Engineering Escherichia coli to assimilate beta-alanine as a major carbon source

The threat of global plastic waste accumulation has spurred the exploration of plastics derived from biological sources. A well-known example is polyester made of 1,3-propanediol (1,3-PDO). However, there is no known pathway to assimilate 1,3-PDO into the central carbon metabolism, posing a potential challenge to upcycling such plastic wastes. Here, we proposed that the 1,3-PDO assimilation pathway could pass through malonate semialdehyde (MSA) as an intermediate. Since MSA is a toxic aldehyde, beta-alanine was chosen as a surrogate substrate in this study to construct the lower part of the proposed pathway. To this end, we successfully engineered E. coli MG1655 to assimilate beta-alanine as the major carbon source. beta-alanine could be easily converted into MSA using a beta-alanine/pyruvate transaminase from Pseudomonas aeruginosa (PaBapt). However, the subsequent step to generate acetyl-CoA from MSA was unknown. After a series of phenotype screenings, adaptive laboratory evolution and transcriptomic analysis, two CoA-acylating MSA dehydrogenases from Vibrio natriegens (VnMmsD), were found to be able to complete the metabolic pathway. Optical density at 600 nm (OD600) of the resulting strain E. coli BA02 could reach 4.5 after 96 h. Two approaches were subsequently used to improve its performance. First, PaBapt and both VnMmsDs were expressed from a single plasmid to mitigate antibiotic stress. Second, a native 3-hydroxy acid dehydrogenase (EcYdfG) was disrupted to address the carbon loss to 3-hydroxypropionate (3-HP) production from MSA. OD600 of the best-performing strain E. coli BA07. could reach 6 within 24 h using 5 g/L beta-alanine. The construction of E. coli BA07. lays a solid foundation to establishing a 1,3-PDO assimilation pathway.

Automated summary

The threat of global plastic waste accumulation has led to the exploration of bioplastics. A proposed pathway for assimilating 1,3-propanediol (1,3-PDO) into central carbon metabolism was investigated using beta-alanine as a surrogate substrate. The study successfully engineered E. coli to assimilate beta-alanine and identified two enzymes from Vibrio natriegens that completed the metabolic pathway.