(619g) Improving Phenotypes of Escherichia coli at the Post-Transcriptional Level By Engineering Poly(A) Polymerase I

In E. coli, poly(A) polymerase I (PAP I, encoded by pcnB) is identified to play a crucial role in the process of RNA degradation by polyadenylation of RNAs. It was first found to be capable of maintaining the copy number of plasmid pBR322 through promoting the decay of antisense RNA (RNA I), which inhibits the replication of plasmids. Later, more than 90% of E. coli open reading frames transcribed during exponential growth were claimed to undergo some degree of polyadenylation by PAP I. Since pcnB mutants have shown different affinity to ribonucleotides and RNA, it is possible to engineering the phenotype of E. coli at the post-transcriptional level by engineering PAP I. In this study, two important mutations (Cys99Arg, Asp205Asn) were found to cause the inactivity of PAP I, respectively, by comparing the fluorescence intensity of mCherry and sfGFP constitutively expressed on the plasmid in pcnB mutant strains against the native ones. Of these two mutations, the latter belongs to the recognized catalytic core pocked, while the former has never been reported. An error-prone pcnB library and the site-saturation mutagenesis library of pcnB at these two sites were constructed and transformed into E. coli to help the generation of variants with different transcriptome. Results from isobutanol tolerance were provided as proof-of-concept. To gain insight into the molecular biological base of tolerance, we are performing transcriptional analysis to quantify the influence of the enriched mutations to the gene expression profiling of strains.