Enhancing acetic acid and 5‐hydroxymethyl furfural tolerance of C. saccharoperbutylacetonicum through adaptive laboratory evolution

Abstract

In this study, adaptive laboratory evolution (ALE) was applied to isolate four strains of Clostridium saccharoperbutylacetonicum able to grow in the presence of hemicellulosic hydrolysate inhibitors unsupported by the parental strain. Among them, isolate RAC-25 presented the best fermentative performance, producing 22.1 g/L of ABE and 16.7 g/L of butanol. Genome sequencing revealed a deletion in the arabinose transcriptional repressor gene (araR) and a mutation in the anti-sigma factor I that promoted a downregulation of sigI. Gene expression analysis indicated high expression of genes related to H +-pumps (ATP synthases), proline biosynthesis (gamma phosphate reductase) and chaperonins (Grol), suggesting an integrated mechanism that is probably coordinated by the repression of sigI. Therefore, in addition to highlighting the power of ALE for selecting robust strains, our results suggest that sigI and araR may be interesting gene targets for increased tolerance toward inhibitor compounds relevant for lignocellulosic biofuels production.

Strain evolution and novel downstream processing with integrated catalysis enable highly efficient co-production of 1,3-Propanediol and organic acid esters from crude glycerol

“…Thus the strategy employed here makes the system as simple as possible and highly efficient as well. Last but most importantly, the time needed for 1 adaptation cycle (OD600 from 0.5 to 1.5) is significantly reduced compared to those reported in the manual ALE experiment (>12 h for 1 cycle) (Liang et al, 2020 ;Zhang et al, 2019 ;Alves et al, 2021 ). An example of the automatic ALE process for 30 g/L crude glycerol is presented in Fig.…”

“…Adaptive laboratory evolution (ALE) is widely used to obtain robust microbes with high tolerance for different environmental stresses. However, the achievement of a stably improved phenotype which can fully withstand a specific stress factor or inhibitor usually requires a long-term ALE with great efforts Alves et al, 2021 ). In order to increase the efficiency, long-term ALE of C. pasteurianum C8 was conducted in a home-made automatic evolutionary system integrated with the real-time monitoring and control of cell growth in this study.…”

“…However, an obvious disadvantage of the traditional ALE experiment is that it requires the passaging of cells manually for many generations to enrich favorable genetic changes at the expense of a lot of manpower. Moreover, the timing of cell passage in ALE is usually not well-determined due to the lack of real-time monitoring of cell growth (Zhang et al, 2019 ;Alves et al, 2021 ). Therefore, it is necessary to develop a smart ALE device which can regulate the cell growth and conduct long-term ALE in an automatic manner for achieving better and more stable crude glycerol tolerance of C. pasteurianum.…”

Strain evolution and novel downstream processing with integrated catalysis enable highly efficient coproduction of 1,3‐propanediol and organic acid esters from crude glycerol

“…ALE is widely used to obtain robust microbes with high tolerance for different environmental stresses. However, the achievement of a stably improved phenotype which can fully withstand a specific inhibitor usually requires a long‐term ALE with great efforts (Alves et al, 2021; A. H. Zhang et al, 2019). To increase the efficiency, long‐term ALE of C. pasteurianum C8 was conducted in a home‐made automatic evolutionary system integrated with the real‐time monitoring and control of cell growth in this study.…”

“…However, an obvious disadvantage of the traditional ALE experiment is that it requires the passaging of cells manually for many generations to enrich favorable genetic changes at the expense of a lot of manpower. Moreover, the timing of cell passage in ALE is usually not well‐determined due to the lack of real‐time monitoring of cell growth (Alves et al, 2021; A. H. Zhang et al, 2019). Therefore, it is necessary to develop a smart ALE device which can regulate the cell growth and conduct long‐term ALE in an automatic manner for achieving better and more stable crude glycerol tolerance of Clostridium pasteurianum .…”