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

Cao, H., Villatoro-Hernandez, J., Weme, R. D. O., Frenzel, E. & Kuipers, O. P., Sep-2018, In : Metabolic Engineering. 49, p. 143-152 10 p.

Research output: Contribution to journalArticleAcademicpeer-review

  • Haojie Cao
  • Julio Villatoro-Hernandez
  • Ruud Detert Oude Weme
  • Elrike Frenzel
  • Oscar P. Kuipers

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 completely solved to achieve significant 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 β-galactosidase expression and screening system and consecutive rounds of mutagenesis, we identified mutant variants of both CodY and CcpA that in conjunction increased production levels up to 290%. RNA-Seq and electrophoretic mobility shift assay (EMSA) 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.

Original languageEnglish
Pages (from-to)143-152
Number of pages10
JournalMetabolic Engineering
Publication statusPublished - Sep-2018


  • Bacillus subtilis, CcpA, CodY, Global transcription machinery engineering (gTME), High-throughput screening, Heterologous proteins, GRAM-POSITIVE BACTERIA, CODY-BINDING SITES, CHAIN AMINO-ACIDS, GENE-EXPRESSION, TRANSCRIPTION MACHINERY, CATABOLITE CONTROL, ESCHERICHIA-COLI, CCPA, OPERON, DNA

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