Metabolic engineering of Escherichia coli for improving shikimate synthesis from glucose
Bioresource Technology, 2014
Xianzhong Chen et al., Lab of Industrial Biotechnology, Jiangnan University, China
Johannes Bartuli Seminar II Studiengang Biotechnologie Overall objectives
● Shikimic acid (or shikimate)
● Reducing limitation of shikimate by utilizing microbial production
Goal:
● Development of an overproducing strain of Escherichia coli by metabolic engineering
Shikimic acid pathway
● Pathway in bacteria and plants for aromatic amino acids and other aromatic compounds
● Shikimate as an intermediate is only found in very low concentrations
Shikimate as precursor for Tamiflu
● Tamiflu is chemically synthesized using Shikimate as precursor
● Tamiflu slows the spread of influenza virus between cells
● 2004, 2007 and 2009: stockpiling in fear of a possible pandemic leading to a production shortage of Tamiflu
● Switch from classical shikimate extraction to microbial production
Metabolic engineering of E. coli for shikimate production
● Metabolic pathway leading to shikimate is well investigated
● Several approaches have been developed to overproduce shikimate
In this publication:
● Systems metabolic engineering of a newly obtained strain of E. coli - able to grow quickly on glucose and/or xylose
Metabolic engineering of E. coli for shikimate production
● Inactivation of genes by deletion - aroK and aroL for shikimate kinase
Metabolic engineering of E. coli for shikimate production
● Inactivation of genes by deletion - ptsG encoding the glucose phosphotransferase system
Metabolic engineering of E. coli for shikimate production
● Inactivation of genes by deletion - ydiB for quinic acid/shikimate dehydrogenase
Metabolic engineering of E. coli for shikimate production
● Inactivation of genes by deletion - aroK and aroL for shikimate kinase - ptsG encoding the phosphotransferase system - ydiB for quinic acid/shikimate dehydrogenase - pykF for pyruvate kinase I - ackA-pta for acetate kinase A and phosphate acetyltransferase
● Deletion of genes introduced successively to create mutants SA1 to SA5
Metabolic engineering of E. coli for shikimate production
● Evaluation of the engineered strains showed - increased shikimate production - decrease byproduct formation
Metabolic engineering of E. coli for shikimate production
● Overexpression of three critical genes - mutated aroG for DAHP synthase
Metabolic engineering of E. coli for shikimate production
● Overexpression of three critical genes - mutated aroG for DAHP synthase
- tktA encoding transketolase I
- ppsA for phosphoenolpyruvate synthase
● Expression by using a low-copy-number plasmid
Enhanced shikimate production during 27 hours shake flask experiments
Enhanced shikimate production during 50 hours bioreactor experiments
● Fed batch process using a 7 L fermenter
● Optimizing process by maintaining a constant glucose concentration as well as the dissolved oxygen level
Conclusion
● Elimination of specific genes increased shikimate accumulation and resulted in byproduct formation at lower concentrations
● Expression of critical genes improved production
● Yield of 0.29 g/g glucose still lower than the maximum theoretical yield of 0.83 g/g under optimal conditions
● Shikimate production level still lower than the previously reported
→ productivity could be further enhanced trough metabolic engineering and process optimization
Literature
● Xianzhong et al., Metabolic engineering of Escherichia coli for improving shikimatesynthesis from glucose, Bioresource Technology, 2014
● Ghosh et al., Production of shikimic acid, Biotechnol. Adv., 2012
● Escalante et al., Metabolic engineering for the production of shikimic acid in an evolved Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system, Microb. Cell Fac., 2010
● Flores et al., Growth Recovery on Glucose under AerobicConditions of anEscherichia coliStrain Carryinga Phosphoenolpyruvate:CarbohydratePhosphotransferase System Deletion byInactivatingarcAand Overexpressing the GenesCoding for Glucokinase and Galactose Permease, J. Mol. Microbiol. Biotechnol., 2007
● Flores et al., Pathway Engineering for the Production of Armatic Compounds in Escherichia Coli, J. Mol.Microbiol. Biotechnol., 1996
● Krämer et al., Metabolic engineering for microbial production of shikimic acid, Metabol. Eng., 2003
● Herrmann et al., The shikimate pathway, Annu. Rev. Plant Biol., 1999
● Inaoko et al., Glucose Uptake Pathway-Specific Regulation of Synthesis of Neotrehalosadiamine, a Novel Autoinducer Produced in Bacillus subtilis, J. Bacteriol, 2007
Thank you for your attention!
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