Improving Organic Solvent Tolerance Of Escherichia Coli By Global Transcription Machinery Engineeirng

Whole-cell biocatalysts are often applied in aqueous/organic biphase system to improvethe solubility of substrate/product as well as their inhibitory effect on cells. However, mostorganic solvents are toxic to microbial cell.In this study, global transcription machinery engineering (gTME) was used to improvethe organic-solvent tolerance (OST) of Escherichia coli as a model strain. The rpoD gene,encoding sigma70, was subjected to error-prone PCR to construct the mutant library. Afterscreening, E. coli JM109carrying mutants C9and B1capable of tolerating69%(v/v)cyclohexane and1.2%(v/v) butanol respectively were obtained. Total protein expressiondifference of E. coli JM109carrying C9under different solvent treatments was investigatedby2D-PAGE. Among204proteins high-abundant proteins,43proteins showed differentexpression level over2-time were analyzed by MALDI-TOF/TOF. Finally,22proteins weresuccessfully identified. Nineteen proteins involved in nucleic acid、amino acid、glucosemetabolism, transporter and porin protein, were up-regulated, while the remaining3proteins(dipeptide transporter, thiol peroxidase and porin protein ompC) were down-regulated. Sixgenes (gapA, sdhB, pepB, yfgM, dppA and bcp gene) encoding4up-regulated proteins(glyceraldehyde-3-phosphate dehydrogenase A, succinate dehydrogenase, FeS subunit,aminopeptidase B and hypothetical protein YFGM) and2down-regulated proteins (dipeptidetransporter, thiol peroxidase) were knocked out from genome of E. coli JM109to evaluatetheir OST-related properties. Meanwhile, complementary expression of gapA, sdhB, pepB,yfgM was also carried out. Our results indicate that3genes (gapA, sdhB and pepB) playcritical roles in OST of E. coli. Glyceraldehyde-3-phosphate dehydrogenase A is involved inglycolysis process and could produce pyruvic acid and ATP. Succinate dehydrogenase FeSsubunit is involved in TCA cycle and could provide coenzyme for succinate dehydrogenasewhich catalyzes the synthesis of fumarate and ATP. Therefore, up-regulated expression ofgapA and sdhB could increase ATP level in cells, providing more energy under solvent stress.The OST-related mechanism of pepB remains unknown. Gene knockout of dppA wasconfirmed to improve OST of E. coli, as dipeptide transporter may responsible for organicsolvent transportation into cytoplasm.Additionally, an important gene mmsB (encodeing3-hydroxyisobutyrate dehydrogenase)identified from pseudomonas putida JUCT1in our previous study, was integrated to E. coliJM109chromosome based on Red-mediated recombination. And the strain E. coliJM109(DE3)(ΔendA::mmsB) showed significant higher OST for various organic solvents,especially butyl acetate and butanol. pET-kmCR carrying a carbonyl reductase from K.marxianuswas was transformed into E. coli JM109(DE3)(ΔendA::mmsB) and JM109(DE3)(as control). The recombinant strain was successfully applied in bioreduction of OPBE to(R)-HPBE in an aqueous/butyl acetate (1:1) biphasic system, giving a yield twice as much asthe control.This study could provide useful knowledge for the genetic engineering of microbialstrains with excellent OST-phenotypes for industrial applications in nonaqueous biocatalysis and biofuel production.