RESUMEN
The potential of the frequently encountered (ßα)8-barrel fold to acquire new functions was tested by an approach combining random mutagenesis and selection in vivo. For this purpose, the genes encoding 52 different phosphate-binding (ßα)8-barrel proteins were subjected to error-prone PCR and cloned into an expression plasmid. The resulting mixed repertoire was used to transform different auxotrophic Escherichia coli strains, each lacking an enzyme with a phosphate-containing substrate. After plating of the different transformants on minimal medium, growth was observed only for two strains, lacking either the gene for the serine phosphatase SerB or the phosphoserine aminotransferase SerC. The same mutants of the E. coli genes nanE (encoding a putative N-acetylmannosamine-6-phosphate 2-epimerase) and pdxJ (encoding the pyridoxine 5'-phosphate synthase) were responsible for rescuing both ΔserB and ΔserC. Unexpectedly, the complementing NanE and PdxJ variants did not catalyze the SerB or SerC reactions in vitro. Instead, RT-qPCR, RNAseq, and transcriptome analysis showed that they rescue the deletions by enlisting the help of endogenous E. coli enzymes HisB and HisC through exclusive up-regulation of histidine operon transcription. While the promiscuous SerB activity of HisB is well-established, our data indicate that HisC is promiscuous for the SerC reaction, as well. The successful rescue of ΔserB and ΔserC through point mutations and recruitment of additional amino acids in NanE and PdxJ provides another example for the adaptability of the (ßα)8-barrel fold.
Asunto(s)
Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Triosa-Fosfato Isomerasa/química , Triosa-Fosfato Isomerasa/genética , Proteínas Bacterianas/genética , Sitios de Unión , Carbohidrato Epimerasas/genética , Cristalización , Proteínas de Escherichia coli/genética , Histidinol-Fosfatasa/química , Ligasas/genética , Espectroscopía de Resonancia Magnética , Metaboloma , Fosfoserina/química , Plásmidos/genética , Mutación Puntual , Pliegue de Proteína , Estructura Secundaria de Proteína , Transaminasas/química , Transaminasas/genéticaRESUMEN
Here, we modified the multiple cloning sites from commonly used expression vectors to create a new suite of cloning plasmids that simplify and speed up cloning procedures in Escherichia coli. Each of our standardized plasmids contains two BsaI restriction sites, allowing for highly efficient cloning of genes and bringing their expression under control of either a T7 (pET21a_BsaI, pET28a_BsaI, and pMAL-c5T_BsaI) or T5 promoter (pUR22 and pUR23). Another plasmid in our suite (pTNA_BsaI) allows for generation of large gene libraries containing >108 variants, which can be constitutively expressed in selection experiments using metabolic complementation of auxotrophic E. coli strains. Coupling restriction and ligation with the BsaI restriction enzyme minimizes hands-on time, while the need for only three different primers to clone a target gene into the six different vectors keeps overall cloning costs low.
Asunto(s)
Clonación Molecular/métodos , Escherichia coli/genética , Biblioteca de Genes , Vectores Genéticos/genética , Secuencia de Bases , Enzimas de Restricción del ADN/metabolismo , Escherichia coli/metabolismo , Vectores Genéticos/metabolismo , Plásmidos/genética , Plásmidos/metabolismo , Regiones Promotoras GenéticasRESUMEN
It has been postulated that the ubiquitous (ßα)8-barrel enzyme fold has evolved by duplication and fusion of an ancestral (ßα)4-half-barrel. We have previously reconstructed this process in the laboratory by fusing two copies of the C-terminal half-barrel HisF-C of imidazole glycerol phosphate synthase (HisF). The resulting construct HisF-CC was stepwise stabilized to Sym1 and Sym2, which are extremely robust but catalytically inert proteins. Here, we report on the generation of a circular permutant of Sym2 and the establishment of a sugar isomerization reaction on its scaffold. Our results demonstrate that duplication and mutagenesis of (ßα)4-half-barrels can readily lead to a stable and catalytically active (ßα)8-barrel enzyme.