• Isobutanol-producing S. cerevisiae strain for industrial application. • Isobutanol-tolerant strain developed from conventional mutagenesis. • CRISPR-Cas9-mediated BAT1 knockout to enhance isobutanol production. • CRISPR-Cas9 avoids the presence of plasmids and antibiotic markers. • Strain highly increased isobutanol yield, potential for bio-alcohol production.

Background and Aim: Antimicrobial resistance (AMR) resulting from antibiotic misuse in livestock poses a growing threat to animal and human health. The development of sustainable probiotic alternatives supports the United Nations Sustainable Development Goals (SDGs) for zero hunger (SDG 2), good health and well-being (SDG 3), and responsible consumption and production (SDG 12). This study aimed to isolate, characterize, and perform genomic analysis of lactic acid bacteria (LAB) from swine feces exhibiting antibacterial activity against pathogenic Escherichia coli, to explore their potential as eco-friendly probiotic feed additives. Materials and Methods: Thirty fecal samples were collected from slaughtered crossbred pigs in Thailand. LAB isolates were screened for antibacterial activity against five E. coli pathotypes (Enteroaggregative E. coli, enterohemorrhagic E. coli, enteroinvasive E. coli, enterotoxigenic E. coli, and enteropathogenic E. coli) and assessed for acid and bile tolerance, adhesion capacity, and gastrointestinal survival. Two promising isolates (ATP111 and ATP210) were subjected to whole-genome sequencing and bioinformatics analyses for genes related to antimicrobial production, stress tolerance, virulence, and AMR. Results: Among 93 initial isolates, Lactiplantibacillus argentoratensis ATP111 and Weissella cibaria ATP210 exhibited broad-spectrum inhibition against all E. coli pathotypes. Both strains survived under pH 2.5 and 1% bile conditions, showing 74.39% and 66.90% survival, respectively, in simulated gastrointestinal conditions. Genomic analyses revealed the presence of genes encoding bacteriocins, polyketide synthases, terpenes, and multiple stress-response proteins, supporting their resilience and antimicrobial functionality. Importantly, both genomes lacked virulence and AMR genes, confirming biosafety for probiotic use. Conclusion: The integrated phenotypic and genomic evidence positions L. argentoratensis ATP111 and W. cibaria ATP210 as safe, effective, and sustainable probiotic candidates for swine health management. Their application as antibiotic alternatives aligns with SDG 3 (good health and well-being), SDG 12 (responsible consumption and production), and SDG 15 (life on land), contributing to reduced antibiotic dependence and improved livestock sustainability. Future in vivo validation is recommended to confirm efficacy and support global AMR mitigation efforts. Keywords: antimicrobial resistance, Escherichia coli, Lactiplantibacillus argentoratensis, Probiotics, SDG 12 (responsible consumption and production), SDG 15 (life on land), SDG 2 (zero hunger), SDG 3 (good health and well-being), sustainability, swine health, Weissella cibaria.

Thai traditional rice wine, known as Sato, is produced from glutinous rice using a mixed-culture starter called Lookpang. This traditional method often suffers from variability and inconsistent flavor profiles. To address these issues, the study aimed to improve the quality of Thai traditional rice wine by utilizing a defined starter culture composed of high-fatty acid ethyl esters (FAEEs)-producing Saccharomyces cerevisiae and Aspergillus oryzae. The fermentation process followed a methodology similar to that used in Japanese sake production, aiming to achieve more consistent quality and flavor profiles in the final product. Furthermore, this study involved fermenting rice wine using low-cost Thai rice to enhance its value. The parental Saccharomyces cerevisiae strain, 312WT, was isolated from Thai Sugar Industry Co., Ltd., and demonstrated fermentation performance comparable to the commercial wine yeast strain EC-1118. Subsequently, conventional mutagenesis was applied to 312WT, resulting in a mutant strain, 312/6, which overproduces fatty acid ethyl esters (FAEE). Rice wine fermented with the 312/6 strain exhibited the highest concentrations of ethyl hexanoate (28.55 ± 7.91 µg/kg, OAV = 29) and ethyl octanoate (207.22 ± 51.65 µg/kg, OAV = 41). These compounds contribute sweet and sour apple flavors to beverages, respectively. Genomic DNA analysis, compared to the parental strain 312WT, demonstrated that 312/6 harbored several mutations in the fatty acid ethyl ester (FAEE) biosynthesis pathway genes (FAS1, FAS2, ACC1, EHT1, and EEB1). These genetic changes could account for the overproduction of FAEEs.

Post-weaning diarrhea caused by pathogenic Escherichia coli is a significant economic challenge in global swine production. Rising antimicrobial resistance linked to traditional antibiotics necessitates safe, sustainable alternatives. This study isolated and characterized host-associated Bacillus species from the gastrointestinal tracts of healthy pigs to evaluate their probiotic potential. Fifty-nine Bacillus isolates were screened for safety, gastrointestinal tract survivability, mucosal adhesion, and antimicrobial efficacy. Several isolates demonstrated robust tolerance to simulated gastrointestinal stress (acidic pH and bile salts), non-hemolytic profiles, and strong mucosal adhesion. Multiple isolates displayed inhibitory activity against pathogenic Escherichia coli in vitro. The most promising candidate, Bacillus subtilis 1BP1Co1, underwent Nanopore whole-genome sequencing. Genomic profiling confirmed its safety, revealing a complete absence of known virulence factors and high-risk antimicrobial resistance genes. Furthermore, genome annotation identified multiple biosynthetic gene clusters responsible for the production of antimicrobial secondary metabolites, including surfactin, fengycin, bacillaene, and subtilosin A, which mechanistically supports its pathogen-inhibitory properties. Collectively, these findings highlight indigenous Bacillus strains, particularly Bacillus subtilis 1BP1Co1, as promising probiotic candidates with the potential to be developed to mitigate Escherichia coli-associated enteric disorders in swine production systems.