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ACS Synth Biol ; 13(3): 969-972, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38340070


Serratia marcescens has garnered increasing attention as a promising host for valuable compound production. However, the lack of an efficient gene regulation toolkit severely hampers its applications. Here, a library of stationary phase promoters was screened in S. marcescens HBA7 using RNA-seq and RT-qPCR, revealing a 43-fold regulatory range with the red fluorescent protein mKate2 as the reporter. The ß-galactosidase was employed to demonstrate the universality in driving the expression of different proteins. The wide-ranging utility of these promoters in different hosts was demonstrated in Escherichia coli. Moreover, to assess their potential application, the strongest promoter, P2, was employed to express the swrW gene, resulting in a roughly 20-fold increase in serrawettin W1 production in S. marcescens HBQA7ΔswrW. In summary, this study successfully constructed a gradient-strength stationary phase promoter library, providing an effective toolkit for gene regulation and secondary metabolite production in diverse prokaryotes, including S. marcescens and E. coli.

Escherichia coli , Serratia marcescens , Serratia marcescens/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Regiões Promotoras Genéticas/genética , Regulação da Expressão Gênica
Artigo em Inglês | MEDLINE | ID: mdl-38225812


Clostridium aceticum DSM1496 is an acid-resistant strain in which ornithine decarboxylase (ODC) plays a crucial role in acid resistance. In this study, we expressed ODC derived from C. aceticum DSM1496 in Escherichia coli BL21 (DE3) and thoroughly examined its enzymatic properties. The enzyme has a molecular weight of 55.27 kDa and uses pyridoxal-5'-phosphate (PLP) as a coenzyme with a Km  = 0.31 mM. ODC exhibits optimal activity at pH 7.5, and it maintains high stability even at pH 4.5. The peak reaction temperature for ODC is 30°C. Besides, it can be influenced by certain metal ions such as Mn2+ . Although l-ornithine serves as the preferred substrate for ODC, the enzyme also decarboxylates l-arginine and l-lysine simultaneously. The results indicate that ODC derived from C. aceticum DSM1496 exhibits the ability to produce putrescine, cadaverine, and agmatine through decarboxylation. These polyamines have the potential to neutralize acid in an acidic environment, facilitating the growth of microorganisms. These significant findings provide a strong basis for further investigation into the acid-resistant mechanisms contributed by ODC.

Biotechnol Appl Biochem ; 69(4): 1428-1437, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34148265


In this study, the 3-quinuclidinone reductase from Agrobacterium tumefaciens (AtQR) was modified by site-directed mutagenesis. And we further obtained a saturation mutant library in which the residue 197 was mutated. A single-point mutation converted the wild enzyme that originally had no catalytic activity in reduction of ethyl 4-chloroacetoacetate (COBE) into an enzyme with catalytic activity. The results of enzyme activity assays showed that the seven variants could asymmetrically reduce COBE to ethyl (S)-4-chloro-3-hydroxybutyrate ((S)-CHBE) with NADH as coenzyme. In the library, the variant E197N showed higher catalytic efficiency than others. The E197N was optimally active at pH 6.0 and 40°C, and the catalytic efficiency (kcat /Km ) for COBE was 51.36 s-1 ·mM-1 . This study showed that the substrate specificity of AtQR could be changed through site-directed mutagenesis at the residue 197.

Agrobacterium tumefaciens , Oxirredutases , Acetoacetatos , Agrobacterium tumefaciens/genética , Cinética , Mutagênese Sítio-Dirigida , Quinuclidinas , Especificidade por Substrato