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Library selection with a randomized repertoire of (βa)8-barrel enzymes results in unexpected induction of gene expression

ABSTRACT: Library selection with a randomized repertoire of (βa)8-barrel enzymes results in unexpected induction of gene expression

PROVIDER: PRJNA550996 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR9600920_1.fastq.gz	Fastqsanger.gz
	SRR9600920_2.fastq.gz	Fastqsanger.gz
	SRR9600921_1.fastq.gz	Fastqsanger.gz
	SRR9600921_2.fastq.gz	Fastqsanger.gz
	SRR9600912_1.fastq.gz	Fastqsanger.gz

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Project description:The (βa)8-barrel enzyme fold is frequently encountered in nature and catalyzes a multitude of different reactions. It is therefore interesting to evaluate the evolutionary potential of this fold by laboratory experiments. Along these lines, we tested the ability of the (βa)8-barrel to acquire new catalytic functions by an approach combining random mutagenesis and selection in vivo. The genes encoding 52 different phosphate-binding (βa)8-barrel proteins were randomized by error-prone PCR and cloned into an expression plasmid. The resulting mixed repertoire was used to transform different auxotrophic Escherichia coli (E. 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 variants of point-mutated E. coli genes nanE (encoding a putative N-acetlymannosamine-6-phosphate 2-epimerase) and pdxJ (encoding the pyridoxine 5’-phosphate synthase) were responsible for rescuing both DserB and DserC. Remarkably, all selected PdxJ variants have acquired a 10-residue C-terminal extension from the cloning vector, that is essential for the rescue of DserB and DserC. Unexpectedly, the complementing NanE and PdxJ variants did not catalyze the SerB or Serc reactions in vitro. However, 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 indicates that HisC is promiscuous for the SerC reaction, as well. The successful rescue of DserB and DserC through point mutations and recruitment of additional amino acids in NanE and PdxJ provides another example for the adaptability of the (βa)8-barrel fold. To our knowledge, this is the first example of (βa)8-barrels functioning not as enzymes, but by activating transcription via an unknown mechanism.

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Library selection with a randomized repertoire of (βa)8-barrel enzymes results in unexpected induction of gene expression

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