Jason (Haojie) Cao
Bacillus subtilis is extensively applied as a model organism for the high-level production of heterologous proteins of interest. The traditional strategies for increasing the use of this microbial cell factory always focused on the regional modification of rate-limiting components or steps. However, the longstanding problem for low productivity of the expression hosts, metabolic burden, has rarely been solved for strain improvements. To address this issue, we developed a tailor-made system, in which rational approaches and combinatorial methods involving mutagenesis and selection were utilized for overexpressing heterologous proteins. Specifically, we systematically rewired metabolic fluxes through the random mutagenesis of global transcriptional regulator CodY and CcpA, and the high-throughput screening for enhanced β-galactosidase production was performed on mutant libraries. By doing this, the selected best phenotype with single amino acid substitutions within the DNA-binding HTH domain of CcpA and CodY could reach an increase of 2-fold in overproduction of reporter protein (β-galactosidase). Transcriptome and gel mobility shift analysis revealed that these two site mutations profoundly altered the overall binding specificity to the respective regulon genes. Importantly, this transcriptome perturbation also affected the expression of these two regulatory proteins themselves, which accordingly lead to the slight further repression of the carbon core metabolism and dramatic de-repression of nitrogen metabolism. Consequently, these two central metabolic networks which are intertwined by the feedback regulation of branched-chain amino acids (BCAAs), was well balanced by the optimization of flux distributions.
1. Increase the use of Bacillus subtilis as a microbial cell factory by the global transcription machinery engineering (gTME) of central metabolic networks.
2. Further reveal the interactions between central metabolic pathways.
Global transcription machinery engineering (gTME), random mutagenesis, high-throughput screening, EMSA, Western Blotting, RNA-seq
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