Precision Quantification and Rational Regulation of Protein Expression with Bicistronic Cassette for Efficient Biotin...

Area of Science:

• Synthetic Biology
• Metabolic Engineering
• Biotechnology

Background:

• Metabolic imbalances due to suboptimal enzyme expression hinder target product synthesis in microbial cell factories.
• Current enzyme expression screening methods are often trial-and-error, limiting efficiency and applicability.

Purpose of the Study:

• To develop a quantitative and efficient strategy for screening enzyme expression levels in microbial cell factories.
• To optimize the expression of the rate-limiting enzyme BioB in biotin synthesis using a novel bicistronic design (BCD) library.

Main Methods:

• Development of a bicistronic design (BCD) library enabling a 992-fold enzyme expression range.
• Quantitative screening using fluorescence intensity correlation with protein abundance (r = 0.96).
• Two-step screening process: expression profiling and focused selection, requiring minimal tests.

Main Results:

• Identification of optimal BCD strengths (BCD6 and BCD7) for different genetic contexts (bio operon vs. bio + isc operons).
• Achieved 1.47-fold and 3.03-fold increases in biotin titer with the optimized strains.
• Confirmed significant increases in BioB protein abundance (2.38-fold and 2.71-fold) via Western Blot analysis.

Conclusions:

• The BCD library provides a versatile and rational approach for tuning enzyme expression in microbial cell factory construction.
• This strategy overcomes limitations of traditional screening methods, enabling efficient optimization for enhanced product yields.
• Pioneering application of BCD for fine-tuning BioB expression significantly improved biotin production, demonstrating its broad utility.