Chen, Rachel

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Engineering Conferences International

Rachel ChenSchool of Chemical and Biomolecular Engineering Georgia Institute of TechnologyBioenergy III, Canary Island, May 2011 E. coli as biocatalyst for bioenergy and biorefinery Metabolic engineering has been successful in making E. coli  to churn out anything desirableLactate   succinate xylitol pyruvatePolyhydroxylalkoatePHA Need to take on polymeric substrates (cellulose/hemicellulose) to reduce costGlucoseXyloseE. coli Ethanol  (iso)butanol hydrocarbonConsolidated Production for cost reductionConsolidated Traditional  Single step accomplished in a reactor as single unit operation Multi-step accomplished as separate unit operations Enzyme production Requires biocatalyst to multitask  Enzymatic hydrolysis Fermentation (product formation) Objectives: Demonstrate a binary strategy to achieve direct fermentation of xylan to ethanol Hemicellulose: 20-40% of lignocelluloseSix types of enzymes are needed for complete hydrolysis of xylanFirst design of the binary system1st strain as hemicellulase producer/secretorEnzyme activity(U/ml)pCRAXE-T5XylTotal Extracellular Secretion percentage (%)Xylanase 0.28 0.26 93Acetylxylanesterase (AXE) 2.76 2.62 95PLac RBSpCRAXE-XylAXEEcoRVRBS TatS 6His Xyl11AXbaIPT5ApR KmRpUC ori2nd strain to convert oligosaccharides to ethanolEnzymes Activity (U/L)β-Xylosidase (β-Xyl) 1,616.5±17.02α-Arabinofuranosidase 46.30±6.64From Klebsiella pneumoniae2nd strain: converting xylooligosaccharide to ethanol0 h 48 hKO11KO11/ pBBKXYNKO11KO11/ pBBKXYNCell growth(A600) 1±0.08 1±0.11 3.02±13 4.74±0.35Reducing Sugar(g/L)1.74±0.13 1.74±0.15 4.39±0.25 1.49±0.17Ethanol (g/L) N.D. N.D. 1.30±0.12 2.91±0.18Binary vs. Single strain vs. acid hydrolysis 24 h fermentationResidual reducing sugar(g/L)Ethanol (g/L)Ethanol yield (%)Binary 1.71±0.06 2.84±0.13 54.5Single strains 0.84±0.07 1.95±0.14 37.6Acid hydrolysate 1.16±0.06 4.38±0.11 84.1Improvement on ethanol yieldCaseAfter 36h induction After 24h fermentationReducing sugar (g/L)Hydrolysis yield (%)Ethanol (g/L)Ethanol yield (%)A(3 enzymes) 3.35±0.11 32.7 2.84±0.13 54.5B 3.35±0.13 32.7 3.29±0.12 63.1C 3.87±0.12 37.8 3.11±0.14 59.6D 3.92±0.06 38.3 3.22±0.13 61.7E (6 enzymes) 3.95±0.25 38.6 3.71±0.27 71.1Xylan to ethanolWhy not 100% yield?Xylooligosaccharides with 4 or more xylose units were not utilizedConclusions Binary strategy is a viable option for consolidated bioprocessing of lignocellulose. Modular design allows easy extension to other biofuel and biorefinery products. Inclusion of all enzymes necessary to de-polymerize lignocellulose and judicious division of labor are important to achieve high yield. Hydrolysis of xylan to smaller units outside the cells or identifying a transporter for uptake larger xylooligosaccharides is important for further improvement. Co-cultivation of two strainsEthanol (g/L) 2.02.42.8Induction time (h)0 10 20 30 40Ethanol (g/L)0.00.40.81.21.6AcknowledgementsShara McClendon    Hyun-Dong Shin              Trinh Vo                   

Engineering an E. coli Binary Culture for Consolidated Production of Biofuel From Hemicelluloses

https://dc.engconfintl.org/cgi/viewcontent.cgi?article=1042&amp;context=bioenergy_iii

Engineering an E. coli Binary Culture for Consolidated Production of Biofuel From Hemicelluloses

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Engineering Conferences International