Efficient 2,3-Butanediol Production from Ethanol by a Modified Four-Enzyme Synthetic Biosystem

Area of Science:

• Biocatalysis and metabolic engineering
• Synthetic biology for chemical production
• Sustainable chemistry and bio-based chemicals

Background:

• 2,3-butanediol (2,3-BD) is a valuable bio-based platform chemical.
• Previous work established a four-enzyme system for ethanol to 2,3-BD conversion.
• Formolase (FLS) exhibited low catalytic efficiency, limiting the system's performance.

Purpose of the Study:

• To engineer formolase (FLS) for improved catalytic efficiency.
• To develop an enhanced cell-free biosystem for 2,3-BD production.
• To optimize 2,3-BD synthesis from ethanol using enzymatic catalysis.

Main Methods:

• Protein engineering of FLS by modifying its tunnel and substrate binding pocket.
• Creation of single-point and combinatorial FLS variants.
• Implementation of a cell-free biosystem utilizing the engineered FLS (FLS:I28V/L482E) for ethanol upgrading.

Main Results:

• Engineered FLS variants demonstrated superior catalytic efficiency and substrate tolerance compared to wild-type.
• The cell-free biosystem with FLS:I28V/L482E achieved a 2,3-BD concentration of 1.39 M (124.83 g/L).
• High productivity (5.94 g/L/h) and yield (92.7%) were obtained via fed-batch fermentation.

Conclusions:

• Enzymatic engineering of FLS is effective in enhancing catalytic performance.
• The modified cell-free enzymatic system offers a promising route for sustainable 2,3-BD production.
• This approach presents a cost-competitive alternative for industrial 2,3-BD manufacturing.