Elimination of metabolic pathways to all traditional fermentation products increases ethanol yields in Clostridium thermocellum

Abstract

Clostridium thermocellum has the natural ability to convert cellulose to ethanol, making it a promising candidate for consolidated bioprocessing (CBP) of cellulosic biomass to biofuels. To further improve its CBP capabilities, a mutant strain of C. thermocellum was constructed (strain AG553; C. thermocellum Δhpt ΔhydG Δldh Δpfl Δpta-ack) to increase flux to ethanol by removing side product formation. Strain AG553 showed a two- to threefold increase in ethanol yield relative to the wild type on all substrates tested. On defined medium, strain AG553 exceeded 70% of theoretical ethanol yield on lower loadings of the model crystalline cellulose Avicel, effectively eliminating formate, acetate, and lactate production and reducing H2 production by fivefold. On 5 g/L Avicel, strain AG553 reached an ethanol yield of 63.5% of the theoretical maximum compared with 19.9% by the wild type, and it showed similar yields on pretreated switchgrass and poplar. The elimination of organic acid production suggested that the strain might be capable of growth under higher substrate loadings in the absence of pH control. Final ethanol titer peaked at 73.4mM in mutant AG553 on 20 g/L Avicel, at which point the pH decreased to a level that does not allow growth of C. thermocellum, likely due to CO2 accumulation. In comparison, the maximum titer of wild type C. thermocellum was 14.1mM ethanol on 10 g/L Avicel. With the elimination of the metabolic pathways to all traditional fermentation products other than ethanol, AG553 is the best ethanol-yielding CBP strain to date and will serve as a platform strain for further metabolic engineering for the bioconversion of lignocellulosic biomass.

Improved growth rate in Clostridium thermocellum hydrogenase mutant via perturbed sulfur metabolism

“…However, wild-type C. thermocellum is limited by its low ethanol yield and titer, producing acetate, lactate, H 2 , formate, free amino acids, and other compounds as additional fermentation products [2, 3]. Recent development of genetic tools for C. thermocellum [4–9] has enabled the construction of numerous targeted mutants, eliminating acetate, lactate, formate, and H 2 production [4, 7, 10–14]. One of the mutations, C. thermocellum ΔhydG , eliminated a hydrogenase maturase protein involved in assembly of the [FeFe]-active site of three of the four hydrogenases in C. thermocellum .…”

LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313

“…To date, the majority of C. thermocellum genetic engineering studies have focused on improving fuel production (24)(25)(26)(27)(28)(29)(30), but little genetic work has targeted understanding regulatory pathways. Here, we combine use of gene deletions with transcriptomics and DNA binding assays to gain insights into the regulatory networks of the three LacI transcription factors in the genome of C. thermocellum DSM1313.…”

An integrated approach: advances in the use ofClostridiumfor biofuel

“…The same researchers added an artificial electron carrier (methyl viologen) which shifted the metabolic flux from acid to alcohol production, producing a high biofuel yield (0.39 g/g) from cellulose, comparable to ethanol yield from corn dextrose by yeast fermentation. A mutant strain of C. thermocellum, constructed to increase flux to ethanol by removing Yang et al,2015 side product formation, showed a two-to three-fold increase in ethanol yield relative to the wild type on all substrates tested (Papanek, Biswas, Rydzak, & Guss, 2015). A problem encountered with using lignocellulosic feedstock is the toxic compounds generated by its degradation which are inhibitory to clostridial growth.…”