Discovery of design strategies for enabling pyrolytic sugars tolerance and utilization by Escherichia coli

Biorenewable biomass has been extensively utilized as an appealing source of carbon and energy for the production of biochemicals and biofuels via biological fermentation to meet the increasing demand of petroleum-based products. A variety of biomass resources have been used such as dedicated crops and wastes including agriculture residue and forestry. Lignocellulosic biomass is consisting of hemicellulose, cellulose and lignin, which is either composed of polysaccharides or phenolic compounds that cannot be metabolized by microorganisms directly. Thus, deconstruction of biomass into fermentable sugar monomers is of great importance. There are a variety of physical or chemical pretreatment methods that have been developed, including the use of acids, alkali, steam, oxidants and high pressure. Fast pyrolysis, which is a type of thermochemical processing, is used in this study. It is an attractive approach to produce pyrolytic sugar syrup due to the advantages such as the flexibility of the feedstocks and the rapidness of reactions. However, the common issue of this bioconversion platform is that a variety of co-products are formed from the pretreatment process, such as phenolic compounds, aldehydes, which have been proved inhibitory to biocatalysts. Therefore, to improve the tolerance and utilization of biocatalysts to the biomass-derived sugars is necessary for increasing the production of target compounds. To overcome this challenge, several strategies have been performed to enable biocatalysts to survive the deterious living environment. The first approach is to reduce the toxicity of the biomass-derived sugars by removing the inhibitors. For example, detoxification of the sugars by the chemical treatments with alkali, oxidants or physical treatments with organic solvents have been developed. In this study, sodium hydroxide and calcium hydroxide were used to form precipitants in order to remove some inhibitors in the pyrolytic sugars. The detoxified sugars have been proved to be more fermentable by performing the fermentations and evaluating E. coli cell membrane integrity and fluidity. Encapsulation of microorganisms, which is aimed to protecting the cells from the inhibitors in the pyrolytic sugars by selectively enabling the nutrients to permeate the porous polymers entrapping the cells, is an alternative method to “remove” the toxic compounds. In this study, calcium alginate, which is in the form of beads, is employed to enclose the cells from the pyrolytic sugars. The ethanol production results demonstrate that the encapsulation of cells helps to improve the tolerance of E. coli KO11 to pyrolytic sugars at a concentration as high as of 1.8% (w/v). In additional to “removal” of inhibitors, the other approach is to improve the performance of biocatalysts in the pyrolytic sugars. Since some inhibition mechanisms have already been well characterized, rational engineering of biocatalysts such as omics analysis, membrane modification can be employed to improve or produce the desired phenotypes. However, the pyrolytic sugars in this study are very complex and containing plenty of compounds that are still unknown. Therefore, directed evolution, which is more straightforward and effective here, was used for enhancing the resistance of two biocatalysts, ethanol-producing E. coli KO11 and lactic acid-producing E. coli SZ194, to the pyrolytic sugars. By long-term sequential transfers, the two evolved strains TJE1 and TJL1 obtained possess 2.6-fold and 4.4-fold the tolerance of parent strains respectively. Subsequently, we characterized the membrane properties between parent and evolved strains to study the changes of cell membrane caused by pyrolytic sugars since most of the inhibitory compounds in the pyrolytic sugars are hydrophobic. It showed that evolved TJE1 obtained strengthened membrane from directed evolution in terms of membrane integrity and fluidity. However, the membrane composition was only slightly changed compared to parent. Besides, we also found that the biofilm and extracellular polymeric substances were greatly decreased in both evolved E. coli strains. In additional, reverse engineering, which is to identify the inhibition mechanism and yield a roadmap by studying the mutations occurred during directed evolution, is applied to reproduce the desired phenotypes of increased tolerance. In this study, csrA, the global carbon storage regulator, was identified with one point mutation within DNA sequence leading to one amino acid change. This mutation was found in both evolved E. coli strains, which inspired us to investigate its effects on the increased tolerance. Here, we did gene switching in parent KO11 and evolved SZ194, followed by a series of assays according to the functionalities of csrA. The results demonstrated that this mutation contributed to the reduction of flagellation, biofilm formation and extracellular polymeric substances. In addition, this mutation was found improving the membrane integrity and maintaining membrane fluidity as well. By introducing this mutation into a model E. coli, MG1655, we found that this mutation was contributing to the increased resistance to certain hydrophobic compounds to varying degrees