| The objective of the present study was to gain some important insights into the inner mechanism of nontargeted approach derived riboflavin-overproducing mutant, to identify of the particular genetic element(s) responsible for the enhanced performance, and to reconstruct a strain with stable performance. In this work, laboratory stock mutagenesis-derived riboflavin-producing strain Bacillus subtilis 24/pMX45 was chosen to conducted genome sequencing, RNA-Seq and inverse metabolic engineering to analyze specific chromosomal mutations. Reintroduction of beneficial mutations into wild type strain resulted in an engineered strain with recovered phenotype and clear genetic background. Based on the reconstructed strain with beneficial mutations, we tried to analyze the mechanism of beneficial mutations.An integrated RNA-Seq was conducted for identification of differentially expressed genes in B. subtilis 24/pMX45, so as to correlate gene transcriptomic profiles with genotype data. As the result, 945 genes showed significant variations at the genomewide transcriptional level between B. subtilis 24/pMX45 and control strain(483 upregulated genes and 462 downregulated genes). Although rib operon was overexpressed, genes involved in purine operon, one carbon unit metabolism, glucose transport system and central carbon metabolism were generally repressed. It was consistent with the phenotype that low specific growth rate and low specific riboflavin produced rate in B. subtilis 24/pMX45.In order to identify genomic changes responsible for riboflavin overproduction, we sought clues in the genome sequencing and transcriptome changes accompanying the phenotype changes. Putative beneficial mutations found in mutant were introduced into parent strain to evaluate their effects on riboflavin production through iterative cycle process. By using markerless mutation delivery system, a series of mutant strains carrying the desired mutations were constructed and investigated in fermentation medium by shake-flask cultivation. At least seven specific mutations in various cellular pathways were identified to be beneficial for riboflavin overproduction, including RibC(G199D), ribD+(G+39A), PurA(P242L), CcpN(A44S), YhcF(R90*), YwaA(Q68*), and YvrH(R222Q) with various contribution. Several mutations were first identified to endow B. subtilis with the capability to accumulate riboflavin. BSW54/pMX45 represented the 980±22.85 mg/L riboflavin production with most beneficial mutations included and plasmid pMX45. Although such beneficial mutations work well in a combined effect, the achieved ribo?avin yields still fall short of the parent mutant B. subtilis 24/pMX45(1211.79±38.47 mg/L). Seven beneficial mutations recovered approximately 80% riboflavin of production in B. subtilis 24/pMX45.RibC(G199D), ribD+(G+39A) resulted in the deregulation of rib operon which direct account for riboflavin overproduction. Moreover, transcriptional regulator CcpN(A44S) in central carbon metabolism redirected carbon flux towards the pentose phosphate pathway for riboflavin overproduction. In addition, inactivation of adenylosuccinate synthetase [PurA(P242L)] led to block of IMP to AMP conversion, which is favorable for riboflavin precursors GTP biosynthesis. Most importantly, beneficial mutation YvrH(R222Q) was first identified to partially lose its function and deregulated the expression of LytC(N-acetylmuramoyl-L-alanine amidase). High activity of LytC resulted in the cell wall damage and probably influenced the cell permeability, which eventually prompted riboflavin production. For an extended application of this approach, we successfully increased 5-aminolevulinic acid production in an engineered Corynebacterium glutamicum by overexpression of Nacetylmuramoyl-L-alanine amidase. This might provide a new universal strategy for biochemicals, especially for those whose biosynthesis was strictly regulated in a feedback or allosteric inhibition manner. |
PDF Full Text Request