Strain Development of Acetogen, Eubacterium limosum KIST612 for Carbon Monoxide-Based Ethanol Production

Abstract

Acetogens are capable of fixing inorganic gases to acetate via Wood-Ljungdahl pathway. Due to their potential to produce value-added metabolites, such as organic acids with/without bio-alcohols, acetogens have received attention in the field of biorefinery. Several efforts are being made to construct genetic toolbox in order to engineer acetogens with the final goal to manipulate a catabolic metabolism to enhance product yields. However, up to now, successful genetic engineering systems are limited to a few acetogens. Eubacterium limosum KIST612 is one of the promising acetogen of which the syngas-utilizing ability had been well studied as well as the genomic data are available. In this research, metabolic engineering was applied to develop the KIST612 that produces a non-native ethanol from carbon monoxide. For development of the strain, it is necessary to understand metabolic properties and bioenergetics and to construct appropriate genetic systems. The KIST612 is one of the few acetogens that can produce butyrate from carbon monoxide. The butyrate production is engaged in the energy conservation of the strain. A genome-guided analysis was used to delineate the pathway of butyrate formation, the enzymes involved and the potential coupling to ATP synthesis. Oxidation of CO is catalyzed by the CO dehydrogenase/acetyl-CoA synthase and coupled to the reduction of ferredoxin. Oxidation of reduced ferredoxin is catalyzed by the Rnf complex in a Na+-dependent manner. Consistent with the finding of a Na+-dependent Rnf complex is the presence of a conserved Na+-binding motif in the c subunit of the ATP synthase. As an activity of the butyryl-CoA dehydrogenase, it was confirmed that the reduction of crotonyl-CoA to butyryl-CoA by oxidation of NADH was coupled to reduction of ferredoxin. It can be postulated that the butyryl-CoA dehydrogenase uses flavin-based electron bifurcation to reduce ferredoxin, which is consistent with the finding of etfA and etfB genes next to it. As a specific characteristic of KIST612, the strain exhibits metabolic versatility depending on the energy source. This metabolic versatility is well represented in the butyrate production of the strain. To assess the versatile regulation on the metabolism, the transcriptional dynamics of KIST612 was explored across different carbon/energy/electron sources and growth phases. A total of 4,579 genes were identified; among them, more than half were differentially transcribed under various growth conditions. These differentially expressed genes were classified into six categories based on their patterns of transcriptional regulation. Pathway enrichment analysis of the differentially expressed genes identified an unexpected feature for both energy metabolism and butyrate synthesis, in that KIST612 has two distinct butyrate formation routes: i) acetate-assimilatory butyrate formation, which dominates under autotrophic condition; and ii) dissimilatory butyrate formation with de-phosphorylation of butyryl phosphate, which dominates under heterotrophic condition. The foreign gene introduction and expression system was developed, as a genetic toolbox for KIST612. The compacted size of shuttle vector backbone was constructed for the strain by modifying pJIR418 that the stable replication was confirmed. In addition, the transcriptomics-based candidate selection of native constitutive promoter and the β-glucuronidase (GUS) reporter gene assay enabled to sort one strong and constitutive native promoter H2 (Prbo) for the strain. Then, based on the genetic system of the strain, the recombinant vector (pECPH2::adhE), that the heterologous gene encoding bi-functional aldehyde/alcohol dehydrogenase (AdhE) with H2 (Prbo) promoter was inserted in the shuttle vector for KIST612, was constructed and it was introduced into the strain. The successful transformation and expression of heterologous AdhE gene enabled to produce non-native ethanol from the strain. The AdhE transformants were cultivated on both heterotrophic glucose- and autotrophic CO- substrate conditions, repectively. As a result of product profiling, KIST612 transformants produced non-native ethanol without any butyrate production in the both conditions. The native butyrate production of KIST612 was inhibited by competition with ethanol production through the heterotrophic expression of AdhE, even without any genome editing. In addition, the transformants produced ethanol with clear re-assimilation of acetate in only autotrophic CO condition, similar to the feature of butyrate production in wild-type KIST612. Interestingly, the AdhE1 transformants produced 27 mM of ethanol consuming 12.9 mmols of CO with complete re-assimilation of acetate, in CO fed-batch condition. In conclusion, the KIST612 was successfully engineered to ethanol producer, as well it was confirmed the possibility of product unification into ethanol by CO fermentation of the strain.