rDNA-mediated multicopy integration and gene dosage quantification system for microbial zeaxanthin biosynthesis.

Area of Science:

• Microbial biotechnology
• Synthetic biology
• Metabolic engineering

Background:

• Industrial zeaxanthin production faces challenges with cost and environmental impact from plant extraction and chemical synthesis.
• Microbial biosynthesis presents a sustainable alternative, but optimizing gene dosage for complex pathways is difficult.
• Efficiently integrating multiple genes and assessing their dosage effects is crucial for pathway optimization.

Purpose of the Study:

• To develop a novel genetic system, rNTS, for one-step, high-copy integration of multiple genes into microbial genomes.
• To enable direct and quantitative assessment of gene dosage effects on biosynthetic pathway performance.
• To optimize zeaxanthin microbial biosynthesis through precise gene copy number control.

Main Methods:

• Development of the rNTS genetic system targeting the ribosomal non-transcribed spacer region for gene integration.
• Utilizing Fluorescence Intensity Ratios (FIRs) for quantitative estimation of gene copy number and selection of optimal gene dosage.
• Employing Cre/LoxP system for marker removal and iterative gene integrations to achieve high copy numbers.
• Application of the rNTS system to optimize the biosynthesis of zeaxanthin, focusing on the rate-limiting enzyme CrtZ.

Main Results:

• The rNTS system demonstrated efficient one-step, high-copy integration of up to 40 single-gene copies or multiple genes totaling 20 copies.
• Iterative integration using the Cre/LoxP system allowed for achieving up to 32 gene copies.
• Optimization of zeaxanthin biosynthesis using rNTS led to titers of 1.10 g/L (glucose fermentation) and 1.19 g/L (methanol-induced fermentation).
• The system provided quantitative gene copy number readability and facilitated rapid identification of optimal enzyme dosages.

Conclusions:

• The rNTS genetic system offers an efficient and versatile tool for high-copy gene integration and quantitative gene dosage assessment in microbial systems.
• This strategy significantly advances microbial biosynthetic pathway optimization, particularly for complex compounds like zeaxanthin.
• The rNTS approach provides a sustainable and scalable solution for industrial biotechnology, enhancing eco-friendly production methods.