Chromosomal mutations in Escherichia coli that improve tolerance to nonvolatile side‐products from dilute acid treatment of sugarcane bagasse

Abstract

Lignocellulosic biomass provides attractive nonfood carbohydrates for the production of ethanol, and dilute acid pretreatment is a biomass‐independent process for access to these carbohydrates. However, this pretreatment also releases volatile and nonvolatile inhibitors of fermenting microorganisms. To identify unique gene products contributing to sensitivity/tolerance to nonvolatile inhibitors, ethanologenic Escherichia coli strain LY180 was adapted for growth in vacuum‐treated sugarcane bagasse acid hydrolysate (VBHz) lacking furfural and other volatile inhibitors. A mutant, strain AQ15, obtained after approximately 500 generations of growth in VBHz, grew and fermented the sugars in a medium with 50% VBHz. Comparative genome sequence analysis of strains AQ15 and LY180 revealed 95 mutations in strain AQ15. Six of these mutations were also found in strain SL112, an independent inhibitor‐tolerant derivative of strain LY180. Among these six mutations, null mutations in mdh and bacA were identified as contributing factors to VBHz tolerance in strain AQ15, based on the genetic and physiological analysis. The deletion of either gene in strain LY180 increased tolerance to VBHz from approximately 30–50% (vol/vol). Considering the location and physiological role of the two enzymes in the cell, it is likely that the two enzymes contribute to the VBHz sensitivity of ethanologenic E. coli by different mechanisms.

Extrapolation of design strategies for lignocellulosic biomass conversion to the challenge of plastic waste

“…Reverse engineering of evolved strains can be used to propose rational strain engineering designs. This was demonstrated by the Ingram group in regards to furfural tolerance (Miller et al., 2009 ; Turner et al., 2011 ; Wang et al., 2013 ) and hydrolysate (Geddes et al., 2011 b; Shi et al., 2020 ; Shi et al., 2016 ).…”

Microbial adaptive evolution

“…With the development of bioinformatics, different omics approaches have been used for understanding and predicting the molecular mechanisms of bacterial temporary and permanent responses to different environments. These omics are used independently to study bacterial adaptation to a specific environment, such as identifying genes related to tolerance to lignocellulosic acid hydrolysates by genome sequencing (Shi et al, 2020 ), and capturing expression patterns of growth-dependent genes in Salmonella and Pseudomonas by transcriptome analysis (Imdahl et al, 2020 ).…”

“…For instance, Dr. Ingram's laboratory adapted two separate cultures of an ethanologenic E. coli strain LY180 for growth in lignocellulosic acid hydrolysate over hundreds of generations. After genome sequence analysis, six common mutations were found in two lineages, strains AQ15 and SL112, indicating parallel evolution (Shi et al, 2020 ). A second example is provided by Burkholderia dolosa , a pathogen that can cause disease in humans.…”

Improving acetic acid and furfural resistance of Saccharomyces cerevisiae by regulating novel transcriptional factors revealed via comparative transcriptome

“…Lignocellulose biomass has high cellulose and hemicellulose content. Fermenting it into bioethanol is an effective way to cope with resource and energy crisis (Shi et al 2020;Cheah et al 2020). Pretreatment and hydrolysis are needed to break down the structure of lignocellulosic biomass and release the fermentable sugars (mainly glucose and xylose) before it could be fermented by Saccharomyces cerevisiae (Du et al 2020;Zhou et al 2018).…”