used in pharmaceutical, nutraceutical, and food industries. Lactulose can be enzymatically produced from lactose by cellobiose 2-epimerase (CEase). Several studies have already focused on the food-grade expression of CEase, but they are all aimed at the biosynthesis of epilactose. Herein, we reported for the first time the biosynthesis of lactulose using the recombinant food-grade Bacillus subtilis. Lactulose biosynthesis was optimized by varying lactulose-producing CEases and expression vectors. Caldicellulosiruptor saccharolyticus CEase and pP43NMK were determined to be the optimal CEase and expression vector. Fine-tuning of CEase expression was investigated by screening a beneficial N-terminal coding sequence. After fed-batch cultivation, the highest fermentation isomerization activity reached 11.6 U/mL. Lactulose was successfully produced by the broth of the engineered B. subtilis with a yield of 52.1 %.

De Novo Biosynthesis of Difucosyllactose by Artificial Pathway Construction and α1,3/4-Fucosyltransferase Rational Design in Escherichia coli.

Difucosyllactose (DFL) is a significant and plentiful oligosaccharide found in human breast milk. In this study, an artificial metabolic pathway of DFL was designed, focusing on the de novo biosynthesis of GDP-fucose from only glycerol. This was achieved by engineering Escherichia coli to endogenously overexpress genes manB, manC, gmd, and wcaG and heterologously overexpress a pair of fucosyltransferases to produce DFL from lactose. The introduction of α-1,2-fucosyltransferase from Helicobacter pylori (FucT2) along with α-1,3/4-fucosyltransferase (HP3/4FT) addressed rate-limiting challenges in enzymatic catalysis and allowed for highly efficient conversion of lactose into DFL. Based on these results, molecular modification of HP3/4FT was performed based on computer-assisted screening and structure-based rational design. The best-performing mutant, MH5, containing a combination of five mutated sites (F49K/Y131D/Y197N/E338D/R369A) of HP3/4FT was obtained. The best strain BLC09-58 harboring MH5 yielded 45.81 g/L of extracellular DFL in 5-L fed-batch cultures, which was the highest titer reported to date.

Highly-efficient in vivo production of lacto-N-fucopentaose V by a regio-specific α1,3/4-fucosyltransferase from Bacteroides fragilis NCTC 9343.

Lacto-N-fucopentaose V (LNFP V) is a typical human milk pentasaccharide. Multi-enzymatic in vitro synthesis of LNFP V from lactose was reported, however, microbial cell factory approach to LNFP V production has not been reported yet. In this study, the biosynthetic pathway of LNFP V was examined in Escherichia coli. The previously constructed E. coli efficiently producing lacto-N-tetraose was used as the starting strain. GDP-fucose pathway module and a regio-specific glycosyltransferase with α1,3-fucosylation activity were introduced to realize the efficient synthesis of LNFP V. The α1,3/4-fucosyltransferase from Bacteroides fragilis was selected as the best enzyme for in vivo biosynthesis of LNFP V from nine candidates, with the highest titer and the lowest by-product accumulation. A beneficial variant K128D was obtained to further enhance LNFP V titer using computer-assisted site-directed mutagenesis. The final strain EW10 could produce 25.68 g/L LNFP V by fed-batch cultivation, with the productivity of 0.56 g/L·h.

Identification and Thermostability Modification of the Mesophilic L-asparaginase from Limosilactobacillus secaliphilus.

L-asparaginase (L-ASNase, E.C.3.5.1.1) could effectively inhibit the formation of acrylamide (AA) by hydrolyzing the AA precursor L-asparagine. However, most of the L-ASNases showed a relatively weak thermostability, posing a big threat on the application of enzyme at high processing temperatures. Here, the recombinant L-ASNase from mesophilic bacteria Limosilactobacillus secaliphilus was identified for the first time. The recombinant enzyme exhibited its optimal activity at pH 8.0 and 60 ℃. Additionally, the thermostability of L. secaliphilus L-ASNase was enhanced by site-directed mutagenesis after multiple sequence alignment. Ten mutants were reasonably constructed, among which the single-point mutants L24Y, S55T, and V155S showed more than 1 ℃ elevated Tm value compared to the wild-type enzyme. In addition, the half-life of mutant at 40, 50, and 55 ℃ was 376.7 min, 62.1 min, and 18.7 min, much higher than that of wild-type enzyme. The molecular dynamic simulation showed that compared to the wild-type enzyme, the structural stability of V155S was greatly strengthened due to the lower RMSF and RMSD value as well as a decreased total energy compared to that of the wild-type enzyme. The results were positive and provided some useful information for the thermostability modification of L-ASNase.

Application of molecular dynamics simulation in the field of food enzymes: improving the thermal-stability and catalytic ability.

Enzymes can produce high-quality food with low pollution, high function, high acceptability, and medical aid. However, most enzymes, in their native form, do not meet the industrial requirements. Sequence-based and structure-based methods are the two main strategies used for enzyme modification. Molecular Dynamics (MD) simulation is a sufficiently comprehensive technology, from a molecular perspective, which has been widely used for structure information analysis and enzyme modification. In this review, we summarize the progress and development of MD simulation, particularly for software, force fields, and a standard procedure. Subsequently, we review the application of MD simulation in various food enzymes for thermostability and catalytic improvement was reviewed in depth. Finally, the limitations and prospects of MD simulation in food enzyme modification research are discussed. This review highlights the significance of MD simulation and its prospects in food enzyme modification.

Engineering Escherichia coli for high-level production of lacto-N-fucopentaose I by stepwise de novo pathway construction.

Lacto-N-fucopentaose I (LNFP I) is an abundant and important fucosylated human milk oligosaccharide (HMO). Here, an efficient LNFP I-producing strain without by-product 2'-fucosyllactose (2'-FL) was developed by advisable stepwise de novo pathway construction in Escherichia coli. Specifically, the genetically stable lacto-N-triose II (LNTri II)-producing strains were constructed by the multicopy integration of ß1,3-N-acetylglucosaminyltransferase. LNTri II can be further converted to lacto-N-tetraose (LNT) by LNT-producing ß1,3-galactosyltransferase. The de novo and salvage pathways of GDP-fucose were introduced into highly efficient LNT-producing chassis. Specific α1,2-fucosyltransferase was verified to eliminate by-product 2'-FL, and binding free energy of the complex was analyzed to explain the product distribution. Subsequently, further attempts aiming to improve α1,2-fucosyltransferase activity and the supply of GDP-fucose were carried out. Our engineering strategies enabled the stepwise de novo construction of strains that produced up to 30.47 g/L of extracellular LNFP I, without accumulation of 2'-FL, and with only minor intermediates residue.

Mutations in the Different Residues between Dextransucrase Gtf-DSM and Reuteransucrase GtfO for the Investigation of Linkage Specificity Determinants.

The dextransucrase Gtf-DSM has 99.3% sequence identity with the reuteransucrase GtfO, and only 11 out of 1045 residues are different between their N-terminally truncated recombinant forms. Gtf-DSM is capable of synthesizing a dextran with 1% (α1 → 2), 6% (α1 → 4), 24% (α1 → 3), and 69% (α1 → 6) linkages, while GtfO produces a reuteran with 21% (α1 → 6) and 79% (α1 → 4) linkages. In this work, using recombinant Gtf-DSM and GtfO as templates, parallel substitutions targeting these 11 distinguishing residues were performed to investigate their linkage specificity determinants. The combinatorial mutation (I937L/D977A/D1083V/Q1086K/K1087G) at the acceptor binding subsites +1 and +2 nearly converted the linkage specificity of Gtf-DSM to that of GtfO. Surprisingly, all of the individual or combinatorial mutations in four residues from domains IV and V of Gtf-DSM significantly altered the linkage specificity of Gtf-DSM. Additionally, all mutations in the 11 distinguishing residues of Gtf-DSM resulted in a dramatically reduced transferase/hydrolysis activity ratio, which was closer to that of GtfO. These mutation results suggested that the linkage specificity differences between Gtf-DSM and GtfO are determined by the distinct micro-physicochemical environments, formed by the concerted action of a series of residues not only from the acceptor binding subsites +1 and +2 but also from domains IV and V.

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

The 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.

Directionally modulating the product chain length of an inulosucrase by semi-rational engineering for efficient production of 1-kestose.

Microbial inulosucrase as a transfructosylation tool has been used to produce inulin and inulin-type fructooligosaccharides with various polymerization degrees. Tailor-made oligosaccharides could be generated by inulosucrase via chain length modulation. In this study, a semi-rational design based on the modeled structure of Lactobacillus reuteri 121 inulosucrase was carried out to screen and construct variants. The residues Arg541 and Arg544 were determined to be significant to the product chain elongation of L. reuteri 121 inulosucrase. The variant R544W altered the product specificity of inulosucrase and produced short-chain fructooligosaccharides with 1-kestose as the main component. Molecular dynamic simulations verified an increased binding free energy of variant R544W with 1-kestose than the wild-type enzyme with 1-kestose. After optimization, 1-kestose and total short-chain fructooligosaccharides production reached approximately 206 g/L and 307 g/L, respectively. This study suggests the great potential of variant R544W in the biotransformation from sucrose to functional sugar.

Characterization of a Novel Mannose Isomerase from Stenotrophomonas rhizophila and Identification of Its Possible Catalytic Residues.

D-Mannose has great value in the treatment of chronic diseases. D-Mannose isomerase can catalyze the bioconversion of D-fructose to D-mannose. Therefore, a novel D-mannose isomerase gene (Strh-MIase) from Stenotrophomonas rhizophila strain IS26 was expressed, purified, and characterized for the industrial production of D-mannose. The specific activities of the Strh-MIase for D-mannose and D-fructose were 437.5 ± 0.8 U/mg and 267.2 ± 0.7 U/mg. Its optimal temperature and pH were 50 °C and 7.0. The enzymatic bioconversion produced 25 g/L D-mannose from concentration D-fructose (100 g/L) in 6 h by recombinant Strh-MIase, resulting in a final yield of 25%. Sodium phosphate inhibition has little influence on D-mannose production when a high concentration of D-fructose is used as substrate. We demonstrate that the metal ions improve the efficiency of D-mannose isomerase because of the enhancement of its thermostability. Moreover, the possible catalytic residues of Strh-MIase were identified by site-directed mutagenesis.

Molecular Characterization of a Mesophilic Cellobiose 2-Epimerase That Maintains a High Catalytic Efficiency at Low Temperatures.

Cellobiose 2-epimerase (CE) can catalyze bioconversion of lactose to its prebiotic derivative epilactose. The catalytic property of a novel CE from Treponema brennaborense (Trbr-CE) was investigated. Trbr-CE showed the highest catalytic efficiency of epimerization toward lactose among all of the previously reported CEs. This enzyme's specific activity could reach as high as 208.5 ± 5.3 U/mg at its optimum temperature, which is 45 °C. More importantly, this enzyme demonstrated a considerably high activity at low temperatures, suggesting Trbr-CE as a promising enzyme for industrial low-temperature production of epilactose. This structurally flexible enzyme exhibited a comparatively high binding affinity toward substrates, which was confirmed by both experimental verification and computational analysis. Molecular dynamics (MD) simulations and binding free energy calculations were applied to provide insights into molecular recognition upon temperature changes. Compared with thermophilic CEs, Trbr-CE presents a more negative enthalpy change and a higher entropy change when the temperature drops.

A new strategy for co-composting dairy manure with rice straw: Addition of different inocula at three stages of composting.

In considering the impact of inoculation time and the characteristics of composting material and inoculants on the usefulness of inoculation, a new composting strategy has been proposed and studied, in which three inocula were inoculated at three stages of composting process respectively: inoculum A (Thermoactinomyces sp. GF1 and GF2) was inoculated before fermentation to increase or maintain high temperature of pile, inoculum B (Coprinus cinerea and Coprinus comatus) was inoculated after thermophilic phase to promote degradation of lignin, and inoculum C (Trichoderma harzianum and Rhizopus oryzae) was inoculated after 30-day fermentation to promote degradation of cellulose. The results showed that the inoculations could significantly enhance the temperature of pile and the degradation of lignocelluloses. When inocula A, B, and C were inoculated into pile, temperature increased from 25°C to 65°C, from 33°C to 39°C and from 33°C to 38°C respectively and 35% lignin and 43% cellulose had been degraded in inoculated pile compared to the degradation of 15% lignin and 25% cellulose in control pile. As a result, the C/N ratio dropped more rapidly degraded in the inoculated pile (reached 20 after 33-day fermentation) than that in the control pile (reached 21.7 after 45-day fermentation). In addition, the volume loss in inoculated pile (76.5%) was higher than that in control pile (53.2%). The study, therefore, indicated that inoculating proper microorganisms at appropriate time improved the composting process and our new composting strategy would be propitious to the co-composting dairy manure with rice straw.