Improved n-Butanol Production from Clostridium cellulovorans by Integrated Metabolic and Evolutionary Engineering

Abstract

Clostridium cellulovorans DSM 743B offers potential as a chassis strain for biomass refining by consolidated bioprocessing (CBP). However, its n-butanol production from lignocellulosic biomass has yet to be demonstrated. This study demonstrates the construction of a coenzyme A (CoA)-dependent acetone-butanol-ethanol (ABE) pathway in C. cellulovorans by introducing adhE1 and ctfA-ctfB-adc genes from Clostridium acetobutylicum ATCC 824, which enabled it to produce n-butanol using the abundant and low-cost agricultural waste of alkali-extracted, deshelled corn cobs (AECC) as the sole carbon source. Then, a novel adaptive laboratory evolution (ALE) approach was adapted to strengthen the n-butanol tolerance of C. cellulovorans to fully utilize its n-butanol output potential. To further improve n-butanol production, both metabolic engineering and evolutionary engineering were combined, using the evolved strain as a host for metabolic engineering. The n-butanol production from AECC of the engineered C. cellulovorans was increased 138-fold, from less than 0.025 g/liter to 3.47 g/liter. This method represents a milestone toward n-butanol production by CBP, using a single recombinant clostridium strain. The engineered strain offers a promising CBP-enabling microbial chassis for n-butanol fermentation from lignocellulose.IMPORTANCE Due to a lack of genetic tools, Clostridium cellulovorans DSM 743B has not been comprehensively explored as a putative strain platform for n-butanol production by consolidated bioprocessing (CBP). Based on the previous study of genetic tools, strain engineering of C. cellulovorans for the development of a CBP-enabling microbial chassis was demonstrated in this study. Metabolic engineering and evolutionary engineering were integrated to improve the n-butanol production of C. cellulovorans from the low-cost renewable agricultural waste of alkali-extracted, deshelled corn cobs (AECC). The n-butanol production from AECC was increased 138-fold, from less than 0.025 g/liter to 3.47 g/liter, which represents the highest titer of n-butanol produced using a single recombinant clostridium strain by CBP reported to date. This engineered strain serves as a promising chassis for n-butanol production from lignocellulose by CBP.

Keywords: Clostridium; adaptive laboratory evolution; consolidated bioprocessing; metabolic engineering; n-butanol.

FIG 1

Integrated metabolic and evolutionary engineering of Clostridium cellulovorans DSM 743B for n-butanol production from AECC by CBP. A CoA-dependent acetone-butanol-ethanol (ABE) pathway composed of adhE1 and ctfA-ctfB-adc (red font) from C. acetobutylicum ATCC 824 was constructed in C. cellulovorans. In parallel, C. cellulovorans was grown in medium with a serial enrichment of n-butanol to evolve and reinforce its butanol tolerance. To maximize butanol production, metabolic and evolutionary engineering were combined, using the evolved strain as a host for metabolic engineering.

FIG 2

Overexpression of adhE1 enabled C. cellulovorans to produce more n-butanol than overexpression of adhE2. (A) Effects of adhE1 and adhE2 introduction on butanol production. (B) Comparison of butyraldehyde dehydrogenase activities and butanol dehydrogenase activities between the ZQW02 and ZQW03 strains. (C and D) Fermentation profiles of ZQW01 and ZQW03, respectively, with AECC as the sole carbon source. The data in panels A and B are the means and standard deviations of three replicates (***, P ≤ 0.001; **, P ≤ 0.01; t test).

FIG 3

Overexpressing ctfAB-adc in ZQW03 dries n-butanol production by reassimilating butyrate. (A) Effects of ctfAB-adc and bukI introduction on n-butanol production and butyrate reassimilation. (B to D) Batch fermentation profile of ZQW05 with AECC as the sole carbon source. The data in panel A are the means and standard deviations of three replicates (**, P ≤ 0.01; t test).

FIG 4

Adaptive laboratory evolution improved n-butanol tolerance of C. cellulovorans. (A) The evolution process of ZQW10. ZQW10 was subcultured in medium supplemented with stepwise enrichment of butanol (3, 6, 9, and 12 g/liter) to evolve and strengthen its n-butanol tolerance. (B) Comparison of n-butanol tolerance levels of mutant strains derived from ZQW10, ZQW20, and ZQW30. Due to evolution, ZQW23, ZQW25, ZQW33, and ZQW35 can tolerate higher concentrations of n-butanol than ZQW13 and ZQW15. In addition, the n-butanol tolerance levels of ZQW33 and ZQW35 were superior to those of ZQW23 and ZQW25.

FIG 5

The dendrogram of genetically modified C. cellulovorans strains in this study.

FIG 6

Improved butanol tolerance led an enhancement in n-butanol production. (A) Effects of laboratory-adaptive evolution on n-butanol production. Mutants ZQW33 and ZQW35 that were derived from evolved strain ZQW30 produced much more n-butanol than the mutants ZQW13 and ZQW15 that were derived from ZQW10. (B to D) Metabolic profile of batch fermentation of ZQW35 with AECC as the sole carbon source. The data in panel A are the means and standard deviations of three replicates (***, P ≤ 0.001; t test).

FIG 7

Two-stage pH control strategy to improve n-butanol production further. (A) Effects of two-stage pH control strategy on n-butanol and butyrate production. (B to D) Metabolic profile of batch fermentation of ZQW35 using a two-stage pH control strategy with AECC as the sole carbon source. The data in panel A are the means and standard deviations of three replicates (***, P ≤ 0.001; **, P ≤ 0.01; t test).

FIG 8

Integrated metabolic and evolutionary engineering enhanced n-butanol production of C. cellulovorans from AECC.