Intein-mediated temperature control for complete biosynthesis of sanguinarine and its halogenated derivatives in yeast.

While sanguinarine has gained recognition for antimicrobial and antineoplastic activities, its complex conjugated structure and low abundance in plants impede broad applications. Here, we demonstrate the complete biosynthesis of sanguinarine and halogenated derivatives using highly engineered yeast strains. To overcome sanguinarine cytotoxicity, we establish a splicing intein-mediated temperature-responsive gene expression system (SIMTeGES), a simple strategy that decouples cell growth from product synthesis without sacrificing protein activity. To debottleneck sanguinarine biosynthesis, we identify two reticuline oxidases and facilitated functional expression of flavoproteins and cytochrome P450 enzymes via protein molecular engineering. After comprehensive metabolic engineering, we report the production of sanguinarine at a titer of 448.64 mg L-1. Additionally, our engineered strain enables the biosynthesis of fluorinated sanguinarine, showcasing the biotransformation of halogenated derivatives through more than 15 biocatalytic steps. This work serves as a blueprint for utilizing yeast as a scalable platform for biomanufacturing diverse benzylisoquinoline alkaloids and derivatives.

Engineering Substrate Promiscuity of Nucleoside Phosphorylase Via an Insertions-Deletions Strategy.

Nucleoside phosphorylases (NPs) are the key enzymes in the nucleoside metabolism pathway and are widely employed for the synthesis of nucleoside analogs, which are difficult to access via conventional synthetic methods. NPs are generally classified as purine nucleoside phosphorylase (PNP) and pyrimidine or uridine nucleoside phosphorylase (PyNP/UP), based on their substrate preference. Here, based on the evolutionary information on the NP-I family, we adopted an insertions-deletions (InDels) strategy to engineer the substrate promiscuity of nucleoside phosphorylase AmPNPΔS2V102 K, which exhibits both PNP and UP activities from a trimeric PNP (AmPNP) of Aneurinibacillus migulanus. Furthermore, the AmPNPΔS2V102 K exerted phosphorylation activities toward arabinose nucleoside, fluorosyl nucleoside, and dideoxyribose, thereby broadening the unnatural-ribose nucleoside substrate spectrum of AmPNP. Finally, six purine nucleoside analogues were successfully synthesized, using the engineered AmPNPΔS2V102 K instead of the traditional "two-enzymes PNP/UP" approach. These results provide deep insights into the catalytic mechanisms of the PNP and demonstrate the benefits of using the InDels strategy to achieve substrate promiscuity in an enzyme, as well as broadening the substrate spectrum of the enzyme.

Alcohol acyltransferases for the biosynthesis of esters.

Esters are widely used in food, energy, spices, chemical industry, etc., becoming an indispensable part of life. However, their production heavily relies on the fossil energy industry, which presents significant challenges associated with energy shortages and environmental pollution. Consequently, there is an urgent need to identify alternative green methods for ester production. One promising solution is biosynthesis, which offers sustainable and environmentally friendly processes. In ester biosynthesis, alcohol acyltransferases (AATs) catalyze the condensation of acyl-CoAs and alcohols to form esters, enabling the biosynthesis of nearly 100 different kinds of esters, such as ethyl acetate, hexyl acetate, ethyl crotonate, isoamyl acetate, and butyl butyrate. However, low catalytic efficiency and low selectivity of AATs represent the major bottlenecks for the biosynthesis of certain specific esters, which should be addressed with protein molecular engineering approaches before practical biotechnological applications. This review provides an overview of AAT enzymes, including their sequences, structures, active sites, catalytic mechanisms, and metabolic engineering applications. Furthermore, considering the critical role of AATs in determining the final ester products, the current research progresses of AAT modification using protein molecular engineering are also discussed. This review summarized the major challenges and prospects of AAT enzymes in ester biosynthesis.

Image Registration for Visualizing Magnetic Flux Leakage Testing under Different Orientations of Magnetization.

The Magnetic Flux Leakage (MFL) visualization technique is widely used in the surface defect inspection of ferromagnetic materials. However, the information of the images detected through the MFL method is incomplete when the defect (especially for the cracks) is complex, and some information would be lost when magnetized unidirectionally. Then, the multidirectional magnetization method is proposed to fuse the images detected under different magnetization orientations. It causes a critical problem: the existing image registration methods cannot be applied to align the images because the images are different when detected under different magnetization orientations. This study presents a novel image registration method for MFL visualization to solve this problem. In order to evaluate the registration, and to fuse the information detected in different directions, the mutual information between the reference image and the MFL image calculated by the forward model is designed as a measure. Furthermore, Particle Swarm Optimization (PSO) is used to optimize the registration process. The comparative experimental results demonstrate that this method has a higher registration accuracy for the MFL images of complex cracks than the existing methods.

Development of a Pichia pastoris cell factory for efficient production of germacrene A: a precursor of ß-elemene.

ß-Elemene, an active ingredient found in medicinal plants like turmeric and zedoary, is a sesquiterpene compound with antitumor activity against various cancers. However, its current mode of production through plant extraction suffers from low efficiency and limited natural resources. Recently, there has been an increased interest in establishing microbial cell factories to produce germacrene A, which can be converted to ß-elemene by a one-step reaction in vitro. In this study, we constructed an engineered Pichia pastoris cell factory for producing germacrene A. We rerouted the fluxes towards germacrene A biosynthesis through the optimization of the linker sequences between germacrene A synthase (GAS) and farnesyl pyrophosphate synthase (ERG20), overexpression of important pathway genes (i.e., IDI1, tHMG1, and ACS), and multi-copy integration of related expression cassettes. In combination with medium optimization and bioprocess engineering, the final titer of germacrene A in a 1 L fermenter reached 1.9 g/L through fed-batch fermentation. This represents the first report on the production of germacrene A in P. pastoris and demonstrates its advantage in producing terpenoids and other value-added natural products.

Engineering the ß-Fructofuranosidase Fru6 with Promoted Transfructosylating Capacity for Fructooligosaccharide Production.

Levan-type fructooligosaccharides (FOS) exhibit enhanced health-promoting prebiotic effects on gut microbiota. The wild type (WT) of ß-fructofuranosidase Fru6 could mainly yield 6-ketose. Semirational design and mutagenesis of Fru6 were exploited to promote the transfructosylating capacity for FOS. The promising variants not only improved the formation of 6-kestose but also newly produced tetrasaccharides of 6,6-nystose and 1,6-nystose (a new type of FOS), and combinatorial mutation boosted the production of 6-kestose and tetrasaccharides (39.9 g/L 6,6-nystose and 4.6 g/L 1,6-nystose). Molecular docking and molecular dynamics (MD) simulation confirmed that the mutated positions reshaped the pocket of Fru6 to accommodate bulky 6-kestose in a reactive conformation with better accessibility for tetrasaccharides formation. Using favored conditions, the variant S165A/H357A could yield 6-kestose up to 335 g/L, and tetrasaccharides (6,6-nystose and 1,6-nystose) reached a high level of 121.1 g/L (134.5 times of the mutant S423A). The ß-(2,6)-linked FOS may show the potential application for the prebiotic ingredients.

Study on Pore Structure and Fractal Characterization during Thermal Evolution of Oil Shale Experiments.

In order to better study the characteristics of the pore structure and to explore the influence factors of its fractal dimensions during the thermal evolution of oil shale, the immature oil shale (T max = 433 °C, TOC = 28.00%) of the Ordos Basin Extension Group was selected to simulate the whole thermal evolution process from immature to over mature in a semiopen system. Organic geochemical data show that the thermal simulation hydrocarbon generation threshold is between 300 and 400 °C. According to AIP-SEM observation, the pore types of the samples are different in different thermal simulation stages. The fractal dimensions are calculated by low-temperature N2 adsorption data using the fractal Frenkel-Halsey-Hill fractal model. The average surface fractal dimension (D 1) is 2.26, indicating that the pore (<4 nm) surface is relatively smooth. The average pore structure fractal dimension (D 2) is 2.49, indicating that the pore (>4 nm) structure is complex. Through the exploration of the relationship between fractal dimensions and organic geochemistry, whole rock X-ray diffraction, and N2 adsorption data, it is found that fractal dimensions have different degrees of correlation with thermal maturity, mineral composition, TOC content, and pore parameters. Through comprehensive research, it shows that hydrocarbon generation and expulsion, oil and gas cracking, and organic matter carbonization have important effects on the pore structure and fractal characteristics of oil shale.

Analysis of Chemical Composition, Amino Acid Content, and Rumen Degradation Characteristics of Six Organic Feeds.

The current study was designed to investigate the chemical composition, amino acid content, and rumen degradation characteristics (nylon bag method) of six organic feeds to illustrate their feeding values. The feeds analyzed were: corn grain (CG), soybean cake (SC), wheat bran (WB), corn silage (CS), oat hay (OT), and alfalfa hay (AF). Our results showed that the contents of crude protein (CP) (47.46%) and ether extract (EE) (8.23%) in SC were highest. The contents of neutral detergent fiber (NDF) (65.00%) and acid detergent fiber (ADF) (39.16%) in OT were highest. The contents of total amino acid (TAA) (42.95%) and essential amino acid (EAA) (19.73%) in SC were highest. Among SC, WB, and CG, the effective degradation rate (ED) of dry matter (DM) is SC (87.89%) > WB (73.32%) > CG (64.25%); the ED value of CP is CG (82.37%) > WB (82.40%) > SC (60.47%). Among CS, OT, and AF, the effective degradation rate (ED) of DM is CS (72.68%) > OT (59.97%) > AF (58.89%); the ED value of CP is AF (76.46%) > CS (72.03%) > OT (71.99%). In conclusion, the chemical composition, amino acid content, and rumen degradation rate of SC and AF were better than those of the other four feeds.

Ginkgo biloba Damo injection combined with troxerutin regulates the TLR4/NF-κB pathway and promotes the recovery of patients with acute cerebral infarction.

OBJECTIVE: Analysis of the effect of Ginkgo biloba Damo injection combined with troxerutin on patients with acute cerebral infarction (ACI) by regulating the TLR4/NF-κB signaling pathway. METHODS: 150 patients with ACI were recruited for a prospective study. These patients were randomly divided into Ginkgo biloba Damo treatment group (n=75) and combined treatment group (Ginkgo biloba Damo + troxerutin, n=75) bilobabiloba. The patients in both groups were treated with conventional treatment. In addition, patients in the Ginkgo biloba Damo treatment group were treated with Ginkgo biloba Damo injection, and those in the combined treatment group were treated with Ginkgo biloba Damo injection combined with troxerutin. The National Institutes of Health Stroke Scale (NIHSS) and Barthel Index (BI) of the two groups were compared. The serum levels of GSH-Px, OX-LDL, CAT, TLR4, NF-κB p65, TNF-α, IL-1ß and IL-6 were detected. The therapeutic effects of the two groups were compared. RESULTS: After treatment, compared with the Ginkgo biloba Damo group, the combined treatment group had a lower NIHSS score and lower expression levels of OX-LDL, TLR4, NF-κB p65, TNF-α, IL-1ß, and IL-6, but higher Barthel score and total effective rate as well as higher serum levels of GSH-Px and CAT (all P<0.05). CONCLUSION: Ginkgo biloba Damo injection combined with troxerutin can improve the neurologic function and activities of daily life in patients with ACI, which can also regulate the TLR4/NF-κB signaling pathway and downstream inflammatory factors to reduce brain tissue inflammation and oxidative stress damage.

Efficient Synthesis of Purine Nucleoside Analogs by a New Trimeric Purine Nucleoside Phosphorylase from Aneurinibacillus migulanus AM007.

Purine nucleoside phosphorylases (PNPs) are promising biocatalysts for the synthesis of purine nucleoside analogs. Although a number of PNPs have been reported, the development of highly efficient enzymes for industrial applications is still in high demand. Herein, a new trimeric purine nucleoside phosphorylase (AmPNP) from Aneurinibacillus migulanus AM007 was cloned and heterologously expressed in Escherichia coli BL21(DE3). The AmPNP showed good thermostability and a broad range of pH stability. The enzyme was thermostable below 55 °C for 12 h (retaining nearly 100% of its initial activity), and retained nearly 100% of the initial activity in alkaline buffer systems (pH 7.0-9.0) at 60 °C for 2 h. Then, a one-pot, two-enzyme mode of transglycosylation reaction was successfully constructed by combining pyrimidine nucleoside phosphorylase (BbPyNP) derived from Brevibacillus borstelensis LK01 and AmPNP for the production of purine nucleoside analogs. Conversions of 2,6-diaminopurine ribonucleoside (1), 2-amino-6-chloropurine ribonucleoside (2), and 6-thioguanine ribonucleoside (3) synthesized still reached >90% on the higher concentrations of substrates (pentofuranosyl donor: purine base; 20:10 mM) with a low enzyme ratio of BbPyNP: AmPNP (2:20 µg/mL). Thus, the new trimeric AmPNP is a promising biocatalyst for industrial production of purine nucleoside analogs.

Excretion and folding of plasmalemma function to accommodate alterations in guard cell volume during stomatal closure in Vicia faba L.

Stomatal movement results in large and repetitive changes in cell volume and consequently surface area. While endocytosis has been extensively studied and is thought to be a major mechanism for accommodating the volume changes as evidenced mainly by fluorescent labelling and confocal imaging, studies at the ultrastructural level in intact guard cells of stomata regulated by natural factors have never been reported. Here, it is reported that excretion and folding of the plasmalemma are critical for accommodating the volume alterations in intact guard cells in Vicia faba L. Using transmission electron microscopy in combination with laser confocal microscopy, it was observed that in fully opened stomata the plasmalemma was smooth and tightly adhered to the cell walls while a whole large vacuole appeared in the cell. In the closed stomata, besides vacuole fragmentation, endocytosis of the tonoplast rather than the plasmalemma commonly occurred. Importantly, in stomata where pore closure was induced by circadian rhythm or CO(2), numerous tiny vesicles were found outside the plasmalemma and, moreover, extensive folding of the plasmalemma could also be found in some regions of the cells. Additionally, an unknown structure was found at the interface between the plasmalemma and cell walls, especially in those areas of the cell where extensive folding occurred, suggesting that plasmalemma turnover is possibly associated with an interaction between the plasmalemma and cell walls. Collectively, the results strongly indicate that excretion and folding of the plasmalemma are critical for the accommodation of the cell volume alterations during stomatal movement.