Recombinant Strains Research Articles

Genome sequences of recombinant African swine fever virus isolated from domestic pigs in Vietnam

ABSTRACT African swine fever is a highly contagious and devastating disease of pigs. Here, we present the draft genome assemblies of three Vietnamese African s wine fever virus (ASFV) isolates: rASF1/2-avac02, rASF1/2-avac03, and rASF1/2-avac07. All isolates were closely related to the Chinese recombinant ASFV strains of genotypes I and II.

Enhanced biosorption of cadmium ions on immobilized surface-engineered yeast using cadmium-binding peptides

A new type of cadmium (Cd) ion cell surface adsorbent was developed by integrating bacteriophage display peptide library technology with cell surface display technology. Cd2+ chelating resin served as the target molecule in screening experiments, leading to the identification of four Cd2+ −binding peptides. These peptides were introduced into Saccharomyces cerevisiae via the pYD1 plasmid using lithium acetate heat shock transformation. Adsorption efficiency tests indicated that the engineered yeasts adsorbed more Cd2+ than the control strain EBY100 when exposed to the same amount of Cd2+. Among these peptides, sequence 3-containing strain was demonstrated to have the highest Cd2+ adsorption efficiency, being 35% higher than the control strain. Additionally, when this recombinant yeast strain was immobilized using sodium alginate, the adsorption efficiency was increased by 55.7% compared to the control strain.

Recent Molecular Epidemiology of Echovirus 11 Throughout North and West Africa Resulted in the First Identification of a Recombinant Strain from an Acute Flaccid Paralysis Case in West Africa

Echovirus 11 has emerged as a major public health concern, causing sepsis in neonates in many European countries in recent years. In Africa, especially West Africa, where resources and diagnostic capacities are limited, only sporadic cases have been reported. To better understand the recent molecular epidemiology of E11 in West Africa, we characterized twenty-three echovirus 11 strains isolated through the acute flaccid paralysis and environmental surveillance systems for polio from 2013 to 2023, using high-throughput sequencing. Our data are noteworthy due to identifying for the first time a recombinant strain from an acute flaccid paralysis case and represent the first focus to date on molecular characterization of echovirus 11 in West Africa. Moreover, our data show that echovirus 11 diverged from 1970 (95% HPD range, 1961–1979) and evolved into four distinct clades, with the virus spread from West Africa to Europe, exhibiting two introductions in France around 2017, from Senegal and Guinea. Furthermore, the in silico analysis reveals four non-conservative amino acid substitutions in the VP1 sequences of the European strains associated with neonatal sepsis in newborns and a conserved amino acid motif in the VP1 protein toward enterovirus genotypes. Our data provide new insights into the epidemiology of echovirus 11 and point to the crucial need to implement specific surveillance programs targeting non-polio enteroviruses for the rapid identification of emerging or re-emerging enterovirus species, particularly in Africa.

Multiple metabolic engineering of Saccharomyces cerevisiae for the production of lycopene

AbstractLycopene is a high‐value‐added tetraterpenoid, which is widely used in cosmetics, medicine, food, and dietary supplements. The intracellular mevalonate pathway of Saccharomyces cerevisiae provides natural precursors for terpenoid product synthesis, so it is an excellent host for the heterologous production of lycopene. In this study, a recombinant strain named L10 with efficient lycopene production capability was constructed through multiple strategies, such as regulating the gene copy number of key enzymes, increasing nicotinamide adenine dinucleotide phosphate supply, and reducing squalene accumulation. Then, considering that intracellular lycopene accumulation can cause cytotoxicity to S. cerevisiae, we attempted to identify a transporter that can efficiently transport lycopene from intracellular to extracellular space. Molecular docking simulations predicted that the ATP‐binding cassette transporter Snq2p may be a potential transporter of lycopene, and its function in promoting lycopene secretion was further determined by overexpression verification. The lycopene secretion titer of the strain L10Z2 overexpressing Snq2p increased to 16.5 times that of the control at the shake‐flask level. After optimizing the galactose regulation system, the intracellular and secreted lycopene production of L11Z2 reached 2113.78 and 26.28 mg/L, respectively, after 150 h fed‐batch culture in a 3‐L bioreactor. This work provides a new research direction for efficient lycopene synthesis in S. cerevisiae cell factory.

Development of a starch-fermenting Zymomonas mobilis strain for bioethanol production

BackgroundBiorefinery using microorganisms to produce biofuels and value-added biochemicals derived from renewable biomass offers a promising alternative to meet our sustainable energy and environmental goals. The ethanologenic strain Zymomonas mobilis is considered as an excellent chassis for constructing microbial cell factories for diverse biochemicals due to its outstanding industrial characteristics in ethanol production, high specific productivity, and Generally Recognized as Safe (GRAS) status. Nonetheless, the restricted substrate range constrains its application.ResultsThe truncated ice nucleation protein InaK from Pseudomonas syringae was used as an autotransporter passenger, and α-amylase was fused to the C- terminal of InaK to equip the ethanol-producing bacterium with the capability to ferment renewable biomass. Western blot and flow cytometry analysis confirmed that the amylase was situated on the outer membrane. Whole-cell activity assays demonstrated that the amylase maintained its activity on the cell surface. The recombinant Z. mobilis facilitated the hydrolysis of starch into oligosaccharides and enabled the streamlining of simultaneous saccharification and fermentation (SSF) processes. In a 5% starch medium under SSF, recombinant strains containing Peno reached a maximum titer of 13.61 ± 0.12 g/L within 48 h. This represents an increase of 111.0% compared to the control strain's titer of titer of 6.45 ± 0.25 g/L.ConclusionsBy fusing the truncated ice nucleation protein InaK with α-amylase, we achieved efficient expression and surface display of the enzyme on Z. mobilis. This fusion protein exhibited remarkable enzymatic activity. Its presence enabled a cost-effective bioproduction process using starch as the sole carbon source, and it significantly reduced the required cycle time for SSF. This study not only provides an excellent Z. mobilis chassis for sustainable bioproduction from starch but also highlights the potential of Z. mobilis to function as an effective cellular factory for producing high-value products from renewable biomass.

Comparison Between Simple Batch and Fed-Batch Bioreactor Cultivation of Recombinant BCG

Background/Objectives: Tuberculosis continues to be a significant global health concern, causing 1.3 million deaths in 2022, particularly affecting children under 5 years old. The Bacillus Calmette-Guérin (BCG) vaccine, developed in 1921, remains the primary defense against tuberculosis but requires modernized production methods. The recombinant BCG-pertussis strain shows potential in providing dual protection against tuberculosis and whooping cough, especially for vulnerable newborns, and enhanced efficacy against bladder cancer. Implementing submerged cultivation techniques for rBCG-pertussis production can offer increased productivity and standardization. Methods: This study explores a fed-batch cultivation strategy with pH-stat control to feed L-glutamic acid through the acid pump into 1 L bioreactor. Three pH values were evaluated for fed-batch and a simple batch without pH control was done for comparison. The viable cell concentration was compared before and after freeze-drying samples harvested during the cultures. Results: L-glutamic acid was identified as the preferred substrate for rBCG-pertussis. While the fed-batch strategy did not enhance the maximum specific growth rate compared to simple batch cultivation, it did improve the specific growth rate after day 4 in the pH 7.4-controlled fed-batch cultures, thereby reducing the cultivation time. Fed-batch cultures controlled at three pH levels exhibited lower optical density than the simple batch, although the viable cell counts were similar. Notably, samples harvested after day 8 from the simple batch cultures showed a reduction in CFU/mL after freeze-drying, whereas all fed-batch samples exhibited high recovery of viable cell counts post lyophilization. Conclusions: The additional glutamate supplied to the fed-batch cultures may have protected the cells during the lyophilization process.

Exploring secretory signal sequences useful in excreting recombinant proteins in Beauveria bassiana as biocontrol fungus.

Entomopathogenic fungi excrete a group of proteins to assimilate nutrients and defeat the host immune defense. Functional secretory signal sequences are needed for efficient secretion of the virulence-related proteins in recombinant strain. In this study, secretome analysis was used to explore the secreted proteins of Beauveria bassiana. Enrichment analysis indicated that B. bassiana secretome was mainly associated with metabolism of glucoside, polysaccharide, extracellular ester compound, and so on. In addition, proteins associated with biogenesis of cellular components were also enriched, including those involved in biogenesis of cell wall and vacuole. Then, four secretory signal sequences were functionally verified with green fluorescent protein as reporter. Finally, a signal sequence was used to excrete three insect venom protein serpins in B. bassiana, in which over-expression of serpin 8 gene resulted in a significant increase in fungal virulence. This study highlights that functional secretory signal sequences are potential molecular elements useful in excretion of virulence-related proteins in insect pathogenic fungi.

A rapid and versatile reverse genetic approach and visualization animal models for emerging zoonotic pseudorabies virus

Pseudorabies virus (PRV), a member of the Alphaherpesvirinae subfamily and a causative pathogen of Aujeszky's disease, has a broad host range including domestic and wild animals. PRV has been reported as a causative agent in patients with acute encephalitis in 2021, which suggests PRV might be a novel animal-origin virus in terms of zoonotic spillover and spread potential. To manage current PRV epidemics in pigs and prepare for future pandemics in other species including humans. Fundamental techniques essential for procuring such knowledge on prevention and therapy of PRV. Here, PRV CD22 strain was isolated and phylogenetic analysis showed that PRV CD22 belongs to the current epidemic strains in China. PRV CD22 was highly lethal to mice and piglets in vivo. Moreover, a rapid and efficient system to generate recombinant PRV was constructed based on PRV CD22 genomic DNA fosmid library. Using this system, a recombinant PRV strain expressing engineered labeling protein was rescued for visualization of viral infection in mouse model. Our study allows the generation of PRV that can be used for downstream treatment analyses. Once experimental or surveillance samples are obtained, PRV can be generated and treated efficiently based on our study.

Genetic diversity and spread of recombinant coxsackievirus A4 in hand, foot, and mouth disease cases in Bangkok, Thailand: 2017–2023

Coxsackievirus A4 (CVA4) has recently become one of the most common causative agents of hand, foot, and mouth disease. The current study investigated the genetic diversity and spread of recombinant CVA4 by analyzing circulating genotypes and recombinant strains in Bangkok, Thailand, from 2017 to 2023. Partial VP1, 3Dpol, and whole genome sequencing of CVA4 samples collected from collaborating hospitals were conducted. Phylogenetic analysis of CVA4 VP1 and 3Dpol genome regions revealed discordance, indicating recombination. The predominant CVA4 genotype was C3, primarily observed in 2019. The predominant genotype in 2017 was C1. D2, commonly found in China, was occasionally observed. In nucleotide similarity analysis, intertypic recombination between CVA4 and EV-A during the evolutionary history of the virus was evident, particularly in the nonstructural region. The estimated emergence of genotypes C1 and C3 in Thailand occurred around 2014, with an evolutionary rate of 5.8 × 10− 3 nucleotide substitutions per site per year. Genotype D2 exhibited notable variability across both the entire genome and the structural protein region compared to genotype C. Monitoring the genetic diversity and circulation of recombinant CVA4 is crucial for identifying newly emerging virus strains, enabling prompt public health responses and containment efforts, and enhancing surveillance in Thailand.

Production and characterization of novel/chimeric sophorose–rhamnose biosurfactants by introducing heterologous rhamnosyltransferase genes into Starmerella bombicola

Glycolipid biosurfactant, sophorolipids (SLs) and rhamnolipids (RLs) can be widely used in agriculture, food and chemical industries. The different physicochemical properties of SLs and RLs, such as hydrophilic lipophilic value (HLB) and critical micelle concentration (CMC), determine they have different application focus. Researchers are still hoping to obtain new glycolipid surfactants with unique surface activities. In this study, we successfully transformed two rhamnosyltransferase genes rhlA and rhlB from Pseudomonas aeruginosa to the sophorolipid-producing Starmerella bombicola CGMGG 1576 to obtain a recombinant strain was SbrhlAB. Two novel components with molecular weight of 554 (C26H50O12) and 536 (C26H48O11) were identified with the ASB C18 column from the fermentation broth of SbrhlAB, the former was a non-acetylated acidic C14:0 glycolipid containing one glucose and one rhamnose, and the latter was an acidic C14:1 glycolipid containing two rhamnoses. With the Venusil MP C18 column, one new glycolipid component was identified as an acidic C18:3 glycolipid with one rhamnose (C24H40O7), which has not been reported before. Our present study demonstrated that novel glycolipids can be synthesized in vivo by reasonable genetic engineering. The results will be helpful to engineer sophorolipid-producing yeast to produce some specific SLs or their derivatives in more rational and controllable way.

Dnajc3 (HSP40) Enhances Axon Regeneration in the Mouse Optic Nerve.

A forward genetics approach was used to identify genomic elements enhancing axon regeneration in the BXD recombinant mouse strains. Axon regeneration was induced by knocking down Pten in retinal ganglion cells (RGCs) using adeno-associated virus (AAV) to deliver an shRNA followed by an intravitreal injection of Zymosan with CPT-cAMP that produced a mild inflammatory response. RGC axons were damaged by optic nerve crush (ONC). Following a 12-day survival period, regenerating axons were labeled by intravitreal injection of Cholera Toxin B (CTB) conjugated with Alexa Fluor 647. Two days later, labeled axons within the optic nerve were examined to determine the number of regenerating axons and the distance they traveled down the optic nerve. The analysis revealed a surprising difference in the amount of axonal regeneration across all 33 BXD strains. There was a 7.5-fold difference in the number of regenerating axons and a 4-fold difference in distance traveled by regenerating axons. These data were used to generate an integral map defining genomic loci modulating the enhanced axonal regeneration. A quantitative trait locus modulating axon regeneration was identified on Chromosome 14 (115 to 119 Mb). Within this locus were 16 annotated genes. Subsequent testing revealed that one candidate gene, Dnajc3 , modulates axonal regeneration. Dnajc3 encodes Heat Shock Protein 40 (HSP40), which is a molecular chaperone. Knocking down Dnajc3 in the high regenerative strain (BXD90) led to a decreased regeneration response, while overexpression of Dnajc3 in a low regenerative strain (BXD34) resulted in an increased regeneration response. These findings suggest that Dnajc3 not only increases the number of regenerating axons, it also increases the distance those axons travel. This may prove to be critical for functional recovery in large mammals, where the distance axons travel to their target is considerably longer than that of the mouse. Thus, Dnajc3 may play a critical role for functional recovery in humans by increasing the number of regenerating axons and the distance the regenerating axons travel.

Metabolic engineering of Mucor circinelloides to improve astaxanthin production.

Astaxanthin is a bioactive natural pigment with antioxidant properties. It has extensive applications within the industrial sector as well as in human and animal health. Mucor circinelloides is a zygomycete fungus that accumulates β-carotene as the main carotenoid compound. M. circinelloides is a well-known model organism among Mucorales for studying carotenogenesis in fungi, which makes it a promising candidate for the biotechnological production of carotenoids. In this study, β-carotene hydroxylase (crtR-B) and ketolase (bkt) genes (codon-optimized) were coexpressed from Haematococcus pluvialis in M. circinelloides using two potent promoters gpd1 and zrt1 respectively to generate an astaxanthin-producing biofactory. Following 72h of cultivation, the recombinant M. circinelloides Mc-57 obtained in this study produced 135 ± 8µg/g of astaxanthin. This is the highest reported amount in M. circinelloides to date. The mRNA levels of crtR-B and bkt in Mc-57 were assayed using RT-qPCR. These levels showed a 5.7-fold increase at 72h and a 5.5-fold increase at 24h, respectively, compared to the control strain. This demonstrated the successful overexpression of both genes, which correlated with the production of astaxanthin in the Mc-57. Moreover, the addition of glutamate (2g/L) and mevalonate (15 mM) resulted in an increase in astaxanthin production in the recombinant strain. The results showed that the combined addition of these metabolic precursors resulted in 281 ± 20µg/g of astaxanthin, which is 2.08-fold higher than the control medium (135 ± 8µg/g). The addition of metabolic precursors also positively impacted the biomass growth of Mc-57, reaching 11.2 ± 0.57g/L compared to 9.1 ± 0.23g/L (control medium). The study successfully addressed the challenge of balancing the accumulation of astaxanthin with biomass growth, which has been regarded as common bottleneck in the metabolic engineering of microbial cells. The development of a recombinant fungal strain of M. circinelloides not only increased astaxanthin content. Additionally, it provided a foundation for further improvement of the biotechnological production of astaxanthin in M. circinelloides.

Genomic epidemiology and immune escape of SARS-CoV-2 recombinant strains circulating in Botswana

Background: SARS-CoV-2 continues to evolve and evade host immunity. We characterized the molecular and mutational landscape of SARS-CoV-2 recombinant strains in Botswana.Methods: We performed genomic, phylogenetic, and immunoinformatic analyses of 5,254 near-complete genomes from 2020 to 2023. We assessed the presence of mutations of interested (MutOI) that may be associated with immune escape in silico.Results: We observed a few recombinant strains in Botswana, with the majority being descendants of Omicron (XBB*), except for XV and XM. Most recombinant sequences corresponded to transmission clusters. Most recombination events occurred within the Receptor Binding Domain (RBD) of the Spike (S) protein. We identified 17 MutOI among different proteins, with the majority occurring at a very low (<4.8 × 10⁻⁵) global prevalence. We also observed S:Q474K, a MutOI in the RBD, that was predicted to escape HLA class I-mediated immune responses. Molecular surveillance is vital to inform early detection and response to potential variants with heightened immune and vaccine breakthrough properties.Conclusions: These results underscore the need for continued molecular surveillance to map the evolutionary landscape of SARS-CoV-2.

Isolation, Molecular Characterisation, and Pathogenicity Analysis of a Novel Recombinant ALV-J Strain Isolated From Chinese Hetian Chickens.

Avian leukosis virus subgroup J (ALV-J) primarily affects poultry, particularly chickens, leading to tumourigenesis and immunosuppression, which results in substantial economic losses. It is important to note that ALV-J is commonly found in indigenous chicken breeds in China, and the virus's vertical transmission characteristics present a significant threat to the preservation of local chicken breeds. The study aimed to investigate the characteristics and effects of the recombinant ALV-J strain LY2021J, with a focus on its genetic composition and its potential influence on virulence and pathogenicity. LY2021J was isolated using DF-1 cells and validated by enzyme-linked immunosorbent assay (ELISA) and IFA. The proviral genome was amplified using segmented PCR and then spliced together using DNASTAR software. Genome-wide genes, including gag, pol, gp85, and long terminal repeat (LTR), were compared. Recombination sites were analysed using RDP5 and SimPlot software. Pathogenicity was evaluated by monitoring symptoms and conducting examinations on SPF chickens. The outbreak of ALV-J in China has caused significant economic losses in the poultry industry. Although largely controlled in white-feather broilers and egg-laying chickens, ALV-J has spread to yellow-feather broilers and local breeds. A strain, LY2021J, isolated from Hetian chickens, showed lower mortality despite severe dysplasia. Genetic analysis revealed high similarity between LY2021J and the Chinese strains JS14NT01 and NX0101, suggesting a shared origin. Recombination with strain ev-1 and specific 3' UTR deletions may explain LY2021J's reduced virulence. Continued monitoring and prevention strategies are essential to mitigate ALV-J's impact.

Regulation of the RCK1 gene on the oxidative tolerance of Saccharomyces cerevisiae

Our previous work indicated that the quorum sensing (QS) effect could regulate the oxidative tolerance of Saccharomyces cerevisiae, and QS may impact oxidative and antioxidative metabolisms of S. cerevisiae by regulating the RCK1 gene. Therefore, this work proposed a reasonable logic that RCK1 could play roles in regulating the oxidative and antioxidative metabolisms of yeast cells. The results presented here suggested that the overexpression of RCK1 has a regulatory effect on the reduction of ROS level and the promotion of oxidative tolerance of S. cerevisiae. The overexpression of RCK1 promoted the ROS generation through activating the MAPK pathway; on the other hand, RCK1-regulated antioxidative metabolism played a more significant role to realize lower ROS level and higher oxidative tolerance of S288c-RCK1 and ΔARO80-RCK1 strains. To improve the fermentation performance of yeast while circumventing metabolic burden, a recombinant strain with over time-controlled overexpression of the RCK1 gene (i.e., S288c'-RCK1 strain) derived from S288c strain was successfully constructed to achieve artificial regulation of yeast oxidative tolerance. Transcriptomics analysis was further performed on both S. cerevisiae wild-type and S288c'-RCK1 strains to identify differentially expressed genes and analyze their functional pathway classification. This work is instructive for artificially modulating the oxidative tolerance of strains to enhance the fermentation performance of yeast.

Novel Intertypic Recombinant Coxsackievirus A2 Containing Specific Amino Acid Mutations in the RNA-Dependent RNA Polymerase Potentially Associated With Its Emergence.

Coxsackievirus A2 (CVA2), a member of enterovirus A species (EV-A), is associated with diverse human diseases and occasionally causes acute gastroenteritis (AGE). In Thailand, CVA2 emerged as the predominant genotype in 2019. The increasing incidence of CVA2, coupled with the limited availability of full-length genomes, highlights the need for more complete genome sequence analysis to facilitate molecular epidemiology study. This study aimed to investigate the molecular epidemiology, evolutionary dynamics, and recombination characteristics of CVA2 associated with AGE in Thailand from 2013 to 2022. A total of 19 full-genome sequences of CVA2 isolated from stool samples of AGE patients in Thailand were characterized and analyzed together with the reference sequences available in the GenBank database. A novel lineage of CVA2 (subgenotype C5) was detected with the potential recombination with CVA10 within the P2 and P3 regions. Specific consensus amino acid mutations, A61S in the VP3 gene and R136K in the 3D (RdRp) gene, were identified in all CVA2 recombinant strains. Additionally, the S45G mutation in the RdRp gene was found to be potentially associated with the emergence of CVA2 infection in 2019. In conclusion, this study reveals potential intertypic recombinant events and specific mutations in CVA2 strains isolated from AGE patients and provides a broader understanding of its evolutionary epidemiology.

Screening, identification, engineering, and characterization of Bacillus-derived α-amylase for effective tobacco starch degradation

In this study, two high-performing α-amylase-producing strains, CK3–5 and A8–1 were successfully isolated and characterized, which were taxonomically confirmed as Bacillus velezensis through whole-genome sequencing and bioinformatics. Bioinformatic sequence analysis and molecular docking revealed the catalytic triad (Asp173-Glu208-Asp274) essential for α-amylase function. Through metabolic engineering, the recombinant strain BAX-5/PT17amy(A8–1)SP002 was developed, which exhibited the highest α-amylase activity of 1440 U/mL upon fermentation optimization, marking a 9.2-fold enhancement over the wild-type strain A8–1, and it successfully degraded 6 % of the starch in the tobacco leaves within 48 h, while the content of 13 harmful substances, including acetamide, pyridine, and acetonitrile, was reduced by 8.6 % to 25.2 %. This study reveals a novel α-amylase gene from B. velezensis and establishes an efficient expression system in B. amyloliquefaciens, offering valuable insights for industrial α-amylase production.

Xanthan gum production in Xanthomonas campestris is increased by favoring the biosynthesis of its monomers

Current efforts to improve xanthan gum (XG) production by Xanthomonas have focused on the growth medium, operating parameters, and downstream steps. However, a key aspect is the development of optimal strains. The present work aimed to investigate the formation of XG monomers, using kinetic and stoichiometric models to identify possible bottlenecks, and to engineer a recombinant strain to overcome such limitations. The galU and ugd genes involved in thebiosynthesis of the UDP-glucose and UDP-glucuronic acid monomers were overexpressed in Xanthomonas campestris pv. campestris. The strains were cultivated in shake flasks and bioreactor. As predicted by in silico analysis, overexpression of the ugd gene resulted in a significant increase in gum synthesis, up to 50% higher volumetric productivity in thebioreactor. To a lesser extent, galU overexpression was also shown to improve product formation. These findings validated the hypothesis that metabolic engineering of the monomer biosynthesis can enhance XG production.

Protein Engineering of Nicotinamide Riboside Kinase Based on a Combinatorial Semirational Design Strategy for Efficient Biocatalytic Synthesis of Nicotinamide Mononucleotides.

Industrial biosynthesis of β-nicotinamide mononucleotide (β-NMN) lacks a highly active nicotinamide riboside kinase for the phosphorylation process. Cumbersome preprocessing steps and excessive ATP addition contribute to its increased cost. To tackle these challenges, a docking combination simulation (DCS) semirational mutagenesis strategy was designed in this study, combining various modification strategies to obtain a mutant NRK-TRA with 2.9-fold higher enzyme activity. Molecular dynamics simulations and structural analysis demonstrate the enhancement of its structural stability. High-density fermentation was achieved through a 5 L fermentation tank, with a titer reaching 208.3 U/mL, the highest in the current report. An ATP-cycling whole-cell catalytic system was employed and optimized by introducing a polyphosphate kinase 2 (PPK2) recombinant strain, and 15.16 g/L β-NMN was obtained through a series of batch transformation experiments. This study provides a new strategy for the efficient screening of highly active enzyme variants and offers a green and promising biotransformation system for NMN production.

Orally administered recombinant Lactobacillus expressing PEDV neutralizing antibody protects piglets against PEDV infection

Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus, causing fatal acute diarrhea in suckling pigs, with mortality rates as high as 100 % in 7-day-old piglets. Due to the challenge of quickly establishing effective active immunity, the main strategy for protecting piglets from PEDV infection relies on antibodies, particularly neutralizing antibodies, to provide passive immune protection. In this study, a recombinant Lactobacillus strain for secreting the Fab fragment of neutralizing antibody against PEDV was constructed (pPG-Fab/J31). The results showed that the Fab antibody was stably expressed by pPG-Fab/J31, and exhibit specific neutralizing effect against PEDV. Then, pPG-Fab/J31 was used for the oral administration of newborn piglets to test the protective effect against PEDV challenge. The findings demonstrated that piglets in the antibody administration group exhibited an alleviation of clinical symptoms, a smaller decrease in weight, significant reduction in viral shedding, and attenuation of intestinal lesions. Additionally, the survival rate of piglets orally administered pPG-Fab/J31 was 100 %. Thus, PEDV neutralizing antibody expressed by recombinant Lactobacillus hold promise as a passive protective candidate, providing a new idea for the prevention and treatment of viral infections.