Combination of Metabolic Pathways for the Production of Acrylic Acid,Propionic Acid and Polyhydroxyalkanoate
Author:Liu Zhi Jie
Supervisor:liu tian zuo
As petroleum becomes scarcer and greenhouse gases accumulate rapidly, production of chemicals from microorganisms by fixing carbon dioxide have very important significance for the sustainable development and improve life quality of human beings. Escherichia coli have been widely used in industry, and produce a lot of bulk chemicals. Acrylic acid and propionic acid, which are important platform chemical, has received significant attention due to its numerous applications. In this study, Escherichia coli were developed for the production of acrylic acid and propionic acid by carbon-dioxide fixation in Escherichia coli by introducing a part of the 3-hydroxypropionate/4-hydroxybutyrate cycle from Metallosphaera sedula DSM 5348.Malonyl-CoA reductase (MCR), malonate semialdehyde reductase (MSR), 3-hydroxypropionyl-CoA synthetase (3HPCS) and 3-hydroxypropionyl-CoA dehydratase (3HPCD) from the 3-hydroxypropionate/4-hydroxybutyrate cycle of Metallosphaera sedula were heterologously expressed in Eshcherichia coli for the first time.3.04 ± 0.23 mg/L of acrylic acid was accumulated in the recombinant strain under shake flask culture condition. Further overexpressed succinyl-CoA synthetase (SCS) from E. coli, the yield was increased to 3.85 ±0.15 mg/L. The yield was increased to 3.99 ± 0.09 mg/L when blocked the acetate formation pathway. The engineered E. coli strain produced acrylic acid at 13.28 ± 0.12 mg/L in 3-L fed-batch fermentation conditions.Next, MCR, MSR,3HPCS,3HPCD and acryloyl-CoA reductase (ACR) from the 3-hydroxypropionate/4-hydroxybutyrate cycle of Metallosphaera sedula were heterologously expressed in Eshcherichia coli for the first time.143.48 ± 9.20 mg/L of propionic acid was accumulated in the recombinant strain under shake flask culture condition. Further overexpressed succinyl-CoA synthetase (SCS) from E. coli, the yield was increased to 312.41 ± 24.41 mg/L.13C labeling exprement shown that 15.1% of the fixed carbon dioxide is from air, and 84.9% is from fermentation of glucose by bacteria. The yield was increased to 335.46 ± 15.28 mg/L when blocked the acetate formation pathway. The engineered E. coli strain produced propionic acid at 1.43 ± 0.03 g/L in 3-L fed-batch fermentation conditions.Medium chain length polyhydroxyalkanoate (mcl-PHA), which is an advanced biomaterial, have important applications in biomedicine. Currently, engineered microorganisms to produce mcl-PHA is one of researching hotspots, it is of great significance that produce mcl-PHA homopolymers directly from microorganisms. The method used to produce mcl-PHA homopolymers at recently is that, use fatty acids as substrate to synthesis the corresponding chain length PHA homopolymers. However, this production method is costly and not conductive to the growth of microorganism. In order to reduce the cost, it is necessary to production of mcl-PHA homopolymers from inexpensive feedstocks. Another work of this study is that engineering fatty acid metabolic pathway of Escherichia coli to produce mcl-PHA homopolymers from glucose.First, Cinnamomum Camphora thioesterase (CCTE) was heterologously expressed in Eshcherichia coli, the recombinant strain acculuated 3-hydroxytetradecanoate. Both fatty acyl-CoA synthetase from E. coli and fatty acid CoA ligase from Pseudomonas putida can catalyze 3-hydroxytetradecanoate to form 3-hydroxyacyl-CoA in E. coli strain. Finally, both PHA synthases PhaC1 and PhaC2 can catalyze 3-hydroxyacyl-CoA to form PHA in E. coli strain. The yield of PHA homopolymer, ploy (3-hydroxytetradecanoate) (PHTD), achieved the highest that is 8.95 ± 0.34 mg/L in the recombinant E. coli strain which co-expressed CCTE, fatty acid CoA ligase and PhaC2 from Pseudomonas putida.