Abstract
BackgroundEngineering efforts targeted at increasing ethanol by modifying the central fermentative metabolism of Clostridium thermocellum have been variably successful. Here, we aim to understand this variation by a multifaceted approach including genomic and transcriptomic analysis combined with chemostat cultivation and high solids cellulose fermentation. Three strain lineages comprising 16 strains total were examined. Two strain lineages in which genes involved in pathways leading to organic acids and/or sporulation had been knocked out resulted in four end-strains after adaptive laboratory evolution (ALE). A third strain lineage recapitulated mutations involving adhE that occurred spontaneously in some of the engineered strains.ResultsContrary to lactate dehydrogenase, deleting phosphotransacetylase (pta, acetate) negatively affected steady-state biomass concentration and caused increased extracellular levels of free amino acids and pyruvate, while no increase in ethanol was detected. Adaptive laboratory evolution (ALE) improved growth and shifted elevated levels of amino acids and pyruvate towards ethanol, but not for all strain lineages. Three out of four end-strains produced ethanol at higher yield, and one did not. The occurrence of a mutation in the adhE gene, expanding its nicotinamide-cofactor compatibility, enabled two end-strains to produce more ethanol. A disruption in the hfsB hydrogenase is likely the reason why a third end-strain was able to make more ethanol. RNAseq analysis showed that the distribution of fermentation products was generally not regulated at the transcript level. At 120 g/L cellulose loadings, deletions of spo0A, ldh and pta and adaptive evolution did not negatively influence cellulose solubilization and utilization capabilities. Strains with a disruption in hfsB or a mutation in adhE produced more ethanol, isobutanol and 2,3-butanediol under these conditions and the highest isobutanol and ethanol titers reached were 5.1 and 29.9 g/L, respectively.ConclusionsModifications in the organic acid fermentative pathways in Clostridium thermocellum caused an increase in extracellular pyruvate and free amino acids. Adaptive laboratory evolution led to improved growth, and an increase in ethanol yield and production due a mutation in adhE or a disruption in hfsB. Strains with deletions in ldh and pta pathways and subjected to ALE demonstrated undiminished cellulolytic capabilities when cultured on high cellulose loadings.
Highlights
Engineering efforts targeted at increasing ethanol by modifying the central fermentative metabolism of Clostridium thermocellum have been variably successful
Holwerda et al Biotechnol Biofuels (2020) 13:40 and pta pathways and subjected to adaptive laboratory evolution (ALE) demonstrated undiminished cellulolytic capabilities when cultured on high cellulose loadings
Whereas ALE resulted in substantially increased ethanol yield in the case of a strain engineered by Argyros et al, it resulted in higher yield of amino acids in the end-strain described in van der Veen et al In this paper, we aimed to identify the mechanisms underlying the divergent ethanol production phenotypes of the lineages described in Argyros et al and in van der Veen et al to evaluate the role and impact of ALE on engineered strains, and to evaluate the extent to which engineering to increase product yield impacts cellulose fermentation capability
Summary
Engineering efforts targeted at increasing ethanol by modifying the central fermentative metabolism of Clostridium thermocellum have been variably successful. Aiming to replicate the success realized with the non-cellulolytic hemicellulose-fermenting thermophile Thermoanaerobacterium saccharolyticum [6, 7], initial metabolic engineering efforts aimed at increasing ethanol yield in C. thermocellum focused on deleting pathways for carbon flux to lactate and acetate Prominent examples of this approach are described in the papers of Argyros et al [8] and van der Veen et al [9]. In both studies, started with the same parent stain (LL345), pathway disruption did not immediately result in changes in ethanol yield, and adaptive laboratory evolution (ALE) was used to give the microorganisms a chance to adjust their metabolism in response to genetic interventions. Whereas ALE resulted in substantially increased ethanol yield in the case of a strain engineered by Argyros et al, it resulted in higher yield of amino acids (and not ethanol) in the end-strain described in van der Veen et al
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