Phosphorus (P) limits the production of maize, one of the major food crops in China. Phosphate-solubilizing bacteria (PSB) have the capacity to solubilize phosphate complexes into plant absorbable and utilizable forms by the process of acidification, chelation, and exchange reactions. In this study, six bacteria, including one Paenibacillus sp. B1 strain, four Pseudomonas sp. strains (B10, B14, SX1, and SX2) and one Sphingobium sp. SX14 strain, were those isolated from the maize rhizosphere and identified based on their 16S rRNA sequences. All strains could solubilize inorganic P (Ca₃(PO₄)₂, FePO₄ and AlPO₄), and only B1 and B10 organic P (lecithin). All strains, except of SX1, produced IAA, and SX14 and B1 showed the highest level. B1 incited the highest increase in root length and the second increase in shoot and total dry weight, shoot length, and total P and nitrogen (N), along with increased root length. In addition, by confocal laser scanning microscopy (CLSM), we found that green fluorescent protein (GFP)-labeled B1 mainly colonized root surfaces and in epidermal and cortical tissue. Importantly, B1 can survive through forming spores under adverse conditions and prolong quality guarantee period of bio-fertilizer. Therefore, it can act as a good substitute for bio-fertilizer to promote agricultural sustainability.

Nitrogen fixation has been established in protokaryotic model Escherichia coli by transferring a minimal nif gene cluster composed of 9 genes (nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV) from Paenibacillus sp. WLY78. However, the nitrogenase activity in the recombinant E. coli 78-7 is only 10 % of that observed in wild-type Paenibacillus. Thus, it is necessary to increase nitrogenase activity through synthetic biology. In order to increase nitrogenase activity in heterologous host, a total of 28 selected genes from Paenibacillus sp. WLY78 and Klebsiella oxytoca were placed under the control of Paenibacillus nif promoter in two different vectors and then they are separately or combinationally transferred to the recombinant E. coli 78-7. Our results demonstrate that Paenibacillus suf operon (Fe–S cluster assembly) and the potential electron transport genes pfoAB, fldA and fer can increase nitrogenase activity. Also, K. oxytoca nifSU (Fe–S cluster assembly) and nifFJ (electron transport specific for nitrogenase) can increase nitrogenase activity. Especially, the combined assembly of the potential Paenibacillus electron transporter genes (pfoABfldA) with K. oxytoca nifSU recovers 50.1 % of wild-type (Paenibacillus) activity. However, K. oxytoca nifWZM and nifQ can not increase activity. The combined assembly of the potential Paenibacillus electron transporter genes (pfoABfldA) with K. oxytoca nifSU recovers 50.1 % of wild-type (Paenibacillus) activity in the recombinant E. coli 78-7. Our results will provide valuable insights for the enhancement of nitrogenase activity in heterogeneous host and will provide guidance for engineering cereal plants with minimal nif genes.

Paenibacillus is a genus of Gram-positive, facultative anaerobic and endospore-forming bacteria. Genomic sequence analysis has revealed that a compact nif (nitrogen fixation) gene cluster comprising 9–10 genes nifBHDKENX(orf1)hesAnifV is conserved in diazotrophic Paenibacillus species. The evolution and function of the orf1 gene within the nif gene cluster of Paenibacillus species is unknown. In this study, a careful comparison analysis of the compositions of the nif gene clusters from various diazotrophs revealed that orf1 located downstream of nifENX was identified in anaerobic Clostridium ultunense, the facultative anaerobic Paenibacillus species and aerobic diazotrophs (e.g., Azotobacter vinelandii and Azospirillum brasilense). The predicted amino acid sequences encoded by the orf1 gene, part of the nif gene cluster nifBHDKENXorf1hesAnifV in Paenibacillus graminis RSA19, showed 60–90% identity with those of the orf1 genes located downstream of nifENX from different diazotrophic Paenibacillus species, but shared no significant identity with those of the orf1 genes from different taxa of diazotrophic organisms. Transcriptional analysis showed that the orf1 gene was expressed under nitrogen fixation conditions from the promoter located upstream from nifB. Mutational analysis suggested that the orf1 gene functions in nitrogen fixation in the presence of a high concentration of O₂.

Zearalenone (ZEN) is a thermostable, lipophilic, non-steroidal estrogenic mycotoxin produced by Fusarium spp. that persistently contaminates food and feed. Its strong estrogenic activity and resistance to conventional detoxification strategies pose significant threats to food safety and human and animal health. Conventional physical and chemical degradation methods often compromise nutritional quality and leave toxic residues. Here we report the engineering of a novel Clonostachys rosea lactone hydrolase, Cr2zen, for efficient ZEN degradation in Pichia pastoris under mild conditions. Native Cr2zen exhibited a protein concentration of 0.076 mg/mL, achieving a degradation rate of approximately 17.9% within 30 min, with kinetic parameters of K_m 75.9 µM and V_max 0.482 µmol/L/s at 30 °C and pH 8.0. By integrating signal peptide screening and codon optimization, we identified Ser-Cr2 as the most effective variant, achieving a rapid 81.53% degradation of 10 ppm ZEN under mild conditions. Fed-batch cultivation in a 7.5 L bioreactor resulted in high cell densities of OD₆₀₀ 332.8 for Ser-Cr2 and 310.8 for Oser-Cr2, with extracellular protein concentrations of 0.62 and 0.79 g/L, respectively. The results demonstrate that signal peptide engineering and codon optimization substantially improved the production of lactone hydrolase in P. pastoris. This study establishes a scalable ZEN degradation under mild conditions in P. pastoris and outlines a strategy to integrate protein and process engineering for enhanced enzymatic mycotoxin degradation.