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

Abstract 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, β-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 β-alanine as the major carbon source. β-alanine could be easily converted into MSA using a β-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 (OD 600 ) of the resulting strain E. coli BA02 could reach 4.5 after 96 hours. Two approaches were subsequently used to improve its performance. First, PaBapt and both VnMmsD s 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. OD 600 of the best performing strain E. coli BA07∆ could reach 6 within 24 hours using 5 g/L β-alanine. The construction of E. coli BA07∆ lays a solid foundation to establishing an 1,3-PDO assimilation pathway.