Polyamines and polyamine transporters increase furfural tolerance of ethanologenic Escherichia coli strain LY180

A library of Sau3AI fragments from Escherichia coli W was screened for promoter activity using a promoterless fucO-ucpA cassette encoding two genes that increase furfural tolerance.  Four different resistant clones were isolated. Each contained a unique combination of small Sau3AI fragments from different regions of the E. coli W chromosome together with a single larger fragment encoding a full length potE (polyamine transporter) gene. Subcloning and further testing revealed that plasmid expression of potE increased furfural tolerance (growth), even without the fucO-ucpA cassette. Microarray comparisons of two furfural-resistant mutants provided further evidence for the importance of polyamine transporters for furfural tolerance. Each mutant had more than 100-fold higher levels of a single polyamine transporter, potE in EMFR9 and puuP in EMFR35. Deletion of either gene (puuP or potE) in the parent (LY180) decreased furfural tolerance. All 8 polyamine transporters in E. coli were cloned and compared.  Four transporter plasmids were beneficial (PotE, PuuP, PlaP and PotABCD), increasing growth in the presence of furfural. Three inhibited growth when inducer was added, even in the absence of furfural. Supplementing AM1 medium with individual polyamines (agmantine, putrescine and cadaverine) also increased furfural tolerance. Combinations of putrescine and plasmids with a putrescine transporters (PotE or PuuP) were more beneficial than either alone. Together, these results demonstrate that plasmid-borne polyamine transporters and polyamine supplements can be used to increase furfural tolerance in E. coli LY180.