Characterization of Clostridium ljungdahlii OTA1: A Non-Autotrophic Hyper Ethanol-Producing Strain

The Clostridium ljungdahlii lab-isolated spontaneous-mutant strain, OTA1, was previously shown to produce twice as much ethanol as the C. ljungdahlii ATCC 55383 strain when cultured in mixotrophic medium containing fructose and syngas. Sequencing identified four unique single nucleotide polymorphisms (SNPs) in the genome of C. ljungdahlii OTA1. Among these, two SNPs were found in genes involved in acetyl-CoA formation from CO/CO2 (acsA and hemL). Homology models of the respective mutated enzymes revealed alterations in the size and hydrogen bonding of the amino acids in their active sites. Failed attempts to grow OTA1 autotrophically suggested that one or both of these mutated genes prevented acetyl-CoA synthesis from CO/CO2 and that acetyl-CoA synthase activity was required for autotrophic growth by C. ljungdahlii. An inoperable Wood-Ljungdahl pathway resulted in higher CO2 and ethanol yields and lower biomass and acetate yields compared to WT for multiple growth conditions including heterotrophic and mixotrophic conditions. The two other SNPs identified in the C. ljungdahlii OTA1 genome were in genes for transcriptional regulators (CLJU_c09320 and CLJU_c18110) and were found to be responsible for deregulated expression of co-localized arginine catabolism and 2-deoxy-D-ribose (the sugar backbone of DNA) catabolism genes. Growth medium supplementation experiments suggested that increased arginine metabolism and 2-deoxy-D-ribose were likely to have minor effects on biomass and fermentation product yields. In characterizing the effects of the SNPs identified in the genome of C. ljungdahlii OTA1, a non-autotrophic C. ljungdahlii strain was discovered that has utility for further physiology and strain performance studies and as a biocatalyst for industrial applications.