Xuehong Zhang

Pickering interfacial catalysis in Pickering emulsions offers a promising platform for heterogeneous catalytic reactions. In the present work, immobilized matrixes with tunable hydrophobicity were fabricated by grafting hydrophilic silica particles with different amounts of octadecyltrichlorosilane (ODTs). By screening the activity of corresponding immobilized lipases, hyperperformance, characterized by both hyperactivity and resistance to product inhibition, had been achieved. Specifically, the immobilized lipase exhibited approximately 4-fold higher activity than that of the free lipase and demonstrated marked resistance to product inhibition, ultimately leading to a final fatty acid concentration approximately three times that obtained with the free lipase. It demonstrates clearly that the hyperperformance of immobilized lipase originates from Pickering interfacial catalysis in W/O Pickering emulsions rather than in O/W Pickering emulsions. The underlying mechanism is attributed to the tuning positioning of lipase at the oil–water interfaces. It was further confirmed by adding Pickering particles as lipase inhibitors and examining the influence of operating parameters (Pickering particle concentration and volume ratio of oil-to-water) on the hyperactivity of immobilized lipase. The hyperperformance of immobilized lipase also provides guidance for selecting hydrophobic matrixes for lipase immobilization, as the hydrophobicity of immobilized matrixes is directly related to the adsorption of immobilized lipase at oil–water interfaces in W/O Pickering emulsions and consequently to the Pickering interfacial catalysis catalyzed by immobilized lipase.

Collagenases can specifically degrade collagen, showing a wide application prospect in food, leather, waste utilization, biotechnology, and other industries. Currently, Hathewaya histolytica is commonly used in industry to produce collagenases, but its application is greatly limited by its pathogenicity. This study first identified five potential Pseudomonas-derived collagenases by sequence alignment. Bioinformatics tools were used to analyze their structures and functions. Heterologous expression of two P. chlororaphis-derived collagenases was achieved in E. coli, and their enzymatic properties were characterized. Bioinformatics analysis shows that the Pseudomonas-derived collagenases had low molecular weights (22.1~50.5 kDa) and good thermal stability (aliphatic index 73.73~88.81). Deletion of P. chlororaphis GP72ANO strain colP1 and colP2 genes had no significant effect on cell growth. The yields of collagenase ColP1 and ColP2 obtained from E. coli BL21(DE3) cultivation broth were 148 mg/L and 322 mg/L, respectively. The optimum temperature of each collagenase was 28 °C, and the soluble collagen activities of ColP1 and ColP2 were up to 42.64 U/mg and 21.21 U/mg, respectively. Collagenase ColP1 had the highest enzyme activity at pH 8, while collagenase ColP2 had the highest enzyme activity at pH 4. Metal ions such as Na⁺, K⁺, Mg²⁺, Ca²⁺, Ni²⁺, and Mn²⁺ inhibited the activity of collagenases to different degrees. This study successfully achieved recombinant expression and preliminary purification of Pseudomonas-derived collagenases in E. coli and explored their function and physicochemical properties.

Pseudomonas chlororaphis, a nonpathogenic plant growth-promoting rhizobacterium, holds immense potential for agricultural and industrial applications due to its ability to biosynthesize bioactive metabolites. However, the lack of efficient genetic tools has hindered its metabolic engineering. In this study, we first characterized an endogenous type I-F CRISPR/Cas system in P. chlororaphis LX24 and established a programmable genome editing toolkit based on this system. Concurrently, the plasmid transformation efficiency of P. chlororaphis LX24 was enhanced by identifying and deleting the restriction-modification systems. We further demonstrated the DNA interference capability with different PAM sequences of the type I-F CRISPR/Cas system, which also exhibited various editing efficiencies ranging from 22 to 87% in P. chlororaphis LX24. By introducing the λ-Red recombination system, the knockout efficiency of the phenazine cluster (8.3 kb) increased by over 9-fold. Next, introducing the sacB-based counterselection marker achieved a 100% plasmid curing success within 36 h. The optimized toolkit was further applied to single-step gene insertion and replacement with 100% success rates. Additionally, we established a CRISPR interference (CRISPRi) system for transcriptional repression in P. chlororaphis LX24 by knocking out nuclease Cas3. Through modulating the induction time and concentration of IPTG, the production of phenazines was reduced to 21-89% within 24 h in P. chlororaphis LX24. Overall, our work developed a convenient and precise genetic tool for the P. chlororaphis LX24, and the methods may also provide a reference for repurposing endogenous CRISPR systems in non-model prokaryotes.

Phenazines are bioactive secondary metabolites with antifungal, anticancer, and insecticidal properties, while hydroxylated derivatives often exhibit enhanced bioactivity. 2‐hydroxyphenazine (2‐OH‐PHZ), which is synthesised by the flavin‐dependent monooxygenase PhzO from phenazine‐1‐carboxylic acid (PCA), shows better bioactivity against the pathogenic fungus Gaeumannomyces graminis vars. tritici . However, the low catalytic efficiency (10%–20% conversion) of PhzO limited 2‐OH‐PHZ production. To boost PhzO activity, engineering flavin reductase (Fre)‐mediated FADH ₂ regeneration was applied to Pseudomonas chlororaphis LX24AE. Remarkably, this approach improved catalytic efficiency from 25% to 40% and increased the production of a novel dihydroxylated derivative. Then, it was first characterised by UPLC‐MS and NMR, and identified as 3,4‐dihydroxyphenazine‐1‐carboxylic acid (3,4‐OH‐PCA). Next, the Fre‐PhzO module through heterologous co‐expression in P. putida KT2440 demonstrated a 4.5‐fold enhancement in hydroxylation efficiency relative to the PhzO mono‐component system, which also confirmed that PhzO and flavin reductase are essential for 3,4‐OH‐PCA biosynthesis. Moreover, in vitro assays further verified that PhzO exhibits FAD‐dependent catalytic promiscuity, simultaneously generating 2‐OH‐PCA and 3,4‐OH‐PCA. Furthermore, in vitro and in vivo assay s demonstrated that substrate concentration affected the distribution of products. Finally, cytotoxicity evaluation of the isolated 3,4‐OH‐PCA was performed, and it showed substantial inhibition against oesophageal cancer TE‐1 cells and human cervical cancer HeLa cells with an IC ₅₀ value of 8.55 μM and 17.69 μM, respectively. This work redefines PhzO as a promiscuous biocatalyst capable of dual hydroxylation, offering a modular platform for engineering bioactive phenazine derivatives.

The biosynthesis of mupirocin, a clinically significant antibiotic produced by Pseudomonas sp. NCIMB 10586, is activated by the N -acyl homoserine lactone (AHL) MupR/I quorum sensing (QS) system. However, to date, limited research has focused on the influence of global regulators such as the GacS/A two-component system (TCS) on the MupR/I QS system or mupirocin biosynthesis. In this study, we characterized the regulatory components of the Gac/Rsm transduction system in the mupirocin-producing model strain NCIMB 10586 and investigated their interconnection with the MupR/I QS circuit and subsequent mupirocin biosynthesis. The production of mupirocin was hampered by either gacS inactivation, gacA inactivation, or the double-mutant of the sRNAs ( RsmY and RsmZ). Similarly, the expressions of mupR and mupI , and AHL synthesis significantly decreased in gacS, gacA, or rsmY/Z mutants, indicating that the GacS/A system stimulates mupirocin biosynthesis via the MupR/I QS system. Five CsrA family proteins, RsmA/E/I/F/N, were found in strain NCIMB 10586, and the single and multiple mutants of rsmA/E/I/F/N showed different phenotypes with respect to mupirocin production. Our results revealed that mupirocin biosynthesis was likely to be negatively regulated by RsmA/E/I, but positively regulated by RsmF. Additionally, the RsmF protein was shown to interact with the 5’ leader of mupR mRNA. In summary, the Gac/Rsm system positively regulates the biosynthesis of mupirocin mainly through the MupR/I QS system, and the model of the regulatory mechanism is proposed. The elucidation of the Gac/Rsm-MupR/I regulatory pathway could help devise ways for improving mupirocin production through genetic engineering. IMPORTANCE: The Gac/Rsm regulatory system plays a global regulatory role in bacterial physiology and metabolism, including secondary metabolism. Mupirocin is a clinically important antibiotic, produced by Pseudomonas sp. NCIMB 10586, whose biosynthesis is activated by the MupR/I quorum sensing system. Global regulators have important impacts on the gene expression of secondary metabolic gene clusters and QS genes, and the GacS/A two-component system is one of the main regulators across Pseudomonas species, which significantly influences antibiotic production. Our study presented that the expressions of QS genes and mup gene cluster were downregulated in gacS, gacA, or rsmY/Z mutants compared to the wild-type. The inactivation of rsmA/E/I/F/N in NCIMB 10586, encoding CsrA family proteins, showed different regulatory traits of mupirocin production, in which the RsmF protein could interact with the 5’ UTR region of mupR mRNA. These findings provide the understanding of the regulatory role of Gac/Rsm on mupirocin biosynthesis and mupR/I QS system and lay foundations for further improving mupirocin production.

Phenazine-1-carboxamide (PCN) has been exploited as a successful biopesticide due to its broad-spectrum antifungal activity. We engineered a PCN-overproducing Pseudomonas chlororaphis strain through overexpressing shikimate pathway genes (aroB, aroQ, aroE, and phzC) and deleting negative regulatory genes (relA, fleQ, and pykF). The optimized strain produced 1.92 g/L PCN with a yield of 0.11 g/g glycerol, the highest titer ever reported by using minimal media. To gain deeper insights into the underlying regulatory network, the final strain and the parental strain were examined using three distinct omic data sets. ¹³C-metabolic flux analysis revealed a substantial flux reconfiguration in the optimized strain, including the activation of the EDEMP cycle, the PP pathway, the glyoxylate shunt, and the shikimate pathway. Metabolomic results indicated that central carbon was rerouted to the shikimate pathway. Transcriptomic data identified global gene expression changes. This study forms the basis for further engineering of strains to achieve outstanding performance.