Theoretical insights into the mechanism underlying aflatoxin B1 transformation by the BsCotA-methyl syringate system.

Global concern exists regarding the contamination of food and animal feed with aflatoxin B1 (AFB1), which poses a threat to the health of both humans and animals. Previously, we found that a laccase from Bacillus subtilis (BsCotA) effectively detoxified AFB1 in a reaction mediated by methyl syringate (MS), although the underlying mechanism has not been determined. Therefore, our primary objective of this study was to explore the detoxification mechanism employed by BsCotA. First, the enzyme and mediator dependence of AFB1 transformation were studied using the BsCotA-MS system, which revealed the importance of MS radical formation during the oxidation process. Aflatoxin Q1 (AFQ1) resulting from the direct oxidation of AFB1 by BsCotA, was identified using ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The results of UPLC-MS/MS and density functional theory calculations indicated that the products included AFQ1, AFB1-, and AFD1-MS-coupled products in the BsCotA-MS system. The toxicity evaluations revealed that the substances derived from the transformation of AFB1 through the BsCotA-MS mechanism exhibited markedly reduced toxicity compared to AFB1. Finally, we proposed a set of different AFB1-transformation pathways generated by the BsCotA-MS system based on the identified products. These findings greatly enhance the understanding of the AFB1-transformation mechanism of the laccase-mediator system.

High-Throughput Screening Techniques for the Selection of Thermostable Enzymes.

The acquisition of a thermostable enzyme is an indispensable prerequisite for its successful implementation in industrial applications and the development of novel functionalities. Various protein engineering approaches, including rational design, semirational design, and directed evolution, have been employed to enhance thermostability. However, all of these approaches require sensitive and reliable high-throughput screening (HTS) technologies to efficiently and rapidly identify variants with improved properties. While numerous reviews focus on modification strategies for enhancing enzyme thermostability, there is a dearth of literature reviewing HTS methods specifically aimed at this objective. Herein, we present a comprehensive overview of various HTS methods utilized for modifying enzyme thermostability across different screening platforms. Additionally, we highlight significant recent examples that demonstrate the successful application of these methods. Furthermore, we address the technical challenges associated with HTS technologies used for screening thermostable enzyme variants and discuss valuable perspectives to promote further advancements in this field. This review serves as an authoritative reference source offering theoretical support for selecting appropriate screening strategies tailored to specific enzymes with the aim of improving their thermostability.

Production of cello-oligosaccharides from corncob residue by degradation-synthesis reactions.

The cellulose-rich corncob residue (CCR) is an abundant and renewable agricultural biomass that has been under-exploited. In this study, two strategies were compared for their ability to transform CCR into cello-oligosaccharides (COS). The first strategy employed the use of endo-glucanases. Although selected endo-glucanases from GH9, GH12, GH45, and GH131 could release COS with degrees of polymerization from 2 to 4, the degrading efficiency was low. For the second strategy, first, CCR was efficiently depolymerized to glucose and cellobiose using the cellulase from Trichoderma reesei. Then, using these simple sugars and sucrose as the starting materials, phosphorylases from different microorganisms were combined to generate COS to a level up to 100.3 g/L with different patterns and degrees of polymerization. Using tomato as a model plant, the representative COS obtained from BaSP (a sucrose phosphorylase from Bifidobacterium adolescens), CuCbP (a cellobiose phosphorylase from Cellulomonas uda), and CcCdP (a cellodextrin phosphorylase from Clostridium cellulosi) were shown to be able to promote plant growth. The current study pointed to an approach to make use of CCR for production of the value-added COS. KEY POINTS: • Sequential use of cellulase and phosphorylases effectively generated cello-oligosaccharides from corncob residue. • Cello-oligosaccharides patterns varied in accordance to cellobiose/cellodextrin phosphorylases. • Spraying cello-oligosaccharides promoted tomato growth.

Characterization of a novel thermostable α-l-arabinofuranosidase for improved synergistic effect with xylanase on lignocellulosic biomass hydrolysis without prior pretreatment.

Utilizing thermostable enzymes in biomass conversion processes presents a promising approach to bypass pretreatment, garnering significant attention from the biorefinery industry. A novel discovered α-l-arabinofuranosidase, Abf4980, exhibits exceptional thermostability by maintaining full activity after 24 h of incubation at 70 °C. It effectively acts on polyarabinosides, cleaving α-1,2- and α-1,3-linked arabinofuranose side chains from water-soluble wheat arabinoxylan while releasing xylose. When synergistically combined with the thermostable bifunctional xylanase/ß-glucanase CbXyn10C from Caldicellulosiruptor bescii at an enzyme-activity ratio of 6:1, Abf4980 achieves the highest degradation efficiency for wheat arabinoxylan. Furthermore, Abf4980 and CbXyn10C demonstrated remarkable efficacy in hydrolyzing unmodified wheat bran and corn cob to generate arabinose and xylooligosaccharides. This discovery holds promising opportunities for improving the efficiency of lignocellulosic biomass conversion into fermentable sugars.

Efficient production of γ-aminobutyric acid using engineered Escherichia coli whole-cell catalyst.

γ-Aminobutyric acid (GABA) has been widely used in the food, feed, pharmaceutical, and chemical industry fields. Previously, we developed a whole-cell catalyst capable of converting L-glutamate (L-Glu) into GABA by overexpressing the glutamate decarboxylase gene (gadz11) from Bacillus sp. Z11 in Escherichia coli BL21(DE3). However, to enhance cell permeability, a freeze-thaw treatment is required, and to enhance GADZ11 activity, pyridoxal 5'-phosphate (PLP) must be added to the reaction system. The aim of this study is to provide a more efficient approach for GABA production by engineering the recombinant E. coli above. First, the inducible expression conditions of the gadz11 in E. coli were optimized to 37 °C for 6 h. Next, an ideal engineered strain was produced via increasing cell permeability by overexpressing sulA and eliminating PLP dependence by constructing a self-sufficient system. Furthermore, an efficient whole-cell biocatalytic process was optimized. The optimal substrate concentration, cell density, and reaction temperature were 1.0 mol/L (the molecular ratio of L-Glu to L-monosodium glutamate (L-MSG) was 4:1), 15 and 37 °C, respectively. Finally, a whole-cell bioconversion procedure was performed in a 3-L bioreactor under optimal conditions. The strain could be reused for at least two cycles with GABA yield, productivity and conversion ratio of 206.2 g/L, 117.8 g/L/h and 100.0%, respectively. This is currently the highest GABA productivity from a mixture of L-Glu and L-MSG reported without the addition of cofactors or additional treatment of cells. This work demonstrates that the novel engineered E. coli strain has the potential for application in large-scale industrial GABA production.

Production of capsaicinoid nonivamide from plant oil and vanillylamine via whole-cell biotransformation.

Capsaicinoids are mostly derived from chili peppers and have widespread applications in food, feed, and pharmacology. Compared with plant extraction, the use of microbial cell factories for capsaicinoids production is considered as a more efficient approach. Here, the biotransformation of renewable plant oil and vanillylamine into capsaicinoid nonivamide was investigated. Nonivamide biosynthesis using nonanoic acid and vanillylamine as substrates was achieved in Escherichia coli by heterologous expression of genes encoding amide-forming N-acyltransferase and CoA-ligase. Through increasing nonanoic acid tolerance of chassis cell, screening key enzymes involved in nonivamide biosynthesis and optimizing biotransformation conditions, the nonivamide titer reached 0.5 g/L. By further integrating a route for conversion of oleic acid to nonanoic acid, nonivamide biosynthesis was finally achieved using olive oil and vanillylamine as substrates, yielding a titer of approximately 10.7 mg/L. Results from this study provide valuable information for constructing highly efficient cell factories for the production of capsaicinoid compounds.

Recombinant expression of IGF-1 and LR3 IGF-1 fused with xylanase in Pichia pastoris.

Insulin-like growth factor-1 (IGF-1) is a pleiotropic protein hormone and has become an attractive therapeutic target because of its multiple roles in various physiological processes, including growth, development, and metabolism. However, its production is hindered by low heterogenous protein expression levels in various expression systems and hard to meet the needs of clinical and scientific research. Here, we report that human IGF-1 and its analog Long R3 IGF-1 (LR3 IGF-1) are recombinant expressed and produced in the Pichia pastoris (P. pastoris) expression system through being fused with highly expressed xylanase XynCDBFV. Furthermore, purified IGF-1 and LR3 IGF-1 display excellent bioactivity of cell proliferation compared to the standard IGF-1. Moreover, higher heterologous expression levels of the fusion proteins XynCDBFV-IGF-1 and XynCDBFV-LR3 IGF-1 are achieved by fermentation in a 15-L bioreactor, reaching up to about 0.5 g/L XynCDBFV-IGF-1 and 1 g/L XynCDBFV-TEV-LR3 IGF-1. Taken together, high recombinant expression of bioactive IGF-1 and LR3 IGF-1 is acquired with the assistance of xylanase as a fusion partner in P. pastoris, which could be used for both clinical and scientific applications. KEY POINTS: • Human IGF-1 and LR3 IGF-1 are produced in the P. pastoris expression system. • Purified IGF-1 and LR3 IGF-1 show bioactivity comparable to the standard IGF-1. • High heterologous expression of IGF-1 and LR3 IGF-1 is achieved by fermentation in a bioreactor.

Production of N-acetylglucosamine from carbon dioxide by engineering Cupriavidus necator H16.

The conversion of CO2 into valuable bioactive substances using synthetic biological techniques is a potential approach for mitigating the greenhouse effect. Here, the engineering of C. necator H16 to produce N-acetylglucosamine (GlcNAc) from CO2 is reported. First, GlcNAc importation and intracellular metabolic pathways were disrupted by the deletion of nagF, nagE, nagC, nagA and nagB genes. Second, the GlcNAc-6-phosphate N-acetyltransferase gene (gna1) was screened. A GlcNAc-producing strain was constructed by overexpressing a mutant gna1 from Caenorhabditis elegans. A further increase in GlcNAc production was achieved by disrupting poly(3-hydroxybutyrate) biosynthesis and the Entner-Doudoroff pathways. The maximum GlcNAc titers were 199.9 and 566.3 mg/L for fructose and glycerol, respectively. Finally, the best strain achieved a GlcNAc titer of 75.3 mg/L in autotrophic fermentation. This study demonstrated a conversion of CO2 to GlcNAc, thereby providing a feasible approach for the biosynthesis of various bioactive chemicals from CO2 under normal conditions..

High Production of Maltooligosaccharides in the Starch Liquefaction Process: A Study on the Hyperthermophilic Mechanism of α-Amylase.

The efficient production of high-value-added bioproducts from starchy substances requires α-amylases with hyperthermophilic properties for industrial starch liquefaction. In this study, two hyperthermophilic α-amylases with significant differences in thermostability, PfAmy and TeAmy, were comparatively studied through structural analysis, domain swapping, and site-directed mutagenesis, finding that three residues, His152, Cys166, and His168, located in domain B were the main contributors to hyperthermostability. The effects of these three residues were strongly synergistic, causing the optimum temperature for the mutant K152H/A166C/E168H of TeAmy to shift to 95-100 °C and stabilize at 90 °C without Ca2+. Compared to PfAmy and TeAmy, the mutant K152H/A166C/E168H, respectively, exhibited 1.7- and 2.5-times higher starch hydrolysis activity at 105 °C and pH 5.5 (10411 ± 70 U/mg) and released 1.1- and 1.7-times more maltooligosaccharides from 1% starch. This work has interpreted the hyperthermophilic mechanism of α-amylase and thereby providing a potential candidate for the efficient industrial conversion of starch to bioproducts.

Engineering Escherichia coli for efficient assembly of heme proteins.

BACKGROUND: Heme proteins, such as hemoglobin, horseradish peroxidase and cytochrome P450 (CYP) enzyme, are highly versatile and have widespread applications in the fields of food, healthcare, medical and biological analysis. As a cofactor, heme availability plays a pivotal role in proper folding and function of heme proteins. However, the functional production of heme proteins is usually challenging mainly due to the insufficient supply of intracellular heme. RESULTS: Here, a versatile high-heme-producing Escherichia coli chassis was constructed for the efficient production of various high-value heme proteins. Initially, a heme-producing Komagataella phaffii strain was developed by reinforcing the C4 pathway-based heme synthetic route. Nevertheless, the analytical results revealed that most of the red compounds generated by the engineered K. phaffii strain were intermediates of heme synthesis which were unable to activate heme proteins. Subsequently, E. coli strain was selected as the host to develop heme-producing chassis. To fine-tune the C5 pathway-based heme synthetic route in E. coli, fifty-two recombinant strains harboring different combinations of heme synthesis genes were constructed. A high-heme-producing mutant Ec-M13 was obtained with negligible accumulation of intermediates. Then, the functional expression of three types of heme proteins including one dye-decolorizing peroxidase (Dyp), six oxygen-transport proteins (hemoglobin, myoglobin and leghemoglobin) and three CYP153A subfamily CYP enzymes was evaluated in Ec-M13. As expected, the assembly efficiencies of heme-bound Dyp and oxygen-transport proteins expressed in Ec-M13 were increased by 42.3-107.0% compared to those expressed in wild-type strain. The activities of Dyp and CYP enzymes were also significantly improved when expressed in Ec-M13. Finally, the whole-cell biocatalysts harboring three CYP enzymes were employed for nonanedioic acid production. High supply of intracellular heme could enhance the nonanedioic acid production by 1.8- to 6.5-fold. CONCLUSION: High intracellular heme production was achieved in engineered E. coli without significant accumulation of heme synthesis intermediates. Functional expression of Dyp, hemoglobin, myoglobin, leghemoglobin and CYP enzymes was confirmed. Enhanced assembly efficiencies and activities of these heme proteins were observed. This work provides valuable guidance for constructing high-heme-producing cell factories. The developed mutant Ec-M13 could be employed as a versatile platform for the functional production of difficult-to-express heme proteins.

Effectiveness of ruminal xylanase with an extra proline-rich C-terminus on lignocellulosic biomass degradation.

The efficient degradation of plant polysaccharides in agricultural waste requires xylanases with high catalytic activity. In this study, the C-terminal proline-rich GH10 xylanase XynA from sheep rumen was investigated using product analysis, structural characterization, truncated and site-directed mutagenesis, molecular dynamics simulation, and application evaluation, revealing that the proline-rich C-terminus contributes to the interaction at the substrate-binding pocket to reduce the binding free energy. Compared to the C-terminally truncated enzyme XynA-Tr, XynA has a more favorable conformation for proton transfer and affinity attack, facilitating the degradation of oligomeric and beechwood xylan without altering the hydrolysis pattern. Moreover, both the reduced sugar yield and weight loss of the pretreated wheat bran, corn cob, and corn stalk hydrolyzed by XynA for 12 h increased by more than 30 %. These findings are important to better understand the relationship between enzyme activities and their terminal regions and suggest candidate materials for lignocellulosic biomass utilization.

Engineering Cupriavidus necator H16 for heterotrophic and autotrophic production of myo-inositol.

Bioconversion of sustainable feedstocks to commodity chemicals is considered as an effective solution for transforming the fossil-based economy into a carbon-neutral model. Here, the CO2-fixing bacterium Cupriavidus necator H16 was exploited for myo-inositol production from renewable substrates. First, by introducing the glucose transportation system, the glucose consumption route was established. Second, two key enzymes involved in myo-inositol biosynthesis were screened and evaluated. A myo-inositol-producing strain was constructed via overexpression of myo-inositol-3-phosphate synthase from Saccharomyces cerevisiae and inositol monophosphatase from Escherichia coli. Finally, carbon flux redirection was achieved through disruption of Entner-Doudoroff pathway and poly(3-hydroxybutyrate) synthesis pathway, resulting in a final myo-inositol production of 520.2, 1076.3 and 1054.8 mg/L from glucose, glycerol and CO2, respectively. The myo-inositol production level from CO2 achieved here set up the record. This study underlines the potential of C. necator to be utilized as microbial factory for upcycling the renewable feedstocks and CO2 to high-value biochemicals.

Direct conversion of carbon dioxide to glucose using metabolically engineered Cupriavidus necator.

Artificial synthesis of glucose, the monomer of starch, from renewable resources and CO2 is a promising method for addressing food crisis and alleviating climate change. Here, the construction of a microbial biocatalyst for glucose production from renewable resources and CO2 was reported. Initially, blocking the glucose catabolic pathway via deletion of glk gene generated a glucose-producing strain of Cupriavidus necator with titers of 24.7, 47.5 and 180.1 mg/L from fructose, glycerol and CO2, respectively. Subsequently, the Entner-Doudoroff pathway and polyhydroxybutyrate biosynthesis pathway were disrupted to further increase glucose accumulation. The maximum glucose titer and yield on biomass from CO2 reached 253.3 mg/L and 91.6 mg/L/OD600, respectively. Finally, the phosphatases that mediate the dephosphorylation of phosphorylated glucose were identified. Overexpression of HAD1 and cbbY2 could enhance glucose titer by 5.5-fold when fructose was used as sole carbon source. This study demonstrates a feasible route for microbial-based synthesis of glucose from CO2.

Metabolic engineering of Pichia pastoris for myo-inositol production by dynamic regulation of central metabolism.

BACKGROUND: The methylotrophic budding yeast Pichia pastoris GS115 is a powerful expression system and hundreds of heterologous proteins have been successfully expressed in this strain. Recently, P. pastoris has also been exploited as an attractive cell factory for the production of high-value biochemicals due to Generally Recognized as Safe (GRAS) status and high growth rate of this yeast strain. However, appropriate regulation of metabolic flux distribution between cell growth and product biosynthesis is still a cumbersome task for achieving efficient biochemical production. RESULTS: In this study, P. pastoris was exploited for high inositol production using an effective dynamic regulation strategy. Through enhancing native inositol biosynthesis pathway, knocking out inositol transporters, and slowing down carbon flux of glycolysis, an inositol-producing mutant was successfully developed and low inositol production of 0.71 g/L was obtained. The inositol production was further improved by 12.7% through introduction of heterologous inositol-3-phosphate synthase (IPS) and inositol monophosphatase (IMP) which catalyzed the rate-limiting steps for inositol biosynthesis. To control metabolic flux distribution between cell growth and inositol production, the promoters of glucose-6-phosphate dehydrogenase (ZWF), glucose-6-phosphate isomerase (PGI) and 6-phosphofructokinase (PFK1) genes were replaced with a glycerol inducible promoter. Consequently, the mutant strain could be switched from growth mode to production mode by supplementing glycerol and glucose sequentially, leading to an increase of about 4.9-fold in inositol formation. Ultimately, the dissolved oxygen condition in high-cell-density fermentation was optimized, resulting in a high production of 30.71 g/L inositol (~ 40-fold higher than the baseline strain). CONCLUSIONS: The GRAS P. pastoris was engineered as an efficient inositol producer for the first time. Dynamic regulation of cell growth and inositol production was achieved via substrate-dependent modulation of glycolysis and pentose phosphate pathways and the highest inositol titer reported to date by a yeast cell factory was obtained. Results from this study provide valuable guidance for engineering of P. pastoris for the production of other high-value bioproducts.

Metabolic engineering of Komagataella phaffii for synergetic utilization of glucose and glycerol.

Komagataella phaffii GS115 is a proven heterologous expression system and has recently been exploited for the production of value-added biochemicals from glucose through metabolic engineering. A major challenge for high-level biochemical production is the appropriate distribution of carbon flux between cell growth and product biosynthesis. In this study, we report the development of a synergetic glucose and glycerol coutilization strategy for K. phaffii, potentially enabling this strain to consume glycerol for growth while conserving more glucose for product formation. First, several potential genes encoding mediator proteins and transcriptional factors that were considered to be associated with carbon catabolite repression in K. phaffii were screened, and deletion of gss1, a glucose sensor, appeared to be able to eliminate the glucose-induced repression of glycerol utilization in a mixed glucose-glycerol medium. Transcriptome comparisons between the parent strain and the Δgss1 mutant were then performed, and the glycerol-metabolism genes that were subjected to glucose regulation were identified. Second, coutilization of glucose and glycerol in K. phaffii was achieved by overexpressing genes relevant to glycerol metabolism, namely, gt1, gut1, and gut2. Furthermore, knockout or knockdown of pfk and zwf genes resulted in a reduction of carbon flux from glucose towards glycolysis and the pentose phosphate pathway. With these efforts, the cell metabolism of the final strain was divided into growth and production modules. This study describes a promising strategy to address the challenge of carbon flux distribution in K. phaffii, and would be valuable in engineering this strain as a versatile fermentation platform for biochemical production.

Engineering a carbohydrate-binding module to increase the expression level of glucoamylase in Pichia pastoris.

BACKGROUND: Glucoamylase is an important industrial enzyme for the saccharification of starch during sugar production, but the production cost of glucoamylase is a major limiting factor for the growth of the starch-based sugar market. Therefore, seeking strategies for high-level expression of glucoamylase in heterologous hosts are considered as the main way to reduce the enzyme cost. RESULTS: ReGa15A from Rasamsonia emersonii and TlGa15B-GA2 from Talaromyces leycettanus have similar properties. However, the secretion level of ReGa15A was significantly higher than TlGa15B-GA2 in Pichia pastoris. To explore the underlying mechanisms affecting the differential expression levels of glucoamylase in P. pastoris, the amino acid sequences and three-dimensional structures of them were compared and analyzed. First, the CBM region was identified by fragment replacement as the key region affecting the expression levels of ReGa15A and TlGa15B-GA2. Then, through the substitution and site-directed mutation of the motifs in the CBM region, three mutants with significantly increased expression levels were obtained. The eight-point mutant TlGA-M4 (S589D/Q599A/G600Y/V603Q/T607I/V608L/N609D/R613Q), the three-point mutant TlGA-M6 (Q599A/G600Y/V603Q) and the five-point mutant TlGA-M7 (S589D/T607I/V608L/N609D/R613Q) have the same specific activity with the wild-type, and the enzyme activity and secretion level have increased by 4-5 times, respectively. At the same time, the expression levels were 5.8-, 2.0- and 2.4-fold higher than that of wild type, respectively. Meanwhile, the expression of genes related to the unfolded protein responses (UPR) in the endoplasmic reticulum (ER) did not differ significantly between the mutants and wild type. In addition, the most highly expressed mutant, TlGA-M7 exhibits rapidly and effectively hydrolyze raw corn starch. CONCLUSIONS: Our results constitute the first demonstration of improved expression and secretion of a glucoamylase in P. pastoris by introducing mutations within the non-catalytic CBM. This provides a novel and effective strategy for improving the expression of recombinant proteins in heterologous host expression systems.

Recombinant expression of hen egg white lysozyme with the assistance of xylanase fusion partner in Pichia pastoris.

Due to its bacteriolytic activity, hen egg white lysozyme (HEWL) is widely used in the feed, food, and pharmaceutical industries. However, its application is hindered by low protein expression levels in microbial expression systems. In this work, a novel fusion protein expression strategy was proposed for increasing the expression level of HEWL. First, HEWL, fused with a highly expressed fusion protein partner xylanase XynCDBFV, is expressed in Pichia pastoris. Secondly, a linker including endogenous protease cleavage sites was introduced between two fusion proteins in order to separate them directly during the secretion process. Finally, the results show that the supernatant of XynCDBFV-HEWL has a higher HEWL expression level and activity compared with HEWL only. It should be noted that the expression of HEWL reaches to about 3.5 g/L, and the activity of HEWL against Micrococcus lysodeikticus reaches to 1.50 × 105 U/mL in a fed-batch fermentation, which is currently the highest level of recombinant expression of an egg white-derived lysozyme. Taken together, we acquired bioactive HEWL for large-scale recombinant production in Pichia pastoris using a novel fusion protein expression strategy, which could then be used for a variety of applications.

Fusion of a proline-rich oligopeptide to the C-terminus of a ruminal xylanase improves catalytic efficiency.

Xylanases are widely used in the degradation of lignocellulose and are important industrial enzymes. Therefore, increasing the catalytic activity of xylanases can improve their efficiency and performance. In this study, we introduced the C-terminal proline-rich oligopeptide of the rumen-derived XynA into XylR, a GH10 family xylanase. The optimum temperature and pH of the fused enzyme (XylR-Fu) were consistent with those of XylR; however, its catalytic efficiency was 2.48-fold higher than that of XylR. Although the proline-rich oligopeptide did not change the enzyme hydrolysis mode, the amount of oligosaccharides released from beechwood xylan by XylR-Fu was 17% higher than that released by XylR. This increase may be due to the abundance of proline in the oligopeptide, which plays an important role in substrate binding. Furthermore, circular dichroism analysis indicated that the proline-rich oligopeptide might increase the rigidity of the overall structure, thereby enhancing the affinity to the substrate and catalytic activity of the enzyme. Our study shows that the proline-rich oligopeptide enhances the catalytic efficiency of GH10 xylanases and provides a better understanding of the C-terminal oligopeptide-function relationships. This knowledge can guide the rational design of GH10 xylanases to improve their catalytic activity and provides clues for further applications of xylanases in industry.

Synergetic Fermentation of Glucose and Glycerol for High-Yield N-Acetylglucosamine Production in Escherichia coli.

N-acetylglucosamine (GlcNAc) is an amino sugar that has been widely used in the nutraceutical and pharmaceutical industries. Recently, microbial production of GlcNAc has been developed. One major challenge for efficient biosynthesis of GlcNAc is to achieve appropriate carbon flux distribution between growth and production. Here, a synergistic substrate co-utilization strategy was used to address this challenge. Specifically, glycerol was utilized to support cell growth and generate glutamine and acetyl-CoA, which are amino and acetyl donors, respectively, for GlcNAc biosynthesis, while glucose was retained for GlcNAc production. Thanks to deletion of the 6-phosphofructokinase (PfkA and PfkB) and glucose-6-phosphate dehydrogenase (ZWF) genes, the main glucose catabolism pathways of Escherichia coli were blocked. The resultant mutant showed a severe defect in glucose consumption. Then, the GlcNAc production module containing glucosamine-6-phosphate synthase (GlmS*), glucosamine-6-phosphate N-acetyltransferase (GNA1*) and GlcNAc-6-phosphate phosphatase (YqaB) expression cassettes was introduced into the mutant, to drive the carbon flux from glucose to GlcNAc. Furthermore, co-utilization of glucose and glycerol was achieved by overexpression of glycerol kinase (GlpK) gene. Using the optimized fermentation medium, the final strain produced GlcNAc with a high stoichiometric yield of 0.64 mol/mol glucose. This study offers a promising strategy to address the challenge of distributing carbon flux in GlcNAc production.

Improving the Substrate Affinity and Catalytic Efficiency of ß-Glucosidase Bgl3A from Talaromyces leycettanus JCM12802 by Rational Design.

Improving the substrate affinity and catalytic efficiency of ß-glucosidase is necessary for better performance in the enzymatic saccharification of cellulosic biomass because of its ability to prevent cellobiose inhibition on cellulases. Bgl3A from Talaromyces leycettanus JCM12802, identified in our previous work, was considered a suitable candidate enzyme for efficient cellulose saccharification with higher catalytic efficiency on the natural substrate cellobiose compared with other ß-glucosidase but showed insufficient substrate affinity. In this work, hydrophobic stacking interaction and hydrogen-bonding networks in the active center of Bgl3A were analyzed and rationally designed to strengthen substrate binding. Three vital residues, Met36, Phe66, and Glu168, which were supposed to influence substrate binding by stabilizing adjacent binding site, were chosen for mutagenesis. The results indicated that strengthening the hydrophobic interaction between stacking aromatic residue and the substrate, and stabilizing the hydrogen-bonding networks in the binding pocket could contribute to the stabilized substrate combination. Four dominant mutants, M36E, M36N, F66Y, and E168Q with significantly lower Km values and 1.4-2.3-fold catalytic efficiencies, were obtained. These findings may provide a valuable reference for the design of other ß-glucosidases and even glycoside hydrolases.