Identification of a novel thermostable transaminase and its application in L-phosphinothricin biosynthesis.

Transaminase (TA) is a crucial biocatalyst for enantioselective production of the herbicide L-phosphinothricin (L-PPT). The use of enzymatic cascades has been shown to effectively overcome the unfavorable thermodynamic equilibrium of TA-catalyzed transamination reaction, also increasing demand for TA stability. In this work, a novel thermostable transaminase (PtTA) from Pseudomonas thermotolerans was mined and characterized. The PtTA showed a high specific activity (28.63 U/mg) towards 2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO), with excellent thermostability and substrate tolerance. Two cascade systems driven by PtTA were developed for L-PPT biosynthesis, including asymmetric synthesis of L-PPT from PPO and deracemization of D, L-PPT. For the asymmetric synthesis of L-PPT from PPO, a three-enzyme cascade was constructed as a recombinant Escherichia coli (E. coli G), by co-expressing PtTA, glutamate dehydrogenase (GluDH) and D-glucose dehydrogenase (GDH). Complete conversion of 400 mM PPO was achieved using only 40 mM amino donor L-glutamate. Furthermore, by coupling D-amino acid aminotransferase (Ym DAAT) from Bacillus sp. YM-1 and PtTA, a two-transaminase cascade was developed for the one-pot deracemization of D, L-PPT. Under the highest reported substrate concentration (800 mM D, L-PPT), a 90.43% L-PPT yield was realized. The superior catalytic performance of the PtTA-driven cascade demonstrated that the thermodynamic limitation was overcome, highlighting its application prospect for L-PPT biosynthesis. KEY POINTS: • A novel thermostable transaminase was mined for L-phosphinothricin biosynthesis. • The asymmetric synthesis of L-phosphinothricin was achieved via a three-enzyme cascade. • Development of a two-transaminase cascade for D, L-phosphinothricin deracemization.

Optimal timing of surgery for gastric cancer after neoadjuvant chemotherapy: a systematic review and meta-analysis.

BACKGROUND: Following neoadjuvant chemotherapy, surgical resection is one of the most preferred treatment options for locally advanced gastric cancer patients. However, the optimal time interval between chemotherapy and surgery is unclear. This review aimed to identify the optimal time interval between neoadjuvant chemotherapy and surgery for advanced gastric cancer. METHODS: Beginning on November 12, 2022, we searched the PubMed, Cochrane Library, Web of Science databases, and Embase.com databases for relevant English-language research. Two authors independently screened the studies, assessed their quality, extracted the data, and analyzed the results. The primary goal was to investigate the relationship between the time interval to surgery (TTS) and long-term survival outcomes for patients. This study has been registered with PROSPERO (CRD42022365196). RESULTS: After an initial search of 4880 articles, the meta-analysis review ultimately included only five retrospective studies. Ultimately, this meta-analysis included 1171 patients, of which 411 patients had TTS of < 4 weeks, 507 patients had TTS of 4-6 weeks, and 253 patients had TTS of > 6 weeks. In survival analysis, patients with TTS of > 6 weeks had poorer overall survival outcomes than patients with TTS of 4-6 weeks (HR = 1.34, 95% CI: 1.03-1.75, P = 0.03). No significant differences were found in terms of disease-free survival the groups. CONCLUSION: Based on the current clinical evidence, patients with locally advanced gastric cancer may benefit better with a TTS of 4-6 weeks; however, this option still needs additional study.

Glutathione-dependent redox homeostasis is critical for chlorothalonil detoxification in tomato leaves.

Glutathione plays a critical role in plant growth, development and response to stress. It is a major cellular antioxidant and is involved in the detoxification of xenobiotics in many organisms, including plants. However, the role of glutathione-dependent redox homeostasis and associated molecular mechanisms regulating the antioxidant system and pesticide metabolism remains unclear. In this study, endogenous glutathione levels were manipulated by pharmacological treatments with glutathione synthesis inhibitors and oxidized glutathione. The application of oxidized glutathione enriched the cellular oxidation state, reduced the activity and transcript levels of antioxidant enzymes, upregulated the expression level of nitric oxide and Ca2+ related genes and the content, and increased the residue of chlorothalonil in tomato leaves. Further experiments confirmed that glutathione-induced redox homeostasis is critical for the reduction of pesticide residues. RNA sequencing analysis revealed that miRNA156 and miRNA169 that target transcription factor SQUAMOSA-Promoter Binding Proteins (SBP) and NUCLEAR FACTOR Y (NFY) potentially participate in glutathione-mediated pesticide degradation in tomato plants. Our study provides important clues for further dissection of pesticide degradation mechanisms via miRNAs in plants.

Development of an aminotransferase-driven biocatalytic cascade for deracemization of d,l-phosphinothricin.

2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO) is the essential precursor keto acid for the asymmetric biosynthesis of herbicide l-phosphinothricin (l-PPT). Developing a biocatalytic cascade for PPO production with high efficiency and low cost is highly desired. Herein, a d-amino acid aminotransferase from Bacillus sp. YM-1 (Ym DAAT) with high activity (48.95 U/mg) and affinity (Km = 27.49 mM) toward d-PPT was evaluated. To circumvent the inhibition of by-product d-glutamate (d-Glu), an amino acceptor (α-ketoglutarate) regeneration cascade was constructed as a recombinant Escherichia coli (E. coli D), by coupling Ym d-AAT, d-aspartate oxidase from Thermomyces dupontii (TdDDO) and catalase from Geobacillus sp. CHB1. Moreover, the regulation of the ribosome binding site was employed to overcome the limiting step of expression toxic protein TdDDO in E. coli BL21(DE3). The aminotransferase-driven whole-cell biocatalytic cascade (E. coli D) showed superior catalytic efficiency for the synthesis of PPO from d,l-phosphinothricin (d,l-PPT). It revealed the production of PPO exhibited high space-time yield (2.59 g L-1 h-1 ) with complete conversion of d-PPT to PPO at high substrate concentration (600 mM d,l-PPT) in 1.5 L reaction system. This study first provides the synthesis of PPO from d,l-PPT employing an aminotransferase-driven biocatalytic cascade.

Jasmonic acid promotes glutathione assisted degradation of chlorothalonil during tomato growth.

Glutathione (GSH) biosynthesis and regeneration play a significant role in the metabolism of chlorothalonil (CHT) in tomatoes. However, the specific regulatory mechanism of GSH in the degradation of CHT remains uncertain. To address this, we investigate the critical regulatory pathways in the degradation of residual CHT in tomatoes. The results revealed that the detoxification of CHT residue in tomatoes was inhibited by buthionine sulfoximine and oxidized glutathione pretreatment, which increased by 26% and 46.12% compared with control, respectively. Gene silencing of γECS, GS, and GR also compromised the CHT detoxification potential of plants, which could be alleviated by GSH application and decreased the CHT accumulation by 33%, 25%, and 21%, respectively. Notably, it was found that the jasmonic acid (JA) pathway participated in the degradation of CHT regulated by GSH. CHT residues reduced by 28% after application of JA. JA played a role downstream of the glutathione pathway by promoting the degradation of CHT residue in tomatoes via nitric oxide signaling and improving the gene expression of antioxidant and detoxification-related enzymes. This study unveiled a crucial regulatory mechanism of GSH via the JA pathway in CHT degradation in tomatoes and offered new insights for understanding residual pesticide degradation.

Enhanced catalytic activity of recombinant transaminase by molecular modification to improve L-phosphinothricin production.

Transaminases catalyze the transfer of an amino group from a donor to a keto group of an acceptor substrate and are applicable to the asymmetric synthesis of herbicide L-phosphinothricin (L-PPT). Here, the important residue sites (C390, I22, V52, R141, Y138 and D239) of transaminase from Salmonella enterica (SeTA) were modified at the adjacency of the substrate-binding pocket to improve the enzyme activity. Among the constructed mutant library, the SeTA-Y138F mutant displayed higher activity than the wild-type enzyme. Compared to the wild-type, SeTA-Y138F showed improved catalytic efficiency with a 4.36-fold increase. The Km and kcat of SeTA -Y138F toward 4-(hydroxy(methyl) phosphoryl)-2-oxobutanoic acid (PPO) were 26.39 mM and 34.28 s-1, respectively. Subsequently, the three-enzyme co-expression system of E. coli BL21 (DE3)/pACYCDuet-SeTA-Y138F/pETDuet-AlaDH-BsGDH was developed by combining a alanine dehydrogenase (AlaDH) to recycle the byproduct of amino donor, a glucose dehydrogenase (BsGDH) for cofactor recycling. Under the optimized conditions, an excellent L-PPT yield of 90.8% was achieved by the whole-cell biotransformation with 500 mM PPO. It exhibited the tri-enzymatic coupling system was potential for effective production of target L-PPT.

Characterization and application of a common epitope recognized by a broad-spectrum C4 monoclonal antibody against capsid proteins of plant potyviruses.

A broad-spectrum monoclonal antibody (C4 MAb) against the capsid proteins (CPs) of plant potyviruses has been generated in previous studies. To clarify which epitope is recognized by this MAb, epitope mapping was performed via phage display library screening and amino acid substitution analysis. Subsequently, a 12-residue epitope in the core region of potyvirus CPs was identified and termed the C4 epitope (WxMMDGxxQxxY/F). This epitope contains tryptophan and tyrosine residues that are crucial for reacting with C4 MAb. The CP of Odontoglossum ringspot tobamovirus (ORSV) separately fused with the C4 epitope of Konjak mosaic potyvirus (KoMV), Zantedeschia mild mosaic potyvirus (ZaMMV), or Dasheen mosaic potyvirus (DsMV) was expressed in a bacterial system and purified. The results of indirect ELISA and Western blotting demonstrated that the C4 epitope of KoMV (Ko) fused to ORSV CP showed the strongest binding affinity to C4 MAb among the three viral epitope tags examined. The binding affinity between Ko tag (WTMMDGEEQIEY) and C4 MAb was determined. To examine the applicability of the Ko tag in planta, GFP and ORSV CP were transiently expressed in Nicotiana benthamiana, and both Ko-tagged proteins were specifically detected using C4 MAb. The Ko tag did not affect the silencing suppressor function of Tomato bushy stunt tombusvirus P19 in N. benthamiana. Furthermore, Ko-tagged EGFP could be successfully expressed, specifically detected and subsequently immunoprecipitated using C4 MAb in a mammalian cell system. Thus, the present study identified a common C4 epitope of potyviruses recognized by the broad-spectrum C4 and PTY 1 MAbs, and the results indicated that the newly designed Ko tag is suitable for application in bacterial, plant, and mammalian cell systems.

A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses.

Potyviruses are major pathogens that often cause mixed infection in calla lilies. To reduce the time and cost of virus indexing, a detection method for the simultaneous targeting of multiple potyviruses was developed by generating a broad-spectrum monoclonal antibody (MAb) for detecting the greatest possible number of potyviruses. The conserved 121-amino-acid core regions of the capsid proteins of Dasheen mosaic potyvirus (DsMV), Konjak mosaic potyvirus (KoMV), and Zantedeschia mild mosaic potyvirus (ZaMMV) were sequentially concatenated and expressed as a recombinant protein for immunization. After hybridoma cell fusion and selection, one stable cell line that secreted a group-specific antibody, named C4 MAb, was selected. In the reaction spectrum test, the C4 MAb detected at least 14 potyviruses by indirect enzyme-linked immunosorbent assay (I-ELISA) and Western blot analysis. Furthermore, the variable regions of the heavy (VH) and light (VL) chains of the C4 MAb were separately cloned and constructed as single-chain variable fragments (scFvs) for expression in Escherichia coli. Moreover, the pectate lyase E (PelE) signal peptide of Erwinia chrysanthemi S3-1 was added to promote the secretion of C4 scFvs into the medium. According to Western blot analysis and I-ELISA, the soluble C4 scFv (VL-VH) fragment showed a binding specificity similar to that of the C4 MAb. Our results demonstrate that a recombinant protein derived from fusion of the conserved regions of viral proteins has the potential to produce a broad-spectrum MAb against a large group of viruses and that the PelE signal peptide can improve the secretion of scFvs in E. coli.

The effect of leptin on the osteoinductive activity of recombinant human bone morphogenetic protein-2 in nude mice.

OBJECTIVE: To investigate the effect of leptin in the model of ectopic bone formation that utilizes subcutaneously implanted recombinant human bone morphogenetic protein-2 (rhBMP-2)-containing type I collagen discs. METHODS: This study was performed in the Clinical Research Center, Southern Medical University, Guangzhou, China from November 2008 to June 2009. A single dose of 20 ul saline (group A), 20 ug leptin (group B), 2 ug rhBMP-2 (group C), 2 ug rhBMP-2 + 20 ug leptin (group D), and type I collagen disks as a carrier were mixed, and subcutaneously implanted into the back of nude mice (n=12). The effect of ectopic bone formation was evaluated by radiography, dual-energy X absorptiometry, biochemical examination of alkaline phosphatase (ALP) activity, histological observation, and semi-quantitative evaluation 4 and 8 weeks after surgery. RESULTS: At 4 and 8 weeks after operation the radiographs, bone mass density, ALP, and histology values showed significant intragroup and intergroup differences, with those at 8 weeks being higher than those at 4 weeks. CONCLUSION: Our results indicated that the sustainedly released material with collagen as a carrier combined treatment with leptin and rhBMP-2 has a very good osteoinductive activity. Leptin is a positive modulator for the osteoinductive efficacy of BMPs, they have synergistic effect. Its mechanisms are probably related to promote the formation of new-vessels and proliferation/ differentiation of many kinds of cells.