Engineering UDP-sugar synthesis pathways for exopolysaccharide biosynthesis in Streptococcus thermophilus AR333

Area of Science:

• Microbiology
• Biotechnology
• Food Science

Background:

• Exopolysaccharides (EPS) from Streptococcus thermophilus improve dairy product quality but are produced at low levels, hindering industrial use.
• Metabolic engineering offers a promising strategy to enhance EPS biosynthesis in S. thermophilus.

Purpose of the Study:

• To systematically investigate the engineering of UDP-sugar synthetic pathways for increased EPS production in S. thermophilus.
• To identify key genes in UDP-sugar metabolism that can be manipulated to boost EPS yields.

Main Methods:

• Engineered Streptococcus thermophilus AR333 by regulating biosynthetic genes of EPS precursors UDP-glucose and UDP-galactose.
• Utilized single and co-overexpression strategies for specific genes involved in UDP-sugar synthesis.
• Performed transcriptional analysis to understand the impact of genetic modifications on EPS gene clusters.

Main Results:

• Single overexpression of eight EPS precursor genes resulted in 7-31% increases in EPS production.
• Overexpression of glk (glucokinase) and galE1 (UDP-galactose-4-epimase) led to significant EPS yields of 275.37 and 288.65 mg L⁻¹, respectively.
• Co-overexpression of lacZ (β-galactosidase) and galE1 achieved a 49% increase, reaching 329.51 mg L⁻¹ EPS, with evidence of precursor and EPS gene cluster upregulation.

Conclusions:

• Engineering UDP-sugar synthesis pathways is an effective strategy for enhancing EPS production in S. thermophilus.
• Specific gene targets like glk, galE1, and lacZ show potential for significant EPS yield improvement.
• This research provides a foundation for optimizing industrial-scale EPS production in S. thermophilus.