(123c) Discovery of Design Strategies for Enabling Pyrolytic Sugars Tolerance and Utilization By Escherichia coli

Lignocellulosic biomass has been extensively studied and utilized to produce a variety of compounds since it has been considered as an alternative source of carbon and energy for the production of biofuels and biochemicals to meet the increasing demands of petroleum-based products. There have been a variety of methods developed to pretreatment biomass for extracting fermentable substrates, however, plenty of organic coproducts are also produced during this process and inhibit the biological utilization. Fast pyrolysis, in our project, was employed to produce sugar syrup from red oak along with aldehydes, ketones, phenolic compounds, and so on, which inhibited the growth and sugar utilization of ethanologenic E. coli KO11. Thus, we developed three strategies to solve this problem. The first is to characterize the impacts of the pyrolytic sugars on cell membrane in terms of membrane fluidity, membrane leakage and membrane composition to study the response of E. coli to the sugars.  The results of membrane analysis showed that the pyrolytic sugars did cause membrane leakage and affect the membrane fluidity of E. coli, which demonstrate that the cell membrane damage may be one of the factors resulting in the inhibition of pyrolytic sugars. The second is to reduce the toxicity of pyrolytic sugars on E. coli by either removing some of the inhibitors in the pyrolytic sugars by alkali treatment or protecting cells from the sugars by the way of encapsulation using Ca alginate. Here we used sodium hydroxide and calcium hydroxide to treat the pyrolytic sugars at high temperature and pH values. The alkali-treated sugars showed less toxicity in terms of both reduced damage on cell membrane and better fermentability. Besides, the cells entrapped into the Ca alginate were still able to produce ethanol at high titer while no growth of the free cells was observed at such concentrations. And the last one is to improve the resistance of E. coli to the pyrolytic sugars by the use of directed evolution. After long-term directed evolution, the tolerance of E. coli KO11 to the pyrolytic sugars was improved and reverse engineering was followed to study the impacts of mutant genes of the tolerant strain obtained from genomic sequencing on the phenotypes of increased tolerance and reproduce it on the KO11 or other microbes.