Biosensor-based growth-coupling as an evolutionary strategy to improve heme export in Corynebacterium glutamicum

Area of Science:

• Microbial Biotechnology
• Metabolic Engineering
• Synthetic Biology

Background:

• Heme, an iron-containing porphyrin, is crucial for the food industry (artificial meat) and medical uses.
• Corynebacterium glutamicum is a promising host for animal-free heme production, but export efficiency needs improvement.
• Engineering heme export pathways is a largely untapped strategy for enhancing microbial heme production.

Purpose of the Study:

• To develop a growth-coupled biosensor for selecting enhanced heme export in Corynebacterium glutamicum.
• To identify genetic targets that improve the expression of the hrtBA heme exporter operon.
• To validate the role of specific genes and mutations in boosting microbial heme production.

Main Methods:

• Designed a biosensor by replacing promoters of growth-regulating genes (pfkA, aceE) with P_hrtB in C. glutamicum.
• Employed plate-based selections and automated fluorescence-activated liquid evolution (fALE) for strain improvement.
• Utilized genome sequencing to identify mutations in high-expression clones and constructed deletion mutants for validation.

Main Results:

• Identified three mutational hotspots: chrS, chrA, and cydD, which significantly increased hrtBA expression.
• Mutations in the ChrSA two-component system and cydD (encoding an ABC-transporter) were confirmed as drivers of enhanced heme export.
• Engineered strains exhibited markedly increased hrtBA expression and elevated cellular heme levels.

Conclusions:

• Biosensor-based growth coupling is effective for selecting improved heme export in microbial hosts.
• Mutations in ChrSA and CydD represent promising targets for enhancing heme production in C. glutamicum.
• This study validates CydDC's role as a bacterial heme transporter and advances strategies for industrial heme biotechnology.