Standard Intein Gene Expression Ramps (SIGER) for Protein-Independent Expression Control

Area of Science:

• Metabolic Engineering
• Synthetic Biology
• Molecular Biology

Background:

• Multigene expression coordination is crucial for developing efficient cell factories.
• Gene ramp features (5'UTR, rare codons, secondary structures) influence translation yield and protein quality by affecting ribosome kinetics.
• Varied gene ramp compositions lead to unpredictable protein expression levels.

Purpose of the Study:

• To introduce the Standard Intein Gene Expression Ramp (SIGER) system for robust control of protein expression.
• To decouple translation initiation from target genes, mitigating issues associated with variable gene ramp sequences.
• To demonstrate SIGER's utility in controlling enzyme ratios within molecular pathways and expressing challenging proteins.

Main Methods:

• Development of the SIGER system utilizing inteins (DnaB and DnaX) to regulate protein expression.
• Generation of sequence-specific gene expression sequences for inteins with defined expression levels.
• In vivo C-terminal protein release by inteins to preserve protein functionality.

Main Results:

• SIGER systems effectively mitigate undesirable effects of gene ramp variations.
• Controlled relative ratios of enzymes involved in molecular pathways were achieved.
• Successful expression of two difficult-to-produce proteins, GumM and CBM73, using the SIGER system.

Conclusions:

• SIGER is a novel tool for precise control of protein expression in metabolic engineering.
• The system enhances predictability of protein yield and quality by managing translation initiation.
• SIGER facilitates the production of complex proteins and optimization of metabolic pathways.