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Alternate Isoleucine Synthesis Pathway in Cyanobacterial Species

¹ Washington University in St. Louis;
² Donald Danforth Plant Science Center

Cyanothece sp. ATCC 51142 is an aerobic N₂-fixing and hydrogen-producing cyanobacterium. Isotopomer analysis of its amino acids reveals an identical labeling profile for leucine and isoleucine when Cyanothece 51142 is grown mixotrophically using [2-¹³C] labeled glycerol as the main carbon source. This indicates that Cyanothece 51142 employs the atypical alternative citramalate pathway for isoleucine synthesis with pyruvate and acetyl-CoA as precursors. Utilization of the citramalate pathway is confirmed by an enzyme assay and LC-MS/MS analysis. Furthermore, the genome sequence of Cyanothece 51142 shows that the gene encoding the key enzyme (threonine ammonia-lyase) in a normal isoleucine pathway is missing. Instead, the cce_0248 gene in Cyanothece 51142 exhibits 53 % identity to the gene encoding citramalate synthase (CimA, GSU1798) from Geobacter sulfurreducens. Reverse transcription PCR also indicates that the cce_0248 gene is expressed and its transcriptional level is lower in the medium with isoleucine than in the isoleucine-free medium. Additionally, a BLAST search for citramalate synthase and threonine ammonia-lyase implies that this alternate isoleucine synthesis pathway may be present in other cyanobacteria, such as Cyanothece and Synechococcus. This suggests that the pathway is more widespread than originally thought, as previous identifications of the citramalate pathway are limited to mostly anaerobic bacteria or archaea. Furthermore, this discovery opens the possibility that such autrotrophic microorganisms may be engineered for robust butanol and propanol production from 2-ketobutyrate, which is an intermediate in the isoleucine biosynthesis pathway.

³ E-mail: yinjie.tang{at}seas.wustl.edu