Yeast Derived LysA2 Can Control Bacterial Contamination in Ethanol Fermentation

Area of Science:

• Biotechnology
• Industrial Microbiology
• Enzyme Engineering

Background:

• Fuel ethanol production faces significant challenges from bacterial contamination, primarily by Lactobacillaceae.
• Current control methods, including antibiotics and acid washing, have drawbacks such as promoting antibiotic resistance and stressing yeast.
• Bacteriophage endolysins, such as LysA2, are lytic enzymes with potential as antimicrobial agents in industrial settings.

Purpose of the Study:

• To evaluate the efficacy of yeast-derived LysA2 for controlling Lactobacillaceae contamination in fuel ethanol fermentations.
• To compare the activity of LysA2 produced in different yeast systems (Pichia pastoris and Saccharomyces cerevisiae).

Main Methods:

• LysA2 was produced intracellularly in Pichia pastoris and its activity was compared to E. coli-produced LysA2.
• The sensitivity of Lactic Acid Bacteria (LAB) to LysA2 was assessed based on their peptidoglycan chemotype.
• Ethanol fermentations using glucose (YPD60) and sugarcane juice were conducted with LysA2-expressing yeast or purified LysA2 in the presence of a susceptible LAB contaminant.

Main Results:

• Intracellular production of LysA2 in Pichia pastoris yielded activity comparable to E. coli-produced LysA2.
• LAB with the A4α peptidoglycan chemotype were most sensitive to LysA2, although some exceptions were noted.
• Pichia-expressed LysA2 (secreted and intracellular) significantly improved ethanol productivity and yields in both YPD60 and sugarcane juice fermentations challenged with LAB.
• LysA2-secreting Saccharomyces cerevisiae controlled contamination in YPD60 but did not notably improve production in sugarcane juice.

Conclusions:

• Yeast-expressed LysA2, either secreted or intracellularly produced, represents a viable alternative for controlling Lactobacillaceae contamination in fuel ethanol fermentation.
• Purified LysA2 or LysA2 secreted by fermenting yeast can enhance ethanol production efficiency.
• Endolysins with broader lytic spectra hold potential to further replace conventional antibiotics and acid washing methods.