Background
4-Hydroxyphenylacetic acid (4HPAA) is an important raw material for the synthesis of drugs, pesticides and biochemicals. Microbial biotechnology would be an attractive approach for 4HPAA production, and cofactors play an important role in biosynthesis.
Results
We developed a novel strategy called cofactor engineering based on clustered regularly interspaced short palindromic repeat interference (CRISPRi) screening (CECRiS) for improving NADPH and/or ATP availability, enhancing the production of 4HPAA. All NADPH-consuming and ATP-consuming enzyme-encoding genes of E. coli were repressed through CRISPRi. After CRISPRi screening, 6 NADPH-consuming and 19 ATP-consuming enzyme-encoding genes were identified. The deletion of the NADPH-consuming enzyme-encoding gene yahK and the ATP-consuming enzyme-encoding gene fecE increased the production of 4HPAA from 6.32 to 7.76 g/L. Automatically downregulating the expression of the pabA gene using the Esa-PesaS quorum-sensing-repressing system further improved the production of 4HPAA. The final strain E. coli 4HPAA-∆yfp produced 28.57 g/L of 4HPAA with a yield of 27.64% (mol/mol) in 2-L bioreactor fed-batch fermentations. The titer and yield are the highest values to date.
Conclusion
This CECRiS strategy will be useful in engineering microorganisms for the high-level production of bioproducts.
Here, we report the complete annotated genome sequence of Bacillus amyloliquefaciens XH7, which is used to produce purine nucleosides in industry. The genome sequence will allow for the characterization of the molecular mechanisms underlying its beneficial properties.Bacillus amyloliquefaciens has been widely used as an industrial producer of primary metabolites, including vitamins and purine nucleosides such as inosine and guanosine (14,17). Purine nucleosides are important intermediates in the food and pharmaceutical industries, where they can be used to synthesize chicken essence (16) and nucleoside antiviral drugs (6, 12) such as ribavirin and acyclovir. Bacillus amyloliquefaciens XH7 is a high-yield purine nucleoside guanosine producer by traditional mutation breeding and has potential for industrial production of purine nucleoside inosine after deletion of the guaB gene. In order to further strain improvement, we report here the complete genome sequence of XH7.The genome was sequenced using the Illumina Solexa GA instrument at Beijing Genomics Institute (BGI) (Shenzhen, China). A library containing 500-bp inserts was constructed. Sequencing was performed with the pair-end strategy of 75-bp reads to produce 629 Mb of filtered sequences, representing a 160-fold coverage of the genome. The sequences were assembled into 76 contigs and 23 scaffolds using the SOAPdenovo package (9).The location and orientation of each scaffold were determined by mapping to the finished reference sequence of B. amyloliquefaciens DSM7 (13) using Mummer 3 (7). Gaps were closed by custom primer walks and long-distance PCR amplification followed by DNA sequencing with an ABI 3730 sequencer. Open reading frames (ORF) were identified by Glimmer 3.02 (4). The resulting translations were used for a BLASTP (1) search against the GenBank NR database, as well as the KEGG (5) and COG (15) databases. tRNA and rRNA genes were identified by tRNAscan-SE (10) and RNAmmer (8), respectively.The complete genomic information of B. amyloliquefaciens XH7 is contained on a single circular chromosome of 3,939,203 bp with an average GC content of 45.82%. A total of 4,204 protein coding genes, 75 tRNA genes, and 7 rrn operons were identified. A total of 25 pseudogenes were also identified, which suggested that B. amyloliquefaciens XH7 was selected by mutagenesis treatment.In performing a comparative analysis of B. amyloliquefaciens XH7 with reference genomes, those of B. amyloliquefaciens strains FZB42 (3) and DSM7 (13), we found some important mutations which may explain the molecular mechanisms for the production of purine nucleosides. Adenylosuccinate synthase gene purA, which blocked the pathway of IMP to AMP, was truncated. The nonsense mutation in GMP reductase gene guaC blocked the pathway of GMP to IMP. Comparative analysis of B. amyloliquefaciens XH7 with the reference genome of Escherichia coli K-12 (2) revealed that the homolog of E. coli purine nucleoside phosphorylase gene gsk was not found in XH7. This feature could make B. amyloliquefaciens ...
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