[Characterization and expression optimization of a highly thermostable hexokinase in Escherichia coli].

Hexokinase is a crucial diagnostic reagent in blood glucose testing, which has high requirements for the enzyme activity and thermal stability. The hexokinases in China mainly rely on imports and are primarily sourced from yeast, with high costs and poor thermal stability, which limit the development of blood glucose diagnostic reagents. Therefore, there is an urgent need for the efficient expression of highly active and thermally stable hexokinases. In this study, an ATP-dependent hexokinase (glucokinase, Glk) from a thermophilic bacterium Glk was heterologously expressed in Escherichia coli BL21(DE3). Glk exhibited high specificity for glucose, dependence on Mg2+, and the highest activity at pH 8.5 and 80 ℃. It retained over 90% activity after storage at 30-37 ℃ for 7 days, demonstrating thermal stability as an alkaline glucose kinase. Subsequently, the factors influencing Glk expression, including culture medium, OD600, final concentration of the inducer, induction temperature, and induction duration, were systematically optimized. The optimization increased the Glk expression by 4.71 folds Glk compared with non-optimized conditions. After purification, Glk exhibited a specific activity of (43.05±2.00) U/mg and the purity ≥98%. In conclusion, the developed expression and purification method for the highly thermostable hexokinase provides more possibilities for overcoming the shortcomings in the preparation of blood glucose diagnostic reagents in China.

A systematic review and meta-analysis:comparing the efficacy of the Ilizarov technique alone with lengthening over a nail for lower extremity bone defects.

PURPOSE: The task faced by surgeons becomes significantly more challenging when they encounter lower extremity bone defects due to a variety of causes requiring lengthening. The most discussed and successful approach is the Illizarov technique, or lengthening over a nail (LON):distraction osteogenesis is also widely performed with monoliteral external fixators and intramedullarylengthening nails have increasingly been used in the last decade. METHODS: The data were collected from PubMed, Cochrane Library, Embase, and the Web of Science for all available studies comparing the outcomes of Ilizarov technique alone and LON technique (from January 1, 1997, to November 30, 2023). The outcomes of interest encompassed the external fixation index (EFI) (month/cm), mean duration of follow-up (MFT) (month), length gained (LG) (cm), consolidation index (CIx) (month/cm), and bone healing index (BHI) (month/cm).Complications include pin tract infection rate (PTI), axial deviation rate (AD), occurrence of intramedullary infection (II), delayed consolidation rate (DC), as well as data categorized into three levels of problems, obstacles, and sequelae based on the severity of complications.Two reviewers independently assessed each study for quality and extracted data. The case-control or respective cohort studies were evaluated using the Newcastle-Ottawa scale (NOS) to determine their techniqueological rigor.The Cochrane Collaboration's risk assessment tool was employed to perform quality evaluations for randomized controlled trials. RESULTS: This review included thirteen studies comprising a total of 629 patients.The external fixation index (month/cm) was significantly smaller in the LON technique compared to the Ilizarov technique alone [Mean Difference(MD) = -29.59, 95% CI -39.68--19.49, P < 0.00001].In terms of the mean follow-up time(month) (MD = -0.92, 95% CI -3.49-1.65, P = 0.57), length gained (cm) (MD = -0.87, 95%CI -2.80-1.07, P = 0.38), consolidation index (month/cm) (MD = 0.66, 95% CI -3.44-4.77, P = 0.75), and bone healing index (month/cm) (MD = -3.33, 95% CI -13.07-6.41, P = 0.5), there were no significant differences observed. The LON technique exhibited a lower incidence of axial deviation [Odds Ratio(OR) = 0.06, 95%CI 0.03-0.16, P < 0.00001] and pin tract infection (OR = 0.30, 95%CI 0.18-0.50, P < 0.00001) compared to the Ilizarov technique alone.The remaining complications, such as intramedullary infection rate (OR = 0.93, 95%CI 0.42-2.06, P = 0.85) and delayed consolidation rate(OR = 0.61, 95%CI 0.20-1.86, P = 0.38), did not exhibit statistically significant differences.Our findings demonstrated that the LON technique results in lower incidences of problems (38.5%vs.58.6%) and sequelae (16.6% vs.30.9%) when compared to the Ilizarov technique alone. However, the rates of obstacles (32.4% vs.32.3%) were comparable between the two methods. CONCLUSIONS: Our findings indicate that patients treated with the LON technique experienced significantly shorter external fixation durations and a lower incidence of complications (e.g., pin tract infections and axial deviation) compared to those treated with the Ilizarov technique alone. Other outcome metrics showed no significant differences between the two techniques. However, the LON technique offers substantial benefits, including reduced external fixation times and increased comfort, which enhance patient compliance. In conclusion, the LON technique is a safe, reliable, and effective method for treating tibial and femoral defects.

Identification and Characterization of an R-M System in Paracoccus denitrifican DYTN-1 to Improve the Plasmid Conjugation Transfer Efficiency.

Paracoccus denitrificans has been identified as a representative strain with heterotrophic nitrification-aerobic denitrification capabilities (HN-AD), and demonstrates strong denitrification proficiency. Previously, we isolated the DYTN-1 strain from activated sludge, and it has showcased remarkable nitrogen removal abilities and genetic editability, which positions P. denitrificans DYTN-1 as a promising chassis cell for synthetic biology engineering, with versatile pollutant degradation capabilities. However, the strain's low stability in plasmid conjugation transfer efficiency (PCTE) hampers gene editing efficacy, and is attributed to its restriction modification system (R-M system). To overcome this limitation, we characterized the R-M system in P. denitrificans DYTN-1 and identified a DNA endonuclease and 13 DNA methylases, with the DNA endonuclease identified as HNH endonuclease. Subsequently, we developed a plasmid artificial modification approach to enhance conjugation transfer efficiency, which resulted in a remarkable 44-fold improvement in single colony production. This was accompanied by an increase in the frequency of positive colonies from 33.3% to 100%. Simultaneously, we cloned, expressed, and characterized the speculative HNH endonuclease capable of degrading unmethylated DNA at 30°C without specific cutting site preference. Notably, the impact of DNA methylase M9 modification on the plasmid was discovered, significantly impeding the cutting efficiency of the HNH endonuclease. This revelation unveils a novel R-M system in P. denitrificans and sheds light on protective mechanisms employed against exogenous DNA invasion. These findings pave the way for future engineering endeavors aimed at enhancing the DNA editability of P. denitrificans.

Rewiring the reductive TCA pathway and glyoxylate shunt of Escherichia coli for succinate production from corn stover hydrolysate using a two-phase fermentation strategy.

Succinate was found extensive applications in the food additives, pharmaceutical, and biopolymers industries. However, the succinate biosynthesis in E. coli required IPTG, lacked NADH, and produced high yields only under anaerobic conditions, unsuitable for cell growth. To overcome these limitations, the glyoxylate shunt and reductive TCA pathway were simultaneously enhanced to produce succinate in both aerobic and anaerobic conditions and achieve a high cell growth meanwhile. On this basis, NADH availability and sugars uptake were increased. Furthermore, an oxygen-dependent promoter was used to dynamically regulate the expression level of key genes of reductive TCA pathway to avoid the usage of IPTG. The final strain E. coli Mgls7-32 could produce succinate from corn stover hydrolysate without an inducer, achieving a titer of 72.8 g/L in 5 L bioreactor (1.2 mol/mol of total sugars). Those findings will aid in the industrial production of succinate.

Engineering two-component systems for advanced biosensing: From architecture to applications in biotechnology.

Two-component systems (TCSs) are prevalent signaling pathways in bacteria. These systems mediate phosphotransfer between histidine kinase and a response regulator, facilitating responses to diverse physical, chemical, and biological stimuli. Advancements in synthetic and structural biology have repurposed TCSs for applications in monitoring heavy metals, disease-associated biomarkers, and the production of bioproducts. However, the utility of many TCS biosensors is hindered by undesired performance due to the lack of effective engineering methods. Here, we briefly discuss the architectures and regulatory mechanisms of TCSs. We also summarize the recent advancements in TCS engineering by experimental or computational-based methods to fine-tune the biosensor functional parameters, such as response curve and specificity. Engineered TCSs have great potential in the medical, environmental, and biorefinery fields, demonstrating a crucial role in a wide area of biotechnology.

Enhancing the activity and succinyl-CoA specificity of 3-ketoacyl-CoA thiolase Tfu_0875 through rational binding pocket engineering.

The 3-ketoacyl-CoA thiolase is the rate-limiting enzyme for linear dicarboxylic acids production. However, the promiscuous substrate specificity and suboptimal catalytic performance have restricted its application. Here we present both biochemical and structural analyses of a high-efficiency 3-ketoacyl-CoA thiolase Tfu_0875. Notably, Tfu_0875 displayed heightened activity and substrate specificity for succinyl-CoA, a key precursor in adipic acid production. To enhance its performance, a deep learning approach (DLKcat) was employed to identify effective mutants, and a computational strategy, known as the greedy accumulated strategy for protein engineering (GRAPE), was used to accumulate these effective mutants. Among the mutants, Tfu_0875N249W/L163H/E217L exhibited the highest specific activity (320% of wild-type Tfu_0875), the greatest catalytic efficiency (kcat/KM = 1.00 min-1mM-1), the highest succinyl-CoA specificity (KM = 0.59 mM, 28.1% of Tfu_0875) and dramatically reduced substrate binding energy (-30.25 kcal mol-1v.s. -15.94 kcal mol-1). A structural comparison between Tfu_0875N249W/L163H/E217L and the wild type Tfu_0875 revealed that the increased interaction between the enzyme and succinyl-CoA was the primary reason for the enhanced enzyme activity. This interaction facilitated rapid substrate anchoring and stabilization. Furthermore, a reduced binding pocket volume improved substrate specificity by enhancing the complementarity between the binding pocket and the substrate in stereo conformation. Finally, our rationally designed mutant, Tfu_0875N249W/L163H/E217L, increased the adipic acid titer by 1.35-fold compared to the wild type Tfu_0875 in shake flask. The demonstrated enzymatic methods provide a promising enzyme variant for the adipic acid production. The above effective substrate binding pocket engineering strategy can be beneficial for the production of other industrially competitive biobased chemicals when be applied to other thiolases.

Improvement of Chalcone Synthase Activity and High-Efficiency Fermentative Production of (2S)-Naringenin via In Vivo Biosensor-Guided Directed Evolution.

Chalcone synthase (CHS) catalyzes the rate-limiting step of (2S)-naringenin (the essential flavonoid skeleton) biosynthesis. Improving the activity of the CHS by protein engineering enhances (2S)-naringenin production by microbial fermentation and can facilitate the production of valuable flavonoids. A (2S)-naringenin biosensor based on the TtgR operon was constructed in Escherichia coli and its detection range was expanded by promoter optimization to 0-300 mg/L, the widest range for (2S)-naringenin reported. The high-throughput screening scheme for CHS was established based on this biosensor. A mutant, SjCHS1S208N with a 2.34-fold increase in catalytic activity, was discovered by directed evolution and saturation mutagenesis. A pathway for de novo biosynthesis of (2S)-naringenin by SjCHS1S208N was constructed in Saccharomyces cerevisiae, combined with CHS precursor pathway optimization, increasing the (2S)-naringenin titer by 65.34% compared with the original strain. Fed-batch fermentation increased the titer of (2S)-naringenin to 2513 ± 105 mg/L, the highest reported so far. These findings will facilitate efficient flavonoid biosynthesis and further modification of the CHS in the future.

Metabolic Pathway Coupled with Fermentation Process Optimization for High-Level Production of Retinol in Yarrowia lipolytica.

Retinol is a lipid-soluble form of vitamin A that is crucial for human visual and immune functions. The production of retinol through microbial fermentation has been the focus of recent exploration. However, the obtained titer remains limited and the product is often a mixture of retinal, retinol, and retinoic acid, necessitating purification. To achieve efficient biosynthesis of retinol in Yarrowia lipolytica, we improved the metabolic flux of ß-carotene to provide sufficient precursors for retinol in this study. Coupled with the optimization of the expression level of ß-carotene 15,15'-dioxygenase, de novo production of retinol was achieved. Furthermore, Tween 80 was used as an extractant and butylated hydroxytoluene as an antioxidant to extract intracellular retinol and prevent retinol oxidation, respectively. This strategy significantly increased the level of retinol production. By optimizing the enzymes converting retinal to retinol, the proportion of extracellular retinol in the produced retinoids reached 100%, totaling 1042.3 mg/L. Finally, total retinol production reached 5.4 g/L through fed-batch fermentation in a 5 L bioreactor, comprising 4.2 g/L extracellular retinol and 1.2 g/L intracellular retinol. This achievement represents the highest reported titer so far and advances the industrial production of retinol.

Dynamic regulation and cofactor engineering of escherichia coli to enhance production of glycolate from corn stover hydrolysate.

Glycolic acid is widely employed in chemical cleaning, the production of polyglycolic acid-lactic acid, and polyglycolic acid. Currently, the bottleneck of glycolate biosynthesis lies on the imbalance of metabolic flux and the deficiency of NADPH. In this study, a dynamic regulation system was developed and optimized to enhance the metabolic flux from glucose to glycolate. Additionally, the knockout of transhydrogenase (sthA), along with the overexpression of pyridine nucleotide transhydrogenase (pntAB) and the implementation of the Entner-Doudoroff pathway, were performed to further increase the production of the NADPH, thereby increasing the titer of glycolate to 5.6 g/L. To produce glycolate from corn stover hydrolysate, carbon catabolite repression was alleviated and glucose utilization was accelerated. The final strain, E. coli Mgly10-245, is inducer-free, achieving a glycolate titer of 46.1 g/L using corn stover hydrolysate (77.1 % of theoretical yield). These findings will contribute to the advancement of industrial glycolate production.

High-Level De Novo Production of (2S)-Eriodictyol in Yarrowia Lipolytica by Metabolic Pathway and NADPH Regeneration Engineering.

(2S)-Eriodictyol, a polyphenolic flavonoid, has found widespread applications in health supplements and food additives. However, the limited availability of plant-derived (2S)-eriodictyol cannot meet the market demand. Microbial production of (2S)-eriodictyol faces challenges, including the low catalytic efficiency of flavone 3'-hydroxylase/cytochrome P450 reductase (F3'H/CPR), insufficient precursor supplementation, and inadequate NADPH regeneration. This study systematically engineered Yarrowia lipolytica for high-level (2S)-eriodictyol production. In doing this, the expression of F3'H/CPR was balanced, and the supply of precursors was enhanced by relieving feedback inhibition of the shikimate pathway, promoting fatty acid ß-oxidation, and increasing the copy number of synthetic pathway genes. These strategies, combined with NADPH regeneration, achieved an (2S)-eriodictyol titer of 423.6 mg/L. Finally, in fed-batch fermentation, a remarkable 6.8 g/L (2S)-eriodictyol was obtained, representing the highest de novo microbial titer reported to date and paving the way for industrial production.