Synthesis of 3-Phenylserine by a Two-enzyme Cascade System with PLP Cofactor.

A two-enzyme cascade system containing ω-transaminase (ω-TA) and L-threonine aldolase (L-ThA) was reported for the synthesis of 3-Phenylserine starting from benzylamine, and PLP was utilized as the only cofactor in these both two enzymes reaction system. Based on the transamination results, benzylamine was optimized as an advantageous amino donor as confirmed by MD simulation results. This cascade reaction system could not only facilitate the in situ removal of the co-product benzaldehyde, enhancing the economic viability of the reaction, but also establish a novel pathway for synthesizing high-value phenyl-serine derivatives. In our study, nearly 95 % of benzylamine was converted, yielding over 54 % of 3-Phenylserine under the optimized conditions cascade reaction.

Influence of Different Ions on Pulse Electrodeposition of CaCO3 Scales in the Simulated Seawater.

In the circulating water system of coastal power plants, various kinds of ions have a great influence on the formation and growth of CaCO3 scales. This paper focuses on investigating the influence of existing ions on the pulse electrodeposition behaviors of CaCO3 scales. Different concentrations of ions, such as Fe3+, Mg2+, PO43- and SiO32-, are introduced to simulate the actual seawater environment, and their influence on the CaCO3 scale deposition behaviors is assessed by linear sweep voltammetry, chronoamperometry, and electrochemical impedance spectroscopy tests. The surface coverage of the CaCO3 scale layer is evaluated through the residual current density and polarization resistance values, while the crystal structure and surface compactness of the layer are confirmed by the scanning electron microscope and X-ray diffractometer tests. Results indicate that high concentrations of Mg2+, Fe3+, and PO43- ions have the most significant inhibitory effect on the pulse electrodeposition of CaCO3 scales, among which the inhibition effect of Mg2+ ions is mainly reflected in the change of crystal morphology of CaCO3, that is, the crystallization growth process is inhibited. The inhibition effect of PO43- ions is mainly reflected in the gradually reduced coverage and density of CaCO3 crystals on the electrode surface, suggesting that the crystallization nucleation process is inhibited, while Fe3+ ions have a certain inhibition effect on both the crystallization nucleation and growth processes. Furthermore, lower concentrations of SiO32- ions also display a significant inhibition effect on the crystallization nucleation and growth process, and the inhibition effect weakens with increased concentration. This study provides a theoretical basis for exploring the removal of ions in the industrial water softening field.

Construction of a Synthetic Microbial Community for Enzymatic Pretreatment of Wheat Straw for Biogas Production via Anaerobic Digestion.

Biological pretreatment is a viable method for enhancing biogas production from straw crops, with the improvement in lignocellulose degradation efficiency being a crucial factor in this process. Herein, a metagenomic approach was used to screen core microorganisms (Bacillus subtilis, Acinetobacter johnsonii, Trichoderma viride, and Aspergillus niger) possessing lignocellulose-degrading abilities among samples from three environments: pile retting wheat straw (WS), WS returned to soil, and forest soil. Subsequently, synthetic microbial communities were constructed for fermentation-enzyme production. The crude enzyme solution obtained was used to pretreat WS and was compared with two commercial enzymes. The synthetic microbial community enzyme-producing pretreatment (SMCEP) yielded the highest enzymatic digestion efficacy for WS, yielding cellulose, hemicellulose, and lignin degradation rates of 39.85, 36.99, and 19.21%, respectively. Furthermore, pretreatment of WS with an enzyme solution, followed by anaerobic digestion achieved satisfactory results. SMCEP displayed the highest cumulative biogas production at 801.16 mL/g TS, which was 38.79% higher than that observed for WS, 22.15% higher than that of solid-state commercial enzyme pretreatment and 25.41% higher than that of liquid commercial enzyme pretreatment. These results indicate that enzyme-pretreated WS can significantly enhance biogas production. This study represents a solution to the environmental burden and energy use of crop residues.

Multiple medicinal chemistry strategies of targeting KRAS: State-of-the art and future directions.

KRAS is the most frequently mutated oncogene and drives the development and progression of malignancies, most notably non-small cell lung cancer (NSCLS), pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC). However, KRAS proteins have maintained the reputation of being "undruggable" due to the lack of suitable deep pockets on its surface. One major milestone for KRAS inhibition was the discovery of the covalent inhibitors bond to the allosteric switch-II pocket of the KRASG12C protein. To date, the FDA has approved two KRASG12C inhibitors, sotorasib and adagrasib, for the treatment of patients with KRASG12C-driven cancers. Researchers have paid close attention to the development of inhibitors for other KRAS mutations and upstream regulatory factors. The KRAS targeted drug discovery has entered a state of rapid development. This article has aimed to present the current state of the art of drug development in the KRAS field. We systematically summarize recent advances in the discovery and optimization processes of direct KRAS inhibitors (including KRASG12C, KRASG12D, KRASG12A and KRASG12R inhibitors), indirect KRAS inhibitors (SOS1 and SHP2 inhibitors), pan-KRAS inhibitors, as well as proteolysis-targetingchimeras degrades and molecular chaperone modulators from the perspective of medicinal chemistry. We also discuss the current challenges and opportunities of KRAS inhibition and hope to shed light on future KRAS drug discovery.

Recent Advances of Lignin Functionalization for High-Performance and Advanced Functional Rubber Composites.

The biomass lignin is the only large-volume renewable feedstock that is composed of aromatics but has been largely underutilized and is sought for valorization as a value-added material. Recent research has highlighted lignin as a promising alternative to traditional petrol-based reinforcements and functional additives for rubber composites. This review summarized the recent advances in the functionalization of lignin for a variety of rubber composites, as well as the compounding techniques for effectively dispersing lignin within the rubber matrix. Significant progress has been achieved in the development of high-performance and advanced functional rubber/lignin composites through carefully designing the structure of lignin-based additives and the optimization of interfacial morphologies. This Review discussed the effect of lignin on composite properties, including mechanical reinforcement, dynamic properties, antiaging performance, and oil resistance, and also the advanced stimuli-responsive performance in detail. A critical analysis for the future development of rubber/lignin composites is presented as concluding remarks.

Computational aided design of a halotolerant CMP kinase for enzymatic synthesis of cytidine triphosphate.

The current biocatalytic method of industrial Cytidine triphosphate (CTP) production suffers from reaction rate loss. It is caused by gradually increasing acetate salt concentration, which inhibits enzyme activities and decreases the final yield. This work gave a possible solution to this problem through computational aided design of CMP kinase (CMPK), an enzyme in the CTP production system, to increase its stability in solution with high acetate salt concentration. Enlightened by the features of natural halophilic enzymes, the basic and neutral surface residues were replaced with acidic amino acids. This protein design strategy effectively increased the activity of CMPK in the working condition (acetate concentration over 1200 mM). The halotolerant CMPK was applied in fed-batch production of CTP. The maximum titer was 201.4 ± 1.6 mM, and the productivity was 12.6 mM L-1 h-1, increased 26.4% and 27.8% from the process using wild-type CMPK, respectively.

Biofilm-Based Biocatalysis for Galactooligosaccharides Production by the Surface Display of ß-Galactosidase in Pichia pastoris.

Galactooligosaccharides (GOS) are one of the most important functional oligosaccharide prebiotics. The surface display of enzymes was considered one of the most excellent strategies to obtain these products. However, a rough industrial environment would affect the biocatalytic process. The catalytic process could be efficiently improved using biofilm-based fermentation with high resistance and activity. Therefore, the combination of the surface display of ß-galactosidase and biofilm formation in Pichia pastoris was constructed. The results showed that the catalytic conversion rate of GOS was up to 50.3% with the maximum enzyme activity of 5125 U/g by screening the anchorin, and the number of the continuous catalysis batches was up to 23 times. Thus, surface display based on biofilm-immobilized fermentation integrated catalysis and growth was a co-culture system, such that a dynamic equilibrium in the consolidated integrative process was achieved. This study provides the basis for developing biofilm-based surface display methods in P. pastoris during biochemical production processes.

Reversible Adsorption and Detachment of Saccharomyces cerevisiae on Thermoresponsive Poly(N-isopropylacrylamide)-Grafted Fibers for Continuous Immobilized Fermentation.

Biofilm-mediated continuous fermentation with cells immobilized has gained much attention in recent years. In this study, thermoresponsive poly(N-isopropylacrylamide)-grafted cotton fibers (PNIPAM-CF) were prepared via an improved surface-initiated atom transfer radical polymerization. The modification process imparted switchable wettability to the surface while maintaining the thermal stability and biocompatibility of the CF. During the ethanol transformation, the rapid, reversible cell adsorption and detachment of Saccharomyces cerevisiae were performed through the modulation of wettability, displaying the enhancement of immobilized biomass and immobilization efficiency from 2.20 g/L and 59.43% to 2.81 g/L and 93.32%, respectively. Moreover, the biofilm adsorption matched well with the Freundlich model, indicating that multilayer adhesion was the main mode of biofilm formation. Based on the accumulation of the biofilm, the fabrication and utilization of PNIPAM-CF improved the efficiency of continuous immobilized fermentation, making the ethanol production reach 26.34 g/L in the sixth batch of fermentation. Meanwhile, wettability regulation further enhanced the reusability of the carrier. Therefore, the findings of this study revealed that the application of smart materials in cell immobilization systems had broad prospects for achieving sustainable and continuous catalysis.

Strategy for the design of a bioreactor for L-lysine immobilized fermentation using Corynebacterium glutamicum.

Biofilm-immobilized fermentation is a novel strategy that has been utilized in L-lysine fermentation. In this study, we describe a strategy for designing bioreactors for immobilized fermentation. We have constructed steel structures in which the carriers can be sewn, forming several star-like structures with different angles, and changing the ventilation robot to the aeration tray. In a 10-L bioreactor, this structure with 12 angles assisted the immobilized system to remedy the gap between free-cell and immobilized fermentation in the conversion rate. In a 50-L bioreactor, this enlarged structure with 16 angles illustrated a 4.61% higher conversion rate than the free-cell fermentation (67.75%) and increased the production by 28.56%. This successful case is the first step towards to industrial production of biofilm-based immobilized fermentation.Key points• The designed steel structure is useful for L-lysine immobilized fermentation in a 10-L bioreactor.• The conversion rate of immobilized fermentation increased from 13.99 to 60.07% and is 1.03% higher than that of the free-cell fermentation.• The conversion rate of the redesigned 50-L bioreactor is higher than that of free-cell fermentation.

Design and optimizing a new CDP-choline in vitro multienzyme producing process starts from d-ribose.

This work designs an in vitro multienzyme system to produce CDP-choline from d-ribose and develop an optimization procedure for one-pot multienzyme catalytic system. The entire process integrated 10 enzymes, and an efficient acetate kinase/acetyl phosphate-based ATP regeneration module was applied. Then, some optimizations to this system were made including selecting optimum enzyme building blocks and improving expression parameters. The process improved the final yield of CDP-choline from 0.2 to 6 g/L (CDP-choline titer 12.2 mM). This new one-pot CDP-choline producing system has a potential for industrial use, and the optimization procedure shed light on improving other one-pot multienzyme system for industrial production of energy rich compounds.

Effect of quorum-sensing molecule 2-phenylethanol and ARO genes on Saccharomyces cerevisiae biofilm.

Biofilms are a form of microbial community that can be beneficial for industrial fermentation because of their remarkable environmental resistance. However, the mechanism of biofilm formation in Saccharomyces cerevisiae remains to be fully explored, and this may enable improved industrial applications for this organism. Although quorum-sensing (QS) molecules are known to be involved in bacteria biofilm formation, few studies have been undertaken with these in fungi. 2-phenylethanol (2-PE) is considered a QS molecule in S. cerevisiae. Here, we found that exogenous 2-PE could stimulate biofilm formation at low cell concentrations. ARO8p and ARO9p are responsible for the synthesis of 2-PE and were crucial to the formation of biofilm. Deletion of the ARO8 and ARO9 genes reduced the content of 2-PE in the early stage of fermentation, reduced ethanol yield and decreased biofilm formation. The expression of FLOp, which is involved in cell adhesion, and the content of extracellular polysaccharides of mutant strains ΔARO8 and ΔARO9 were also significantly reduced. These findings indicate that the production of 2-PE had a positive effect on biofilm formation in S. cerevisiae, thereby providing further key details for studying the formation of biofilm mechanism in the future. KEY POINTS: • Quorum-sensing molecule 2-PE positively affects biofilm formation in S. cerevisiae. • 2-PE synthetic genes ARO8 and ARO9 deletion reduced extracellular polysaccharide. • ARO8 and ARO9 deletion reduced the gene expression of the FLO family.

Biofilm-based fermentation: a novel immobilisation strategy for Saccharomyces cerevisiae cell cycle progression during ethanol production.

Biofilm-based fermentation, as a new immobilisation strategy, is beneficial for industrial fermentation due to its excellent environmental resistance, high productivity and continuous fermentation relative to calcium alginate-immobilised fermentation. These two techniques differ mainly regarding cell stages. Here, we describe the cell phenotype of Saccharomyces cerevisiae biofilm-based fermentation and compare cell cycle stages with those during immobilisation in calcium alginate. Most cells in the biofilm-based fermentation adhered to the cotton-fibre carrier of the biofilm and were in the G2/M phase whereas alginate-embedded cells were in the G1/G0 phase. Deletion of the RIM15 gene, which regulates cell cycle progression according to nutritional status, hampered the cell cycle arrest observed in alginate-embedded cells, enhanced biofilm formation and improved fermentation ability. The improved biofilm formation shown by the rim15△ strain could be attributed to an increase in the expression level of the adhesion protein FLO11 and synthesis of trehalose. These findings suggest that the extracellular environment is mainly responsible for the difference between biofilm-based fermentation and alginate-embedded fermentation, and that RIM15 plays an essential role in cell cycle progression. KEY POINTS: • In the biofilm, S. cerevisiae cell populations were mostly in the G2/M phase while alginate-embedded cells were arrested in the G1/G0 phase. • The RIM15 gene partially influenced the cell cycle progression observed during ethanol fermentation. • Biofilm-based cells were actively adsorbed on the physical carrier. • Biofilm immobilisation could maintain cell division activity explaining its fermentation efficiency.

Isolation and characterization of plant growth-promoting rhizobacteria and their effects on the growth of Medicago sativa L. under salinity conditions.

Plant growth-promoting rhizobacteria are a group of free-living bacteria that colonize plant rhizosphere and benefit plant root growth, thereby increasing host plant to cope with salinity induced stress. The aim of this study was to (1) isolate and characterize auxin-producing bacteria showing a high plant growth-promoting (PGP) potential, and (2) evaluate the PGP effects on the growth of Medicago sativa L under salinity stress (130 mM NaCl). Of thirteen isolates, Bacillus megaterium NRCB001 (NRCB001), B. subtilis subsp. subtilis NRCB002 (NRCB002) and B. subtilis NRCB003 (NRCB003) had the ability to produce auxin, which ranged from 47.53 to 154.38 µg ml-1. The three auxin-producing bacterial strains were shown multiple PGP traits, such as producing siderophore and NH3, showing ACC deaminase activity, solubilize phosphate and potassium. Furthermore, NRCB001, NRCB002, and NRCB003 could survive in LB medium containing 1750 mM NaCl. The three auxin-producing with salinity tolerance strains were selected for further analyses. In greenhouse experiments, when inoculated with NRCB001, NRCB002 and NRCB003, dry weight of alfalfa significantly (P < 0.05) increased by 24.1%, 23.1% and 38.5% respectively, compared with those of non-inoculated control seedlings under normal growth condition. When inoculated with NRCB002 and NRCB003, dry weight of alfalfa significantly (P < 0.05) increased by 96.9 and 71.6% respectively, compared with those of non-inoculated control seedlings under 130 mM NaCl condition. Our results indicated that NRCB002 and NRCB003 having PGP traits are promising candidate strains to develop biofertilizers, especially used under salinity stress conditions.

Knockout of pde gene in Arthrobacter sp. CGMCC 3584 and transcriptomic analysis of its effects on cAMP production.

Arthrobacter sp. CGMCC 3584 is used for the industrial production of cyclic adenosine monophosphate (cAMP). However, because of the paucity of genetic engineering tools for genetic manipulation on Arthrobacter species, only a few metabolically engineered Arthrobacter have been constructed and investigated. In this study, for the first time, we constructed an arpde knockout mutant of Arthrobacter without any antibiotic resistance marker by a PCR-targeting-based homologous recombination method. Our results revealed that the deletion of arpde had little effect on biomass production and improved cAMP production by 31.1%. Furthermore, we compared the transcriptomes of the arpde knockout strain and the wild strain, aiming to understand the capacities of cAMP production due to arpde inactivation at the molecular level. Comparative transcriptomic analysis revealed that arpde inactivation had two major effects on metabolism: inhibition of glycolysis, PP pathway, and amino acid metabolism (phenylalanine, tryptophan, branched-chain amino acids, and glutamate metabolism); promotion of the purine metabolism and carbon flux from the precursor 5'-phosphoribosyl 1-pyrophosphate, which benefited cAMP production.

Effects of Spo0A on Clostridium acetobutylicum with an emphasis on biofilm formation.

Clostridium acetobutylicum is a well-known strain for biofuel production. In previous work, it was found that this strain formed biofilm readily during fermentation processes. Biofilm formation could protect cells and enhance productivities under environmental stresses in our previous work. To explore the molecular mechanism of biofilm formation, Spo0A of C. acetobutylicum was selected to investigate its influences on biofilm formation and other physiological performances. When spo0A gene was disrupted, the spo0A mutant could hardly form biofilm. The aggregation and adhesion abilities of the spo0A mutant as well as its swarming motility were dramatically reduced compared to those of wild type strain. Sporulation was also negatively influenced by spo0A disruption, and solvent production was almost undetectable in the spo0A mutant fermentation. Furthermore, proteomic differences between wild type strain and the spo0A mutant were consistent with physiological performances. This is the first study confirming a genetic clue to C. acetobutylicum biofilm and will be valuable for biofilm optimization through genetic engineering in the future.

RNA accumulation in Candida tropicalis based on cofactor engineering.

Redox cofactors play an important role in biosynthetic and catabolic reactions and the transfer of energy for the cell. Therefore, studying the relationship between cofactor perturbation and metabolism is a useful approach to improve the yield of target products. To study RNA accumulation and metabolism when intracellular cofactor balance was impaired, the water-forming NADH oxidase (NoxE) from Lactococcus lactis and membrane-bound transhydrogenase (PntAB) from Escherichia coli were expressed in Candidatropicalis no. 121, respectively. Expression of noxE significantly decreased the intracellular NADH/NAD+ ratio, but the NADPH/NADP+ ratio did not differ significantly. PntAB increased the intracellular NADH pool, while the NADPH/NADP+ ratio decreased. The perturbation of the cofactors caused a large redistribution of metabolic fluxes. The biomass and RNA content decreased by 11.0% and 10.6% in pAUR-noxE strain, respectively, while the RNA content increased by 5.5% and the biomass showed no signification difference in pAUR-pntAB strain. Expression of noxE and pntAB led to decreases and increases in the ATP concentration and yield of RNA, respectively, which also indicated that ATP plays an important role in the RNA biosynthesis.

Competitive adsorption of vanillin and syringaldehyde on a macro-mesopore polymeric resin: modeling.

Vanillin and syringaldehyde are widely used as flavoring and fragrance agents in the food products. The potential of a macro-mesoporous adsorption resin was assessed for separation of these binary mixtures. This work focuses on modeling of the competitive adsorption behaviors and exploration of the adsorption mechanism. The characterization results showed the resin had a large BET surface area and specific pore structure with hydrophobic properties. By analysis of the physicochemical properties of the solutes and the resin, the separation mechanism was mainly contributed by hydrophobic effect. Subsequently, the competitive Langmuir isotherm model was used to fit the competitive adsorption isotherms. The pore diffusion coefficient was obtained by macropore diffusion model. Afterwards, a mathematical model was established to predict the breakthrough curves of the binary mixture at various operating conditions. The data and model presented are valuable for design and simulation of the continuous chromatographic separation process.

Towards acetone-uncoupled biofuels production in solventogenic Clostridium through reducing power conservation.

Microbial production of butanol by solventogenic Clostridium has long been complicated with the formation of acetone as an unwanted product, which causes poor product yields and creates a most important problem concerning substrate transformation. Intensive attempts concentrate on carbon conversion pathways to eliminate acetone, but have actually achieved little so far. Here, we believe microbial product distribution can largely depend on how the cell plays its energetic cofactors in central metabolism, and demonstrate that by introducing a synthetic 2,3-butanediol synthesis pathway in Clostridium acetobutylicum as an NADH-compensating module to readjust the reducing power at a systems level, the production of acetone can be selectively and efficiently eliminated (< 0.3 g/L). H2 evolution was reduced by 78%, and the total alcohol yield was strikingly increased by 19% to 0.44 g/g glucose, much higher than those yet reported for butanol fermentation. These findings highlight that it is the loss of reducing power rather than typically manipulated solventogenesis genes that dominates acetone formation. Further study revealed that the NADH-module triggered apparent regulation of pathways involved in electron transfer and reducing power conservation. The study also suggested the key to conservation of intracellular reducing power might essentially lie in the intermediate processes in central metabolism that are related to redox partners, butyrate or C4 branches, and possibly NADH and NADPH specificity. This study represents the first effective redox-based configuration of C. acetobutylicum and provides valuable understandings for redox engineering of native Clostridium species towards advanced production of biofuels and alcohols.

Regulating Cofactor Balance In Vivo with a Synthetic Flavin Analogue.

A novel strategy to regulate cofactor balance in vivo for whole-cell biotransformation using a synthetic flavin analogue is reported. High efficiency, easy operation, and good applicability were observed for this system. Confocal laser scanning microscopy was employed to verify that the synthetic flavin analogue can directly permeate into Escherichia coli cells without modifying the cell membrane. This work provides a promising intracellular redox regulatory approach to construct more efficient cell factories.