Boosting heterologous protein production yield by adjusting global nitrogen and carbon metabolic regulatory networks in Bacillus subtilis

Bacillus subtilis is extensively applied as a microorganism for the high-level production of heterologous proteins. Traditional strategies for increasing the productivity of this microbial cell factory generally focused on the targeted modification of rate-limiting components or steps. However, the longstanding problems of limited productivity of the expression host, metabolic burden and non-optimal nutrient intake, have not yet been solved to achieve production strain improvements. To tackle this problem, we systematically rewired the regulatory networks of the global nitrogen and carbon metabolism by random mutagenesis of the pleiotropic transcriptional regulators CodY and CcpA, to allow for optimal nutrient intake, translating into significantly higher heterologous protein production yields. Using a {beta}-galactosidase expression and screening system and consecutive rounds of mutagenesis, we identified mutant variants of both CcpA and CodY that in conjunction increased production levels up to 290%. RNA-Seq and electrophoretic gel mobility shift analyses showed that amino acid substitutions within the DNA-binding domains altered the overall binding specificity and regulatory activity of the two transcription factors. Consequently, fine-tuning of the central metabolic pathways allowed for enhanced protein production levels. The improved cell factory capacity was further demonstrated by the successfully increased overexpression of GFP, xylanase and a peptidase in the double mutant strain.\n\nHighlightsO_LIThe global transcription machinery engineering (gTME) technique was applied to build mutational libraries of the pleiotropic regulators CodY and CcpA in Bacillus subtilis\nC_LIO_LISpecific point mutations within the DNA-binding domains of CodY and CcpA elicited alterations of the binding specificity and regulatory activity\nC_LIO_LIChanges in the transcriptome evoked the reprogramming of networks that gear the carbon and nitrogen metabolism\nC_LIO_LIThe rewired metabolic networks provided a higher building block capacity for heterologous protein production by adjusting the nutrient uptake and channeling its utilization for protein overexpression\nC_LI