Jason (Haojie) Cao

Title

Jason Cao, room 5172.646 tel 2194 email This email address is being protected from spambots. You need JavaScript enabled to view it.

Introduction

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.

 

Aim

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.

Techniques

 Global transcription machinery engineering (gTME), random mutagenesis, high-throughput screening, EMSA, Western Blotting, RNA-seq

References

 1. Westers L, Westers H, Quax WJ (2004) Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism. Biochim Biophys Acta 1694(1-3):299-310.

 2. Sonenshein AL (2007) Control of key metabolic intersections in Bacillus subtilis. Nat Rev Microbiol 5(12):917-927.

 3. Brinsmade SR, et al. (2014) Hierarchical expression of genes controlled by the Bacillus subtilis global regulatory protein CodY. Proc Natl Acad Sci USA 111(22):8227-8232.

 4. Alper H, Stephanopoulos G (2007) Global transcription machinery engineering: a new approach for improving cellular phenotype. Metab Eng 9(3):258-267.

MolGen | RuG | GBB

Copyright © 2015 MolGen. All Rights Reserved.