Strain improvement of Parageobacillus thermoglucosidasius - Continuous mutagenesis and selection to elicit complex phenotypes

  • Maria Vittoria Ortenzi

Student thesis: Doctoral ThesisPhD


Energy production based on fossil fuels is not sustainable and its consequences on the global climate and environment are challenging the safety of our ecosystem. Alternative energy sources have been widely researched, and bioethanol is one of them, but current practice can create competition for resources used as food. Second generation bioethanol is instead produced from inedible lignocellulosic biomass, municipal solid waste or algal biomass. However, a competitive large-scale production system has yet to be put in place.
Among the organisms with advantageous physiological characteristics for production of second generation bioethanol at industrial level, is Parageobacillus thermoglucosidasius. This Gram-positive thermophile can ferment pentoses, hexoses and some polymeric/oligomeric carbohydrates to produce lactate, ethanol, acetate and formate. Genetic engineering of P. thermoglucosidasius has produced a strain (TM242) able to produce mainly ethanol at 92 % of the maximum theoretical ethanol yield of 0.51 g/g glucose. To be economically viable, microbial cell factories have to deliver ethanol titers above 5 % v/v and 90 % of the theoretical yield. However, P. thermoglucosidasius undergoes osmolytic shock at glucose concentrations higher than 50 g/L and shows growth inhibition at concentrations of ethanol above 16 g/L (2 % v/v).
The aim of this project was to improve the tolerance of P. thermoglucosidasius towards glucose and ethanol to make it a robust cell factory. These traits are complex phenotypes controlled by an undefined number of genes and proteins. Strain improvement was pursued using Genome Replication Engineering Assisted Continuous Evolution (GREACE). GREACE is a technique that couples genome-wide mutagenesis (obtained in the original work by inefficient proofreading activity of the E. coli ε subunit of the DNA polymerase during replication) and continous self-selection under environmental stress conditions of increasing stringency. In this study the putative role of P. thermoglucosidasius dnaQ in proofreading was assessed by exploring the mutagenic effect of DnaQ variants created by overlapping PCR after bioinformatic comparison with that from E. coli. The mutation frequency of strains carrying these variants was calculated based on generation of ciprofloxacin resistant mutants, resulting in a set of “mutator strains” with different mutagenic strength. In a initial application, the DnaQ variants were used to evolve glucose tolerance by progressively subculturing the wild type P. thermoglucosidasius NCIMB 11955 at increasing concentrations of glucose. Mutants able to survive at 150 g/L were obtained, their genomes sequenced and analysed, revealing mutations affecting various cellular functions but no obvious pattern. GREACE was then applied using the strongest mutator available (dnaQH207L) to the high ethanol producing strain TM242 with selection in continuous culture at increasing concentrations of ethanol. This produced strains able to tolerate 26.5 g/L (3.36 % v/v) ethanol. Isolates of key bioreactor samples were tested in a high-throughput screen to confirm the phenotype and their genomes were sequenced. Among the numerous mutations observed contributing to survival in high ethanol concentration, the most relevant ones were those relating to acetaldehyde detoxification.
Date of Award16 Jun 2021
Original languageEnglish
Awarding Institution
  • University of Bath
SupervisorDavid Leak (Supervisor) & Susanne Gebhard (Supervisor)


  • Parageobacillus thermoglucosidasius
  • thermophile
  • evolutionary engineering
  • adaptive laboratory engineering
  • ethanol resistance
  • glucose resistance
  • directed evolution
  • mutation frequency
  • chemostat
  • continuous culture
  • NGS sequencing
  • genomes analysis
  • complex phenotypes
  • dnaQ mutants

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