Metabolic changes of the acetogen Clostridium sp. AWRP through adaptation to acetate challenge

Abstract

In this study, we report the phenotypic changes that occurred in the acetogenic bacterium Clostridium sp. AWRP as a result of an adaptive laboratory evolution (ALE) under the acetate challenge. Acetate-adapted strain 46 T-a displayed acetate tolerance to acetate up to 10 g L−1 and increased ethanol production in small-scale cultures. The adapted strain showed a higher cell density than AWRP even without exogenous acetate supplementation. 46 T-a was shown to have reduced gas consumption rate and metabolite production. It was intriguing to note that 46 T-a, unlike AWRP, continued to consume H2 at low CO2 levels. Genome sequencing revealed that the adapted strain harbored three point mutations in the genes encoding an electron-bifurcating hydrogenase (Hyt) crucial for autotrophic growth in CO2 + H2, in addition to one in the dnaK gene. Transcriptome analysis revealed that most genes involved in the CO2-fixation Wood-Ljungdahl pathway and auxiliary pathways for energy conservation (e.g., Rnf complex, Nfn, etc.) were significantly down-regulated in 46 T-a. Several metabolic pathways involved in dissimilation of nucleosides and carbohydrates were significantly up-regulated in 46 T-a, indicating that 46 T-a evolved to utilize organic substrates rather than CO2 + H2. Further investigation into degeneration in carbon fixation of the acetate-adapted strain will provide practical implications for CO2 + H2 fermentation using acetogenic bacteria for long-term continuous fermentation.

Autotrophic adaptive laboratory evolution of the acetogenClostridium autoethanogenumdelivers the gas-fermenting strain LAbrini with superior growth, products, and robustness

“…In acetogens, ALE has improved tolerance towards methanol in Sporomusa ovata (Tremblay et al, 2015), cyanide in Clostridium ljungdahlii (Oswald et al, 2018), benzene, toluene, and xylenes in C. autoethanogenum (Piatek et al, 2020), and acetate in Clostridium sp. AWRP (Kwon et al, 2022). ALE also afforded an Eubacterium limosum strain with higher CO tolerance and faster growth (Jin et al, 2022; Kang et al, 2020).…”

“…In acetogens, ALE has improved tolerance towards methanol in Sporomusa ovata (18), cyanide in Clostridium ljungdahlii (19), benzene, toluene, and xylenes in C. autoethanogenum (20), and acetate in Clostridium sp. AWRP (21). ALE was also recently used in Clostridium carboxidivorans to expand its product spectrum and improve growth on CO 2 +H 2 (22) and in Eubacterium limosum for higher CO tolerance and faster growth (23, 24).…”

Development of a genetic engineering toolbox for syngas-utilizing acetogen Clostridium sp. AWRP

“…Therefore, we determined the strength of several promoters in AWRP under a heterotrophic growth condition (Fig. 2 ): three constitutive promoters P fdx , P pta and P trxA , which were chosen according to the previous transcriptome study during growth on CO 2 + H 2 [ 19 ], one putative inducible promoter P xyl (upstream of DMR38_09110), and two promoters from C. acetobutylicum (P thl and P ptb ) [ 33 – 35 ]. Promoters were cloned upstream of bgaL from C. beijerinckii (Cbei_1236) and a synthetic terminator BBa_B1010 [ 36 , 37 ].…”

“…From previous studies, this bacterium appears to exhibit a different alcohol production metabolism compared to model strains: its ethanol selectivity to acetate could be greatly increased by CO supply [ 17 , 18 ]. Nonetheless, in this strain, there are several questions that remain unclear including its nutritional requirements [ 19 ], which genetic modifications may be required to address. Therefore, we sought to develop procedures for genetic modifications in Clostridium sp.…”

Tolerance in Solventogenic Clostridia for Enhanced Butanol Production: Genetic Mechanisms and Recent Strain Engineering Advances

“…Although those species are not solventogenic, they closely resemble and share some metabolic pathways with solventogenic clostridia. For example, many molecular chaperones (or heat and chemical shock proteins) are upregulated in the acidogenic Clostridium AWRP [12]. In solventogenic clostridial fermentation, such as with Clostridium acetobutylicum ATCC 824, chaperone-like genes such as phaP, as well as others such as pncB and cfa demonstrated elevated expression levels in the presence of acetic and other inhibitors [13].…”