Transcriptome sequencing reveals key metabolic pathways for the synthesis of L-serine from glycerol and glucose in Escherichia coli

The blast resistance allele of OsBsr-d1 does not exist in most japonica rice varieties of Jilin Province in China. The development of Bsr-d1 knockout mutants via CRISPR/Cas9 enhances broad-spectrum resistance to rice blast in Northeast China. Rice blast is a global disease that has a significant negative impact on rice yield and quality. Due to the complexity and variability of the physiological races of rice blast, controlling rice blast is challenging in agricultural production. Bsr-d1, a negative transcription factor that confers broad-spectrum resistance to rice blast, was identified in the indica rice cultivar Digu; however, its biological function in japonica rice varieties is still unclear. In this study, we analyzed the blast resistance allele of Bsr-d1 in a total of 256 japonica rice varieties from Jilin Province in Northeast China and found that this allele was not present in these varieties. Therefore, we generated Bsr-d1 knockout mutants via the CRISPR/Cas9 system using the japonica rice variety Jigeng88 (JG88) as a recipient variety. Compared with those of the wild-type JG88, the homozygous Bsr-d1 mutant lines KO#1 and KO#2 showed enhanced leaf blast resistance at the seedling stage to several Magnaporthe oryzae (M. oryzae) races collected from Jilin Province in Northeast China. Physiological and biochemical indices revealed that the homozygous mutant lines produced more hydrogen peroxide than did JG88 plants when infected with M. oryzae. Comparative RNA-seq revealed that the DEGs were mainly involved in the synthesis of amide compounds, zinc finger proteins, transmembrane transporters, etc. In summary, our results indicate that the development of Bsr-d1 knockout mutants through CRISPR/Cas9 can enhance the broad-spectrum resistance of rice in Northeast China to rice blast. This study not only provides a theoretical basis for disease resistance breeding involving the Bsr-d1 gene in Northeast China, but also provides new germplasm resources for disease-resistance rice breeding.

CRISPR/Cas9-mediated knockout of Bsr-d1 enhances the blast resistance of rice in Northeast China.

This research combined Whole-Genome sequencing, intraspecific comparative genomics and transposon mutagenesis to investigate the menaquinone-7 (MK-7) synthesis potential in Bacillus subtilis natto. First, Whole-Genome sequencing showed that Bacillus subtilis natto BN-P15-11-1 contains one single circular chromosome in size of 3,982,436 bp with a GC content of 43.85 %, harboring 4,053 predicted coding genes. Next, the comparative genomics analysis among strain BN-P15-11-1 with model Bacillus subtilis 168 and four typical Bacillus subtilis natto strains proves that the closer evolutionary relationship Bacillus subtilis natto BN-P15-11-1 and Bacillus subtilis 168 both exhibit strong biosynthetic potential. To further dig for MK-7 biosynthesis latent capacity of BN-P15-11-1, we constructed a mutant library using transposons and a high throughput screening method using microplates. We obtained a YqgQ deficient high MK-7 yield strain F4 with a yield 3.02 times that of the parent strain. Experiments also showed that the high yield mutants had defects in different transcription and translation regulatory factor genes, indicating that regulatory factor defects may affect the biosynthesis and accumulation of MK-7 by altering the overall metabolic level. The findings of this study will provide more novel insights on the precise identification and rational utilization of the Bacillus subtilis subspecies for biosynthesis latent capacity. 

Comparative genomics analysis and transposon mutagenesis provides new insights into high menaquinone-7 biosynthetic potential of Bacillus subtilis natto.

Single-cell protein and recombinant proteins derived from Saccharomyces cerevisiae have been extensively applied in various fields, including food additives, pharmaceuticals, and animal feed. Enhancement of global protein synthesis ability by rational engineering can potentially satisfy the increasing demand, while the complexity of regulation mechanisms in yeast hinders it. This study integrated ARTP mutagenesis, microfluidic screening, and comparative transcriptomic analysis to look insight into the regulation mechanism. Initially, a high-throughput screening method based on dual fluorescent proteins was established for selecting the mutants with increased protein content. After two rounds of ARTP mutagenesis, one yeast mutant was identified with a 10.92% increase in the final protein content, and the yield increased by 18.18%. Subsequent comparative transcriptomic analysis revealed: (1) significant upregulation of key genes involved in the ribosome biosynthesis pathways; (2) increased expression of genes associated with translation factors; (3) downregulation of many energy metabolism pathways. These findings contribute to a deeper understanding of the underlying metabolic mechanisms behind the enhancement of global protein synthesis in yeast. They also provide potential molecular targets for use in reverse metabolic engineering, aimed at constructing highly efficient cell factories for protein synthesis and production. 

Identification of regulation mechanisms associated with enhanced global protein synthesis ability by Saccharomyces cerevisiae via microfluidic screening and transcriptome analysis

Bacillus subtilis is central to Asian fermented soybean foods, including Japanese natto. To explore the genomic boundaries of B. subtilis var. natto, we conducted a comparative pangenome analysis of 42 strains, including the core natto clade (n=26) and its closest relatives. Our analysis revealed a striking evolutionary paradox centered on a single strain isolated from Indian bekang. Core-genome phylogenetic analysis places this bekang strain clearly outside the tight natto clade, with a Nepalese kinema strainbeing its closest systematic neighbor. In stark contrast, quantitative analysis of accessory gene profiles revealed this single bekang strain is the functional nearest neighbor to the natto clade, sharing a highly conserved accessory gene repertoire. This shared profile defines a “natto-type” adaptive strategy (the “broad-sense natto group,” n=27), separating it from other related strains. Analysis of this group-specific repertoire revealed an enrichment of transcriptional regulators and metabolic enzymes. This finding provides a compelling case study (n=1) of polygenic adaptation, suggesting complex evolutionary pathways, such as horizontal gene transfer or selective retention, can drive rapid adaptation across disparate lineages.

Conserved accessory genes link phylogenetically distinct Bacillus subtilis from Japanese and Indian fermented soyfoods

Comparative transcriptome analysis reveals the underlying mechanism for over-accumulation of menaquinone-7 in Bacillus subtilis natto mutant

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Comparative transcriptome analysis reveals the underlying mechanism for over-accumulation of menaquinone-7 in Bacillus subtilis natto mutant