Centralized industrialization of pork in Europe and America contributes to the global spread of Salmonella enterica.

Salmonella enterica causes severe food-borne infections through contamination of the food supply chain. Its evolution has been associated with human activities, especially animal husbandry. Advances in intensive farming and global transportation have substantially reshaped the pig industry, but their impact on the evolution of associated zoonotic pathogens such as S. enterica remains unresolved. Here we investigated the population fluctuation, accumulation of antimicrobial resistance genes and international serovar Choleraesuis transmission of nine pig-enriched S. enterica populations comprising more than 9,000 genomes. Most changes were found to be attributable to the developments of the modern pig industry. All pig-enriched salmonellae experienced host transfers in pigs and/or population expansions over the past century, with pigs and pork having become the main sources of S. enterica transmissions to other hosts. Overall, our analysis revealed strong associations between the transmission of pig-enriched salmonellae and the global pork trade.

A New-Generation Base Editor with an Expanded Editing Window for Microbial Cell Evolution In Vivo Based on CRISPR‒Cas12b Engineering.

Base editors (BEs) are widely used as revolutionary genome manipulation tools for cell evolution. To screen the targeted individuals, it is often necessary to expand the editing window to ensure highly diverse variant library. However, current BEs suffer from a limited editing window of 5-6 bases, corresponding to only 2-3 amino acids. Here, by engineering the CRISPR‒Cas12b, the study develops dCas12b-based CRISPRi system, which can efficiently repress gene expression by blocking the initiation and elongation of gene transcription. Further, based on dCas12b, a new-generation of BEs with an expanded editing window is established, covering the entire protospacer or more. The expanded editing window results from the smaller steric hindrance compared with other Cas proteins. The universality of the new BE is successfully validated in Bacillus subtilis and Escherichia coli. As a proof of concept, a spectinomycin-resistant E. coli strain (BL21) and a 6.49-fold increased protein secretion efficiency in E. coli JM109 are successfully obtained by using the new BE. The study, by tremendously expanding the editing window of BEs, increased the capacity of the variant library exponentially, greatly increasing the screening efficiency for microbial cell evolution.

A metagenomic catalog of the early-life human gut virome.

Early-life human gut microbiome is a pivotal driver of gut homeostasis and infant health. However, the viral component (known as "virome") remains mostly unexplored. Here, we establish the Early-Life Gut Virome (ELGV), a catalog of 160,478 non-redundant DNA and RNA viral sequences from 8130 gut virus-like particles (VLPs) enriched or bulk metagenomes in the first three years of life. By clustering, 82,141 viral species are identified, 68.3% of which are absent in existing databases built mainly from adults, and 64 and 8 viral species based on VLPs-enriched and bulk metagenomes, respectively, exhibit potentials as biomarkers to distinguish infants from adults. With the largest longitudinal population of infants profiled by either VLPs-enriched or bulk metagenomic sequencing, we track the inherent instability and temporal development of the early-life human gut virome, and identify differential viruses associated with multiple clinical factors. The mother-infant shared virome and interactions between gut virome and bacteriome early in life are further expanded. Together, the ELGV catalog provides the most comprehensive and complete metagenomic blueprint of the early-life human gut virome, facilitating the discovery of pediatric disease-virome associations in future.

Engineering hyperthermophilic pullulanase to efficiently utilize corn starch for production of maltooligosaccharides and glucose.

Pullulanase is a starch-debranching enzyme that hydrolyzes side chain of starch, oligosaccharides and pullulan. Nevertheless, the limited activities of pullulanases constrain their practical application. Herein, the hyperthermophilic type II pullulanase from Pyrococcus yayanosii CH1 (PulPY2) was evolved by synergistically engineering the substrate-binding pocket and active-site lids. The resulting mutant PulPY2-M2 exhibited 5-fold improvement in catalytic efficiency (kcat/Km) compared to that of PulPY2. PulPY2-M2 was utilized to develop a one-pot reaction system for efficient production of maltooligosaccharides. The maltooligosaccharides conversion rate of PulPY2-M2 reached 96.1%, which was increased by 5.4% compared to that of PulPY2. Furthermore, when employed for glucose production, the glucose productivity of PulPY2-M2 was 25.4% and 43.5% higher than that of PulPY2 and the traditional method, respectively. These significant improvements in maltooligosaccharides and glucose production and the efficient utilization of corn starch demonstrated the potential of the engineered PulPY2-M2 in starch sugar industry.

Carbapenem-resistant Klebsiella pneumoniae capsular types, antibiotic resistance and virulence factors in China: a longitudinal, multi-centre study.

Epidemiological knowledge of circulating carbapenem-resistant Klebsiella pneumoniae (CRKP) is needed to develop effective strategies against this public health threat. Here we present a longitudinal analysis of 1,017 CRKP isolates recovered from patients from 40 hospitals across China between 2016 and 2020. Virulence gene and capsule typing revealed expansion of CRKP capsule type KL64 (59.5%) alongside decreases in KL47 prevalence. Hypervirulent CRKP increased in prevalence from 28.2% in 2016 to 45.7% in 2020. Phylogenetic and spatiotemporal analysis revealed Beijing and Shanghai as transmission hubs accounting for differential geographical prevalence of KL47 and KL64 strains across China. Moderate frequency capsule or O-antigen loss was also detected among isolates. Non-capsular CRKP were more susceptible to phagocytosis, attenuated during mouse infections, but showed increased serum resistance and biofilm formation. These findings give insight into CRKP serotype prevalence and dynamics, revealing the importance of monitoring serotype shifts for the future development of immunological strategies against CRKP infections.

Structure-oriented engineering of nitrile hydratase: Reshaping of substrate access tunnel and binding pocket for efficient synthesis of cinnamamide.

Cinnamamide and its derivatives are the most common and important building blocks widely present in natural products. Currently, nitrile hydratase (NHase, EC 4.2.1.84) has been widely used in large-scale industrial production of nicotinamide and acrylamide, while its catalytic activity is extremely low or inactive for bulky nitrile substrates such as cinnamonitrile. Therefore, beneficial variant ßF37P/L48P/F51N were obtained from PtNHase of Pseudonocardia thermophila JCM3095 by reshaping of substrate access tunnel and binding pocket, which exhibited 14.88-fold improved catalytic efficiency compared to the wild-type PtNHase. Structure analysis, molecular dynamics simulations and dynamical cross-correlation matrix (DCCM) analysis revealed that the introduced mutations enlarged the substrate access tunnel and binding pocket, enhanced overall anti-correlated movements of enzymes, which would promote product release during the dynamic process of catalysis. In a hydration process, the complete conversion of 5 mM cinnamonitrile was achieved by ßF37P/L48P/F51N in a 50 mL reaction, with cinnamamide yield of almost 100 % and productivity of 0.736 g L-1 h-1. The study demonstrates the co-evolution of substrate access tunnel and binding pocket is an effective strategy, and provides a valuable reference for future research. Furthermore, NHases have huge potential for catalyzing bulky nitriles to form corresponding amides in large-scale industrial production.

Metabolic engineering of glycolysis in Escherichia coli for efficient production of patchoulol and τ-cadinol.

Glucose metabolism suppresses the microbial synthesis of sesquiterpenes with a syndrome of "too much of a good thing can be bad". Here, patchoulol production in Escherichia coli was increased 2.02 times by engineering patchoulol synthase to obtain an initial strain. Knocking out the synthetic pathway for cyclic adenosine monophosphate relieved glucose repression and improved patchoulol titer and yield by 27.7 % and 43.1 %, respectively. A glycolysis regulation device mediated by pyruvate sensing was constructed which effectively alleviated overflow metabolism in a high-glucose environment with 10.2 % greater patchoulol titer in strain 070QA. Without fine-tuning the glucose-feeding rate, patchoulol titer further increased to 1675.1 mg/L in a 5-L bioreactor experiment, which was the highest level reported in E. coli. Using strain 070QA as a chassis, the τ-cadinol titer reached 15.2 g/L, representing the first report for microbial production of τ-cadinol. These findings will aid in the industrial production of sesquiterpene.

Graphene oxide-enhanced colorimetric detection of Mec A gene based on toehold-mediated strand displacement.

Mec A, as a representative gene mediating resistance to ß-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA), allows a new genetic analysis for the detection of MRSA. Here, a sensitive, prompt, and visual colorimetry is reported to detect the Mec A gene based on toehold-mediated strand displacement (TMSD) and the enrichment effect of graphene oxide (GO). The Mec A triggers to generate the profuse amount of signal units of single-stranded DNA (SG) composed of a long single-stranded base tail and a base head: the tail can be adsorbed and enriched on the surface of GO; the head can form a G quadruplex structure to exert catalytic function towards 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid). Therefore, through the enrichment effect of GO, the signal units SG reflects different degrees of signal amplification on different substrates (such as aqueous solution or filter membrane). This strategy demonstrates a broad linear working range from 100 pM to 1.5 nM (solution) and 1 pM to 1 nM (filter membrane), with a low detection limit of 39.53 pM (solution) and 333 fM (filter membrane). Analytical performance in real samples suggests that this developed colorimetry is endowed with immense potential for clinical detection applications.

Creation of Architecturally Minimal Transcriptionally Activating Riboswitches Responsive to Theophylline Reveals an Unconventional Design Strategy.

Riboswitches are noncoding RNA switches that are largely utilized in bacteria and play a significant role in synthetic biology. Nonetheless, their natural counterparts possess lengthy sequences and intricate structures, posing challenges for their modular integration into complex gene circuits. Consequently, it is imperative to develop simplified synthetic riboswitches that can be effortlessly incorporated into gene circuits. The conventional approach to generate synthetic riboswitches entails tedious library construction and extensive screening, which frequently yields suboptimal performance. To overcome this obstacle, alternative methods are urgently needed. In this study, we created a novel approach to designing a diverse set of transcription-activating riboswitches that exhibit high performance and broad compatibility. The strategy involved starting with a synthetic theophylline RNA aptamer and designing an expression platform that forms a transcriptional terminator in its inactive state but switches to an antiterminator when it is activated. Several sequences were designed, constructed, and subjected to virtual screening, resulting in the identification of two transcription-activating riboswitches. These riboswitches were then engineered to reduce the basal leakage and increase the activation level through extending the hairpin region using a screened random sequence. These architecturally minimal synthetic riboswitches were highly adapted to different constitutive promoters in a modular manner, generating a differentially responsive output to theophylline. As a proof-of-principle, the synthetic riboswitches were applied to rewire a synthetic quorum-sensing circuit (QSC). The reprogrammed QSC successfully modulated the temporal responsive profile against the activation. This strategy is expected to expand the variety of high-performance riboswitches that are responsive to different ligands, thereby further facilitating the design of complex genetic circuits.

Discovery and Engineering of a Novel Bacterial L-Aspartate α-Decarboxylase for Efficient Bioconversion.

L-aspartate α-decarboxylase (ADC) is a pyruvoyl-dependent decarboxylase that catalyzes the conversion of L-aspartate to ß-alanine in the pantothenate pathway. The enzyme has been extensively used in the biosynthesis of ß-alanine and D-pantothenic acid. However, the broad application of ADCs is hindered by low specific activity. To address this issue, we explored 412 sequences and discovered a novel ADC from Corynebacterium jeikeium (CjADC). CjADC exhibited specific activity of 10.7 U/mg and Km of 3.6 mM, which were better than the commonly used ADC from Bacillus subtilis. CjADC was then engineered leveraging structure-guided evolution and generated a mutant, C26V/I88M/Y90F/R3V. The specific activity of the mutant is 28.8 U/mg, which is the highest among the unknown ADCs. Furthermore, the mutant displayed lower Km than the wild-type enzyme. Moreover, we revealed that the introduced mutations increased the structural stability of the mutant by promoting the frequency of hydrogen-bond formation and creating a more hydrophobic region around the active center, thereby facilitating the binding of L-aspartate to the active center and stabilizing the substrate orientation. Finally, the whole-cell bioconversion showed that C26V/I88M/Y90F/R3V completely transformed 1-molar L-aspartate in 12 h and produced 88.6 g/L ß-alanine. Our study not only identified a high-performance ADC but also established a research framework for rapidly screening novel enzymes using a protein database.

The metagenomic and metabolomic profile of the gut microbes in Chinese full-term and late preterm infants treated with Clostridium butyricum.

The present study investigated the composition, abundance, and diversity of gut microbes in full-term and late-preterm infants from a medical center in eastern China. A total of 144 genomes of stool samples were captured for 16S rRNA metagenomic analyses. A high abundance of commensal intestinal bacteria was detected in these samples such as Phocaeicola vulgatus, Escherichia coli, and Faecalibacterium prausnitzii, indicating a relatively consistent diversity of gut microbes in the present full-term infants aged 38-40 weeks. However, late preterm infants (n = 50) with mandatory antimicrobials feeding exhibited lower diversity but a higher composition of opportunistic pathogens such as Enterococcus species. Centralized on the situation, we explored the regulatory effect of Clostridium butyricum as probiotics on these late preterm infants. The consumption of C. butyricum did not restore the composition of gut microbes altered by antimicrobials to normal levels, although several opportunistic pathogens decreased significantly after probiotic therapy including Staphylococcus aureus, Sphingomonas echinoides, and Pseudomonas putida. We also compared the effects of day-fed versus night-fed probiotics. Intriguingly, the nighttime feeding showed a higher proportion of C. butyricum compared with probiotic day-feeding. Finally, fecal metabolome and metabolites were analyzed in late preterm infants with (n = 20) or without probiotic therapy (n = 20). The KEGG enrichment analysis demonstrated that vitamin digestion and absorption, synaptic vesicle cycle, and biotin metabolism were significantly increased in the probiotic-treated group, while MSEA indicated that a series of metabolism were significantly enriched in probiotic-treated infants including glycerolipid, biotin, and lysine, indicating the complex effects of probiotic therapy on glutathione metabolism and nutrients digestion and absorption in late preterm infants. Overall, this study provided metagenomic and metabolomic profile of the gut microbes in full-term newborns and late preterm infants in eastern China. Further studies are needed to support and elucidate the role of probiotic feeding in late preterm infants with mandatory antimicrobial treatment.

Distributed genotyping and clustering of Neisseria strains reveal continual emergence of epidemic meningococcus over a century.

Core genome multilocus sequence typing (cgMLST) is commonly used to classify bacterial strains into different types, for taxonomical and epidemiological applications. However, cgMLST schemes require central databases for the nomenclature of new alleles and sequence types, which must be synchronized worldwide and involve increasingly intensive calculation and storage demands. Here, we describe a distributed cgMLST (dcgMLST) scheme that does not require a central database of allelic sequences and apply it to study evolutionary patterns of epidemic and endemic strains of the genus Neisseria. We classify 69,994 worldwide Neisseria strains into multi-level clusters that assign species, lineages, and local disease outbreaks. We divide Neisseria meningitidis into 168 endemic lineages and three epidemic lineages responsible for at least 9 epidemics in the past century. According to our analyses, the epidemic and endemic lineages experienced very different population dynamics in the past 100 years. Epidemic lineages repetitively emerged from endemic lineages, disseminated worldwide, and apparently disappeared rapidly afterward. We propose a stepwise model for the evolutionary trajectory of epidemic lineages in Neisseria, and expect that the development of similar dcgMLST schemes will facilitate epidemiological studies of other bacterial pathogens.

Synergetic engineering of Escherichia coli for efficient production of l-tyrosine.

l-Tyrosine, an aromatic non-essential amino acid, is the raw material for many important chemical products, including levodopa, resveratrol, and hydroxytyrosol. It is widely used in the food, drug, and chemical industries. There are many studies on the synthesis of l-tyrosine by microorganisms, however, the low titer of l-tyrosine limited the industrial large-scale production. In order to enhance l-tyrosine production in Escherichia coli, the expression of key enzymes in the shikimate pathway was up- or down-regulated. The l-tyrosine transport system and the acetic acid biosynthesis pathway were modified to further enhance l-tyrosine production. In addition, the phosphoketolase pathway was introduced in combination with cofactor engineering to redirect carbon flux to the shikimate pathway. Finally, after adaptive laboratory evolution to low pH an optimal strain was obtained. The strain can produce 92.5 g/L of l-tyrosine in a 5-L fermenter in 62 h, with a yield of 0.266 g/g glucose.

Bacterial indole-3-lactic acid affects epithelium-macrophage crosstalk to regulate intestinal homeostasis.

Tryptophan and its derivatives perform a variety of biological functions; however, the role and specific mechanism of many tryptophan derivatives in intestinal inflammation remain largely unclear. Here, we identified that an Escherichia coli strain (Ec-TMU) isolated from the feces of tinidazole-treated individuals, and indole-3-lactic acid (ILA) in its supernatant, decreased the susceptibility of mice to dextran sulfate sodium-induced colitis. Ec-TMU and ILA contribute to the relief of colitis by inhibiting the production of epithelial CCL2/7, thereby reducing the accumulation of inflammatory macrophages in vitro and in vivo. Mechanistically, ILA downregulates glycolysis, NF-κB, and HIF signaling pathways via the aryl hydrocarbon receptor, resulting in decreased CCL2/7 production in epithelial cells. Clinical evidence suggests that the fecal ILA level is negatively correlated with the progression indicator of inflammatory bowel diseases. These results demonstrate that ILA has the potential to regulate intestinal homeostasis by modulating epithelium-macrophage interactions.

Whole genome sequence-based characterization of virulence and antimicrobial resistance gene profiles of Staphylococcus aureus isolated from food poisoning incidents in eastern China.

Staphylococcus aureus is an opportunistic foodborne pathogen occasionally isolated from diarrhea patients. In recent years, increasing studies have reported the detection of S. aureus in food poisoning incidents due to food contamination in the North and South of China. However, the epidemiology and genetic characteristics of S. aureus from food poisoning incidents in Eastern China remain unknown. The present study examined the genetic characteristics, antimicrobial resistance, and virulent factors of multidrug-resistant S. aureus isolated from 22 food poisoning incidents reported by the hospitals and health centers in Eastern China from 2011 to 2021. A total of 117 resistant and enterotoxigenic S. aureus isolates were collected and sequenced, among which 20 isolates were identified as methicillin resistant. Genetic analysis revealed 19 distinct CC/ST types, with CC6, CC22, CC59, CC88, and CC398 being the most frequent variants in methicillin-resistant S. aureus (MRSA). A considerable shift in CC types from CC1 to CC398 between 2011 and 2021 was observed in this study, indicating that CC398 may be the main epidemic strain circulating in the current food poisoning incidents. Additionally, genes for enterotoxins were detected in 55 isolates, with a prevalence of 27.8% (27/97) for methicillin-sensitive variants and 35.0% (7/20) for MRSA. The scn gene was detected in 59.0% of the isolates, demonstrating diverse contaminations of S. aureus among livestock-to-human transmission. Of the 117 isolates, only ten isolates displayed multi-drug resistance (MDR) to penicillin, tetracycline, and macrolides. None of the 117 foodborne S. aureus isolates tested positive for vanA in this study. Together, the present study provided phylogenetic characteristics of S. aureus from food poisoning incidents that emerged in Eastern China from 2011 to 2021. Our results suggested that these diarrhea episodes were hypotonic and merely transient low-MDR infections, however, further research for continued surveillance given the detection of virulence and antimicrobial resistance determinants is required to elucidate the genomic characteristics of pathogenic S. aureus in food poisoning incidents in the context of public health.

Stepwise genetic modification for efficient expression of heterologous proteins in Aspergillus nidulans.

Filamentous fungi are widely used in food fermentation and therapeutic protein production due to their prominent protein secretion and post-translational modification system. Aspergillus nidulans is an important model strain of filamentous fungi, but not a fully developed cell factory for heterologous protein expression. One of the limitations is its relatively low capacity of protein secretion. To alleviate this limitation, in this study, the protein secretory pathway and mycelium morphology were stepwise modified. With eGFP as a reporter protein, protein secretion was significantly enhanced through reducing the degradation of heterologous proteins by endoplasmic reticulum-associated protein degradation (ERAD) and vacuoles in the secretory pathway. Elimination of mycelial aggregation resulted in a 1.5-fold and 1.3-fold increase in secretory expression of eGFP in typical constitutive and inducible expression systems, respectively. Combined with these modifications, high secretory expression of human interleukin-6 (HuIL-6) was achieved. Consequently, a higher yield of secretory HuIL-6 was realized by further disruption of extracellular proteases. Overall, a superior chassis cell of A. nidulans suitable for efficient secretory expression of heterologous proteins was successfully obtained, providing a promising platform for biosynthesis using filamentous fungi as hosts. KEY POINTS: • Elimination of mycelial aggregation and decreasing the degradation of heterologous protein are effective strategies for improving the heterologous protein expression. • The work provides a high-performance chassis host △agsB-derA for heterologous protein secretory expression. • Human interleukin-6 (HuIL-6) was expressed efficiently in the high-performance chassis host △agsB-derA.

Characterization of microbial community and antibiotic resistome in intra urban water, Wenzhou China.

The present study investigated the water quality index, microbial composition and antimicrobial resistance genes in urban water habitats. Combined chemicals testing, metagenomic analyses and qualitative PCR (qPCR) were conducted on 20 locations, including rivers from hospital surrounds (n = 7), community surrounds (n = 7), and natural wetlands (n = 6). Results showed that the indexes of total nitrogen, phosphorus, and ammonia nitrogen of hospital waters were 2-3 folds high than that of water from wetlands. Bioinformatics analysis revealed a total of 1,594 bacterial species from 479 genera from the three groups of water samples. The hospital-related samples had the greatest number of unique genera, followed by those from wetlands and communities. The hospital-related samples contained a large number of bacteria associated with the gut microbiome, including Alistipes, Prevotella, Klebsiella, Escherichia, Bacteroides, and Faecalibacterium, which were all significantly enriched compared to samples from the wetlands. Nevertheless, the wetland waters enriched bacteria from Nanopelagicus, Mycolicibacterium and Gemmatimonas, which are typically associated with aquatic environments. The presence of antimicrobial resistance genes (ARGs) that were associated with different species origins in each water sample was observed. The majority of ARGs from hospital-related samples were carried by bacteria from Acinetobacter, Aeromonas and various genera from Enterobacteriaceae, which each was associated with multiple ARGs. In contrast, the ARGs that were exclusively in samples from communities and wetlands were carried by species that encoded only 1 to 2 ARGs each and were not normally associated with human infections. The qPCR showed that water samples of hospital surrounds had higher concentrations of intI1 and antimicrobial resistance genes such as tetA, ermA, ermB, qnrB, sul1, sul2 and other beta-lactam genes. Further genes of functional metabolism reported that the enrichment of genes associated with the degradation/utilization of nitrate and organic phosphodiester were detected in water samples around hospitals and communities compared to those from wetlands. Finally, correlations between the water quality indicators and the number of ARGs were evaluated. The presence of total nitrogen, phosphorus, and ammonia nitrogen were significantly correlated with the presence of ermA and sul1. Furthermore, intI1 exhibited a significant correlation with ermB, sul1, and blaSHV, indicating a prevalence of ARGs in urban water environments might be due to the integron intI1's diffusion-promoting effect. However, the high abundance of ARGs was limited to the waters around the hospital, and we did not observe the geographical transfer of ARGs along with the river flow. This may be related to water purifying capacity of natural riverine wetlands. Taken together, continued surveillance is required to assess the risk of bacterial horizontal transmission and its potential impact on public health in the current region.

Counteraction of stability-activity trade-off of Nattokinase through flexible region shifting.

The widespread trade-off between stability and activity severely limits enzyme evolution. Although some progresses have been made to overcome this limitation, the counteraction mechanism for enzyme stability-activity trade-off remains obscure. Here, we clarified the counteraction mechanism of the Nattokinase stability-activity trade-off. A combinatorial mutant M4 was obtained by multi-strategy engineering, exhibiting a 20.7-fold improved half-life; meanwhile, the catalytic efficiency was doubled. Molecular dynamics simulation revealed that an obvious flexible region shifting in the structure of mutant M4 was occurred. The flexible region shifting which contributed to maintain the global structural flexibility, was considered to be the key factor for counteracting the stability-activity trade-off. Further analysis illustrated that the flexible region shifting was driven by region dynamical networks reshaping. This work provided deep insight into the counteraction mechanism of enzyme stability-activity trade-off, suggesting that flexible region shifting would be an effective strategy for enzyme evolution through computational protein engineering.