Alanine synthesized by alanine dehydrogenase enables ammonium-tolerant nitrogen fixation in Paenibacillus sabinae T27.

Most diazotrophs fix nitrogen only under nitrogen-limiting conditions, for example, in the presence of relatively low concentrations of NH4+ (0 to 2 mM). However, Paenibacillus sabinae T27 exhibits an unusual pattern of nitrogen regulation of nitrogen fixation, since although nitrogenase activities are high under nitrogen-limiting conditions (0 to 3 mM NH4+) and are repressed under conditions of nitrogen sufficiency (4 to 30 mM NH4+), nitrogenase activity is reestablished when very high levels of NH4+ (30 to 300 mM) are present in the medium. To further understand this pattern of nitrogen fixation regulation, we carried out transcriptome analyses of P. sabinae T27 in response to increasing ammonium concentrations. As anticipated, the nif genes were highly expressed, either in the absence of fixed nitrogen or in the presence of a high concentration of NH4+ (100 mM), but were subject to negative feedback regulation at an intermediate concentration of NH4+ (10 mM). Among the differentially expressed genes, ald1, encoding alanine dehydrogenase (ADH1), was highly expressed in the presence of a high level of NH4+ (100 mM). Mutation and complementation experiments revealed that ald1 is required for nitrogen fixation at high ammonium concentrations. We demonstrate that alanine, synthesized by ADH1 from pyruvate and NH4+, inhibits GS activity, leading to a low intracellular glutamine concentration that prevents feedback inhibition of GS and mimics nitrogen limitation, enabling activation of nif transcription by the nitrogen-responsive regulator GlnR in the presence of high levels of extracellular ammonium.

Design, tuning, and blackbox optimization of laser systems.

Chirped pulse amplification (CPA) and subsequent nonlinear optical (NLO) systems constitute the backbone of myriad advancements in semiconductor manufacturing, communications, biology, defense, and beyond. Accurately and efficiently modeling CPA+NLO-based laser systems is challenging because of the complex coupled processes and diverse simulation frameworks. Our modular start-to-end model unlocks the potential for exciting new optimization and inverse design approaches reliant on data-driven machine learning methods, providing a means to create tailored CPA+NLO systems unattainable with current models. To demonstrate this new, to our knowledge, technical capability, we present a study on the LCLS-II photo-injector laser, representative of a high-power and spectro-temporally non-trivial CPA+NLO system.

Redox cascade reaction for kinetic resolution of racemic α-methylbenzylamine and biosynthesis of α-phenylethanol.

Chiral α-methylbenzylamine and α-phenylethanol are important building blocks for the industrial production of optically active drugs, bioactive compounds. Methods for the simultaneous synthesis of chiral α-methylbenzylamine and α-phenylethanol remain rare. Herein, a biocatalytic redox cascade reaction composed of ω-transaminase, aldo-keto reductase, and glutamate dehydrogenase for chiral α-methylbenzylamine and α-phenylethanol synthesis from racemic α-methylbenzylamine was constructed. A novel ω-transaminase and two different chiral aldo-keto reductases were demonstrated in the cascade reaction. The cosubstrate and redox equivalents were regenerated simultaneously by glutamate dehydrogenase. Using the approach, (R)-α-phenylethanol, (S)-α-phenylethanol, and (R)-α-methylbenzylamine were prepared with excellent stereoselectivity (ee > 99.7%). Furthermore, semi-preparative-scale biotransformation of racemic α-methylbenzylamine was conducted. The production of (R)-α-phenylethanol reached 26.05 mM at 24 h, and the production of (S)-α-phenylethanol reached 25.44 mM at 32 h. Taken together, a novel idea was proposed for the efficient and green synthesis of chiral α-methylbenzylamine and α-phenylethanol, which had great potential for industrial application. KEY POINTS: • Excellent stereoselectivity chiral α-methylbenzylamine and α-phenylethanol were synthesized. • A novel ω-transaminase demonstrated the catalysis toward (S)-α-methylbenzylamine. • Two novel aldo-keto reductases demonstrated the conversion toward acetophenone.

Biosynthesis of Feruloyl Glycerol from Ferulic Acid and Glycerol Through a Two-Enzyme Cascade Reaction.

Feruloyl glycerol (FG) has a variety of biological activities, but the green synthesis methods of FG remain rare. In this study, FG was prepared by a cascade reaction catalyzed by 4-coumarate coenzyme A ligase (4CL) and hydroxycinnamoyl acyltransferase 4 (HCT4). The cascade reaction carried out at solvent water and room temperature is more convenient and greener. Firstly, the product derived from the cascade reaction was characterized by TLC, HPLC, FTIR, and ESI-MS. The results showed that the product was FG. Secondly, the effects of temperature, pH, enzyme ratio, Mg2+ concentration, and CoA concentration on the cascade reaction were investigated. Consequently, the highest reaction rate was obtained at 30 °C, pH 6, an enzyme ratio of 1:3, and Mg2+ concentration of 5 mM. Finally, semi-preparative scale synthesis for FG was conducted. The production of FG reached 35.1 mM at 24 h with the FG conversion of 70.18%. In a word, a novel idea for the efficient and green synthesis of FG was proposed, which had great potential for industrial application.

Assembly of nitrogenase biosynthetic pathway in Saccharomyces cerevisiae by using polyprotein strategy.

Nitrogenase in some bacteria and archaea catalyzes conversion of N2 to ammonia. To reconstitute a nitrogenase biosynthetic pathway in a eukaryotic host is still a challenge, since synthesis of nitrogenase requires a large number of nif (nitrogen fixation) genes. Viral 2A peptide mediated "cleavage" of polyprotein is one of strategies for multigene co-expression. Here, we show that cleavage efficiency of NifB-2A-NifH polyprotein linked by four different 2A peptides (P2A, T2A, E2A, and F2A) in Saccharomyces cerevisiae ranges from ~50% to ~90%. The presence of a 2A tail in NifB, NifH, and NifD does not affect their activity. Western blotting shows that 9 Nif proteins (NifB, NifH, NifD, NifK, NifE, NifN, NifX, HesA, and NifV) from Paenibacillus polymyxa that are fused into two polyproteins via 2A peptides are co-expressed in S. cerevisiae. Expressed NifH from Klebsiella oxytoca NifU and NifS and P. polymyxa NifH fusion linked via 2A in S. cerevisiae exhibits Fe protein activity.

Anti-Hcp1 Monoclonal Antibody Is Protective against Burkholderia pseudomallei Infection via Recognizing Amino Acids at Asp95-Leu114.

Melioidosis, a severe tropical illness caused by Burkholderia pseudomallei, poses significant treatment challenges due to limited therapeutic options and the absence of effective vaccines. The pathogen's intrinsic resistance to numerous antibiotics and propensity to induce sepsis during acute infections further complicate management strategies. Thus, exploring alternative methods for prevention and treatment is crucial. Monoclonal antibodies (mAbs) have emerged as a promising strategy for the prevention and treatment of infectious diseases. This study focused on generating three mAbs (13F1, 14G11, and 15D9) targeting hemolysin-coregulated protein 1 (Hcp1), a protein involved in the type VI secretion system cluster 1 (T6SS1) of B. pseudomallei. Notably, pretreatment with 13F1 mAb significantly reduced the intracellular survival of B. pseudomallei and inhibited the formation of macrophage-derived multinucleated giant cells (MNGCs). This protective effect was also observed in vivo. We identified a sequence of amino acids (Asp95-Leu114) within Hcp1 as the likely binding site for 13F1 mAb. In summary, our findings reveal that 13F1 mAb counteracts infection by targeting Hcp1, offering potential new targets and insights for melioidosis prevention.

Evaluation of early versus delayed laparoscopic cholecystectomy in the treatment of acute cholecystitis.

BACKGROUND/AIMS: The optimal timing of laparoscopic cholecystectomy (LC) in the treatment of acute cholecystitis remains controversial. This retrospective study was undertaken to assess the clinical outcomes, possible advantages and disadvantages of early versus delayed LC for acute cholecystitis. MATERIALS AND METHODS: Records of all patients admitted for acute cholecystitis in whom laparoscopic cholecystectomy was attempted between January 2004 and January 2006, at National Taiwan University Hospital were reviewed. RESULTS: A total of 89 patients were recruited to the study. Of these, 56 patients received early laparoscopic cholecystectomy (ELC), and 33 patients received delayed laparoscopic cholecystectomy (DLC) following conservative therapy. There were no intergroup differences in age, gender, or days of symptoms prior to presentation. Patients undergoing ELC experienced a significantly longer operation time (109 +/- 37.59 minutes versus 77 +/- 25.65 minutes, p < 0.001), more blood loss (76ml versus 28ml, p = 0.006) and a longer post-operation hospital stay (4.5 days versus 2.6 days, p < 0.001). The conversion rate to open cholecystectomy was not significantly different (4/56 versus 2/33, p = 0.84), and there were no biliary tract injury or other major complications in either group. However, patients with ELC had a shorter total hospital stay (4.53 days versus 7.79 days, p < 0.001) and fewer admission times (1 time in ELC versus 2.4 times in DLC, p < 0.001). CONCLUSIONS: Both early and delayed LC appears to be effective and safe in the treatment of acute cholecystitis. Early LC may be more technically demanding and time-consuming, and may be associated with a higher rate of wound infections; however, it also tends to shorten the total length of hospital stay and reduce the risk of repeat cholecystitis. We recommend early LC for acute cholecystitis comparison with delayed LC.

Diazotrophic Paenibacillus beijingensis BJ-18 Provides Nitrogen for Plant and Promotes Plant Growth, Nitrogen Uptake and Metabolism.

Diazotrophic bacteria can reduce N2 into plant-available ammonium (NH4 +), promoting plant growth and reducing nitrogen (N) fertilizer requirements. However, there are few systematic studies on the effects of diazotrophic bacteria on biological N2 fixation (BNF) contribution rate and host plant N uptake and metabolism. In this study, the interactions of the diazotrophic Paenibacillus beijingensis BJ-18 with wheat, maize, and cucumber were investigated when it was inoculated to these plant seedlings grown in both low N and high N soils, with un-inoculated plants as controls. This study showed that GFP-tagged P. beijingensis BJ-18 colonized inside and outside seedlings, forming rhizospheric and endophytic colonies in roots, stems, and leaves. The numbers of this bacterium in the inoculated plants depended on soil N levels. Under low N, inoculation significantly increased shoot dry weight (wheat 86.1%, maize 46.6%, and cucumber 103.6%) and root dry weight (wheat 46.0%, maize 47.5%, and cucumber 20.3%). The 15N-isotope-enrichment experiment indicated that plant seedlings derived 12.9-36.4% N from BNF. The transcript levels of nifH in the inoculated plants were 0.75-1.61 folds higher in low N soil than those in high N soil. Inoculation enhanced NH4 + and nitrate (NO3 -) uptake from soil especially under low N. The total N in the inoculated plants were increased by 49.1-92.3% under low N and by 13-15.5% under high N. Inoculation enhanced activities of glutamine synthetase (GS) and nitrate reductase (NR) in plants, especially under low N. The expression levels of N uptake and N metabolism genes: AMT (ammonium transporter), NRT (nitrate transporter), NiR (nitrite reductase), NR, GS and GOGAT (glutamate synthase) in the inoculated plants grown under low N were up-regulated 1.5-91.9 folds, but they were not obviously changed under high N. Taken together, P. beijingensis BJ-18 was an effective, endophytic and diazotrophic bacterium. This bacterium contributed to plants with fixed N2, promoted plant growth and N uptake, and enhanced gene expression and enzyme activities involved in N uptake and assimilation in plants. However, these positive effects on plants were regulated by soil N status. This study might provide insight into the interactions of plants with beneficial associative and endophytic diazotrophic bacteria.

Combined Assembly and Targeted Integration of Multigene for Nitrogenase Biosynthetic Pathway in Saccharomyces cerevisiae.

Biological nitrogen fixation, a process unique to diazotrophic prokaryote, is catalyzed by the nitrogenase complex. There has been a long-standing interest in reconstituting a nitrogenase biosynthetic pathway in a eukaryotic host with the final aim of developing N2-fixing cereal crops. In this study, we report that a nitrogenase biosynthetic pathway (∼38 kb containing 15 genes) was assembled in two individual one-step methods via in vivo assembly and integrated at δ and HO sites in Saccharomyces cerevisiae chromosome. Of the 15 genes, 11 genes (nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA, nifV, groES, groEL) were from Paenibacillus polymyxa WLY78 and 4 genes (nifS, nifU, nifF, nifJ) were from Klebsiella oxytoca. The 15-gene nitrogenase biosynthetic pathway was correctly assembled and transcribed in the recombinant S. cerevisiae. The NifDK tetramer with an identical molecular weight as that of P. polymyxa was formed in yeast and the expressed NifH exhibited the activity of Fe protein. This study demonstrates that it will be possible to produce active nitrogenase in eukaryotic hosts.