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[政策法规 ] Guidance for the assessment of detoxification processes in feed 进入全文
European Food Safety Authority
Directive 2002/32/EC of the European Parliament and of the Council, in its article 3, provides that the use of products intended for animal feed which contain levels of undesirable substances exceeding the maximum levels laid down in Annex I of that Directive cannot be placed in the European market. It is possible to use acceptable detoxification processes on these products in order to conform with the provisions of Annex I of that Directive. Commission Regulation (EU) 2015/7862 establishes the acceptability criteria for detoxification processes to ensure that the detoxified feed does not endanger animal and public health and the environment and that the characteristics of the feed are not adversely altered by the detoxification process. The Panel on Contaminants in the food chain (CONTAM Panel) has assessed a series of requests for feed detoxification processes since the entry into force of the Regulation (EU) 2015/786 on 1 July 2017. Frequently the information provided is insufficient for the Panel to come to conclusions if the detoxification process fulfils the criteria set in the legislation and additional clarifications are necessary which prolong the risk assessment process. To help the feed business operators in the preparation and submission of the necessary information for the evaluation of feed detoxification processes according to the Regulation, the CONTAM Panel, in its Plenary meeting on 15 December 2022, identified the need for guidance. The guidance will be provided in a statement addressing the implementation of the criteria for the acceptance of detoxification processes applied to products intended for animal feed, based on the Panel's experience gained during the last years while working under the provisions of the abovementioned Regulation.
[学术文献 ] Harnessing generative AI to decode enzyme catalysis and evolution for enhanced engineering 进入全文
National Science Review
Enzymes, as paramount protein catalysts, occupy a central role in fostering remarkable progress across numerous fields. However, the intricacy of sequence-function relationships continues to obscure our grasp of enzyme behaviors and curtails our capabilities in rational enzyme engineering. Generative artificial intelligence (AI), known for its proficiency in handling intricate data distributions, holds the potential to offer novel perspectives in enzyme research. Generative models could discern elusive patterns within the vast sequence space and uncover new functional enzyme sequences. This review highlights the recent advancements in employing generative AI for enzyme sequence analysis. We delve into the impact of generative AI in predicting mutation effects on enzyme fitness, catalytic activity and stability, rationalizing the laboratory evolution of de novo enzymes, and decoding protein sequence semantics and their application in enzyme engineering. Notably, the prediction of catalytic activity and stability of enzymes using natural protein sequences serves as a vital link, indicating how enzyme catalysis shapes enzyme evolution. Overall, we foresee that the integration of generative AI into enzyme studies will remarkably enhance our knowledge of enzymes and expedite the creation of superior biocatalysts.
[学术文献 ] An efficient CRISPRCas9 genome editing system based on a multiple sgRNA processing platform in Trichoderma reesei for strain improvement and enzyme production 进入全文
Biotechnology for Biofuels and Bioproducts
Background The CRISPR/Cas9 technology is being employed as a convenient tool for genetic engineering of the industrially important filamentous fungus Trichoderma reesei. However, multiplex gene editing is still constrained by the sgRNA processing capability, hindering strain improvement of T. reesei for the production of lignocellulose-degrading enzymes and recombinant proteins. Results Here, a CRISPR/Cas9 system based on a multiple sgRNA processing platform was established for genome editing in T. reesei. The platform contains the arrayed tRNA−sgRNA architecture directed by a 5S rRNA promoter to generate multiple sgRNAs from a single transcript by the endogenous tRNA processing system. With this system, two sgRNAs targeting cre1 (encoding the carbon catabolite repressor 1) were designed and the precise deletion of cre1 was obtained, demonstrating the efficiency of sgRNAs processing in the tRNA−sgRNA architecture. Moreover, overexpression of xyr1-A824V (encoding a key activator for cellulase/xylanase expression) at the ace1 (encoding a repressor for cellulase/xylanase expression) locus was achieved by designing two sgRNAs targeting ace1 in the system, resulting in the significantly enhanced production of cellulase (up to 1- and 18-fold on the Avicel and glucose, respectively) and xylanase (up to 11- and 41-fold on the Avicel and glucose, respectively). Furthermore, heterologous expression of the glucose oxidase gene from Aspergillus niger ATCC 9029 at the cbh1 locus with the simultaneous deletion of cbh1 and cbh2 (two cellobiohydrolase coding genes) by designing four sgRNAs targeting cbh1 and cbh2 in the system was acquired, and the glucose oxidase produced by T. reesei reached 43.77 U/mL. Besides, it was found the ER-associated protein degradation (ERAD) level was decreased in the glucose oxidase-producing strain, which was likely due to the reduction of secretion pressure by deletion of the major endogenous cellulase-encoding genes. Conclusions The tRNA−gRNA array-based CRISPR-Cas9 editing system was successfully developed in T. reesei. This system would accelerate engineering of T. reesei for high-level production of enzymes including lignocellulose-degrading enzymes and other recombinant enzymes. Furthermore, it would expand the CRISPR toolbox for fungal genome editing and synthetic biology.
[学术文献 ] Combinatorial optimization of gene expression through recombinase-mediated promoter and terminator shuffling in yeast 进入全文
Nature Communications
Microbes are increasingly employed as cell factories to produce biomolecules. This often involves the expression of complex heterologous biosynthesis pathways in host strains. Achieving maximal product yields and avoiding build-up of (toxic) intermediates requires balanced expression of every pathway gene. However, despite progress in metabolic modeling, the optimization of gene expression still heavily relies on trial-and-error. Here, we report an approach for in vivo, multiplexed Gene Expression Modification by LoxPsym-Cre Recombination (GEMbLeR). GEMbLeR exploits orthogonal LoxPsym sites to independently shuffle promoter and terminator modules at distinct genomic loci. This approach facilitates creation of large strain libraries, in which expression of every pathway gene ranges over 120-fold and each strain harbors a unique expression profile. When applied to the biosynthetic pathway of astaxanthin, an industrially relevant antioxidant, a single round of GEMbLeR improved pathway flux and doubled production titers. Together, this shows that GEMbLeR allows rapid and efficient gene expression optimization in heterologous biosynthetic pathways, offering possibilities for enhancing the performance of microbial cell factories.
[学术文献 ] p-Anisaldehyde suppresses Aspergillus flavus infection and aflatoxin biosynthesis via targeting cell structure and Ap1-regulatory antioxidant system 进入全文
Food Frontiers
Aflatoxin, mainly produced by Aspergillus flavus, is one of the most notorious mycotoxin for its toxicity and carcinogenicity. Despite extensive efforts, effective strategies to control A. flavus and AFB1 contamination remain elusive. Here, we investigate the potential of p-anisaldehyde (AS), an aldehyde derived from plant essential oils, as a natural antifungal agent against A. flavus and its ability to modulate AFB1 biosynthesis. We found that AS exhibited broad-spectrum antifungal activities against Aspergillus spp. and effectively inhibited A. flavus asexual development, AFB1 production, and pathogenicity. AS treatment disrupted the cell surface structure and membrane integrity, as observed by scanning electron microscopy and PI staining. RNA-sequencing analysis revealed that AS significantly altered the expression of genes involved in redox homeostasis, plasma membrane function, and cell cycle progression. Further investigation demonstrated that AS induced a reduction in mitochondrial membrane potential (Δψm) and accumulation of reactive oxygen species (ROS), leading to cell cycle arrest at the G2/M phase. The AS-induced ROS accumulation was found to be mitigated by the superoxide dismutase-mediated antioxidant system, which is regulated by transcriptional factor Ap1. Notably, the Ap1-regulatory ROS detoxification system was also found to be involved in A. flavus pathogenicity and AFB1 production. Overall, these findings provide valuable insights into the inhibitory mechanism of AS against A. flavus, paving the way for its potential application as a natural strategy to mitigate AFB1 contamination in both food and agriculture crops.
[学术文献 ] Effects of host-associated low-temperature probiotics in olive flounder (Paralichthys olivaceus) aquaculture 进入全文
Scientific Reports
This study investigated the effects of supplementation of low-temperature probiotics isolated from the intestines of olive flounder on the growth performance, digestibility, and regulation of intestinal microbiota and the expression of genes related to growth, immunity, and apoptosis in olive flounder. Bacteria showing high growth at approximately 15–20 °C, which is the temperature of olive flounder culture, were isolated and confirmed to be Pseudomonas species through 16S rRNA gene sequence analysis. Whole-genome sequencing revealed that the strain has a 6,195,122 bp single circular chromosome and a guanine–cytosine content of 59.9%. In the feeding trial, supplementation with 1 × 108 CFU/g of the isolate strain positively modulated growth performances, digestive enzyme activity, and gut microbiota composition of olive flounder. RT-qPCR for the comparison of growth, immunity, and apoptosis-related gene expression levels showed no significant differences between the groups. Therefore, the isolated host-associated low-temperature probiotics improved the growth performance of olive flounder by causing positive changes in digestive activity and intestinal microbial composition without affecting host gene expression.
