FROM INDUSTRIAL WASTE GAS TO A GREEN ADDITIVE S y n g a s t o L i q u i r i t i g e n i n NEXT SLIDE
Meet Our Team Exotic Fermentation of Flavonoid Supervisor Team Captain Team member Enrico Orsi Xing Fu Adini Arifah Green Terpene: Sustainable Master student of Biotechnology Master student of Biotechnology production of terpenes by Wageningen University Wageningen University redesigning isoprene biosynthesis 2
Milestones Fermentation 2000BC First fermentation Baking, brewing and cheese making in Egypt 1982 First consumer product from GMO Insulin for human produced by bacteria in America 2004 First PAL gene expressed in microbes The key entry enzyme that produce plant-based product expressed in yeast
Thermophilic strains Extreme PH/salty strains Syngas substrate strains FOSSILE VS. BIOBASED 4 We reaching a tipping point in traditional fermentation Biomass price increasing New energy Oil price dropping Contamination, less subsidy, more competitors...
Liquiritigenin Flavonoid Derivate Pharmacy industry Anti-cancer 1 , anti-virus (HIV) 1* Food and consumer use Natural food additives, cigarette additives Chemical industry Green and non-toxic compound
Innovation Overview From industrial waste gas to high value compound Exotic microbe to work in high Exotic pathway to fix CO 2 /H 2 Gene modification to increase flux temperature Thermoanaerobacter kivui Ability to survive in 70 ° C Able to fix CO2/H2/CO Pathway to Flavonoids ( Liquiritigenin) The pathway is working in E.coli and yeast
PATHWAY TO FLAVONOIDS 2 WLP From syngas to Acyl-CoA From Acetyl-CoA to amino acid metabolism Shikimate Pathway Produce L-tyrosine Liquiritigeni n Final pathway to final product C:H 1.2-3 maximal
INVESTMENT AND RETURN Investment 6 Fermenter: 3M*2 € Gas cleaner: 0.2M*2 € Heat exchanger: 60k*2 € DSP Centrifuge:40k € Ultrafilter:50k € Distillation column: 50k*2 € Cost and Cost of natural syngas 70 € /TCM 7 (36041mol by 65 ° C) Return 2k €/TCM 7* Adding 10% H 2 Production 17k € /TCM Assumption C:H 2 =3 5% carbon flux to product 416kg production 829g/mol 8
FINANCE RETURN Based on 15 years THE MARKET THE ANNUAL REVENUE The current production of liquiritigenin is from plant. Expected production 52t 660 € /kg (98%, China) Net revenue 33 M € PMT:5%, 15 years 20 labours DSP excluded 750 € 34M € 9
Conclusion From Plant to bacteria Big market and high value 70k ton market in China 2017 660 euro per Kg Cheap substrate and promising DSP Possible to get syngas with low price/ subsidy Precursor for flavonoid production Geno tools and yields By developing gene tools Higher yield is possible 10
ATP related questions Flavonoid making cost ATP. T. kivui use Na+ gradient power to make ATP H2 is used to generate power to build Na+ gradient Biegel, E., Schmidt, S., González, J. M., & Müller, V. (2011). Biochemistry, evolution and physiological function of the Rnf complex, a novel ion-motive electron transport complex in prokaryotes. Cellular and molecular life sciences , 68 (4), 613-634.
DSP Liquiritigenin is not soluble in cold water or methanol/ ethanol Crystal in acid liquid Wash by Crystal by Solution in methanol adding EDC Crystal in pH2 MTBE (5v/v) 98% 99.1%
Cost analysis
Further steps to increase flux Gene enzyme operation function effects Resource 2-fold of final Acyl-CoA adhE alcohol/dehydrogenase delete product 8 competing 10-fold of pathway TesB thioesterase II delete product 9 PDH* pyruvate dehydrogenase complex overexpress Malonyl-CoA PGK phosphoglycerate kinase overexpress pathway 60% increase of glyceraldehyde-3-phosphate 10 product GapA dehydrogenase overexpress Malonyl-CoA competing FumC fumarase delete pathway ACS Acyl-CoA synthase in WLP overexpress WLP pathway N/A 11
Reference 1. Ma, J., Fu, N. Y., Pang, D. B., Wu, W. Y., & Xu, A. L. (2001). Apoptosis induced by isoliquiritigenin in human gastric cancer MGC-803 cells. Planta medica , 67 (08), 754-757. 1. *Harada, S. (2005). The broad anti-viral agent glycyrrhizin directly modulates the fluidity of plasma membrane and HIV-1 envelope. Biochemical Journal , 392 (1), 191-199. 2. Metabolic engineering of microorganisms for the synthesis of plant natural products. Journal of biotechnology , 163 (2), 166-178. 3. Flavonoid biosynthesis, wikipidia, https://en.wikipedia.org/wiki/Flavonoid_biosynthesis 4. Chen, G. Q., & Jiang, X. R. (2018). Next generation industrial biotechnology based on extremophilic bacteria. Current opinion in biotechnology , 50 , 94-100. 5. Phillips, J. R., Huhnke, R. L., & Atiyeh, H. K. (2017). Syngas fermentation: a microbial conversion process of gaseous substrates to various products. Fermentation , 3 (2), 28. 6. NEO programme of SenterNovem (October 2005), Bio-ethanol from bio-syngas , TU/e 7. Pei, P., Korom, S. F., Ling, K., & Nasah, J. (2016). Cost comparison of syngas production from natural gas conversion and underground coal gasification. Mitigation and adaptation strategies for global change , 21 (4), 629-643. 7.* INDEPENDENT REPORT TO THE DUTCH GOVERNMENT- Global sustainability objectives require a technological breakthrough, http://www.h2- fuel.nl/en/h2fuel_pdf/independent-report-dutch-government/ 8. Zhou, S., Iverson, A.G., Grayburn, W.S., 2008. Engineering a native homoethanol pathway in Escherichia coli B for ethanol production. Biotechnol. Lett. 30, 335 – 342. 9. Baek, J.M., Mazumdar, S., Lee, S.W., Jung, M.Y., Lim, J.H., Seo, S.W., Jung, G.Y., Oh, M.K., 2013. Butyrate production in engineered Escherichia coli with synthetic scaffolds. Biotechnol. Bioeng. 110, 2790 – 2794. 10. Bhan, N., Xu, P., Khalidi, O., and Koffas, M. A. (2013). Redirecting carbon flux into malonyl-CoA to improve resveratrol titers: proof of concept for genetic inter- ventions predicted by OptForce computational framework. Chem. Eng. Sci. 103, 109 – 114. doi: 10.1016/j.ces.2012.10.009 11. Fast, A. G., & Papoutsakis, E. T. (2018). Functional Expression of the Clostridium ljungdahlii Acetyl-Coenzyme A Synthase in Clostridium acetobutylicum as Demonstrated by a Novel In Vivo CO Exchange Activity En Route to Heterologous Installation of a Functional Wood-Ljungdahl Pathway. Applied and environmental microbiology , 84 (7), e02307-17.
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