工程大肠杆菌制备羟基脂肪酸及1,3-丙二醇氧化转化反应的研究

Hydroxy fatty acids (HFAs) are important chemicals with widely application in biodegradable polymer material, chemical, food and mechanical industries. Recently, bioconversion of HFAs is receiving increased interests as the bioprocesses typically use renewable feedstock and do not generate environmental pollution. The bio-based 1,3-propanediol, also known as an important platform chemical for the production of a variety of derivatives, but its methyl esters usually made from non-renewable oil or coal resources. Recently, much attention has been paid to its bio-based production, and to its oxidative transformation into methyl esters in a green way. As a model microorganism, E. coli possesses the advantage of clear genetic background and is friendly to molecular operations. Engineered E. coli has been widely used in the production of various chemicals. In this study, we reconstructed the metabolic pathway of the host cells and got engineered strains to efficiently produce hydroxy fatty acids from glucose and exogenous fatty acids, respectively. These studies laid the foundation for the large-scale production of hydroxy fatty acids. To modify the native fatty acid synthesis metabolic pathway in E. coli , the heterologous thioesterase genes were engineered to enhance intracellular free fatty acid content; the endogenous fadD gene was knocked out to decrease the fatty acid degradation and thus to enhance the production of FFAs . Once FFAs were accumulated in the biocatalysts, different fatty acid hydroxylases were overexpressed to produce specific chain long hydroxy fatty acids and the biotransformation conditions were optimized. The finally engineered E. coli strain by knocking out the fad D gene and employing the P450 BM3 and 'TesA gave the maximal HFAs production, the hydroxy fatty acid concentration reached 117.0 mg/L under shake-flask conditions. In order to solve the problem of coenzyme NADPH regeneration during reaction, a glucose dehydrogenase (GDH) which is able to regenerate NADP + to NADPH was introduced to further improve the yield. 123.6 mg/L hydroxy fatty acid was finally obtained in shake flask. In addition, an engineered strain was reconstructed to transfer 2.5mM lauric acid as the substrate by coexpressing P450 BM3 and GDH, the conversion rate is 96.2%, and the hydroxy lauric acids reached 529.0 mg/L. As the fatty acid carbon chain extended, the bio-hydroxylation reaction reduced its efficiency. The conventional two-step route to produce methyl esters from alcohols involves an intermediate step to synthesize the carboxylic acids. However, the processes needed a rather long operation and the product recovery is low, An environmentally benign one-pot route to synthesize methyl esters through an efficient oxidative transformation of alcohols is attracting great interests, which includes oxidation (1,3-propanediol oxidized to acid) and esterification (acid and methanol esterification into corresponding methyl ester). Au/CeO 2 catalysts with different crystal CeO 2 as support were prepared by a deposition-precipitation method and tested for the 1,3-propanediol oxidative transformation. The characterization of the different catalysts was carried out by XRD, TEM, H 2 -TPR, NH 3 -TPD and CO 2 -TPD, and the results were related to the catalytic performance. The results show that the catalytic performance of the Au/CeO 2 catalysts depended on the shapes of the support CeO 2 and the acidic-basic property of Au/CeO 2 . These results suggest that the Au/CeO 2 samples are bi-functional catalysts, which possess acid/base catalytic sites and oxidative catalytic site. Their acidic-basic properties could be varied with the CeO 2 shapes. The Au/CeO 2 samples with CeO 2 nanorods and nanopolyhedra as supports contain a certain amount of the stronger acidic and basic sites with broad strength distribution. In the absence of any base, the selectivity of methyl acrylate (41.6% at 92.3% conversion) was greatly improved using the Au/CeO 2 rods ({110} and {100}) as catalysts. To be specific, methyl 3-methoxypropionate was firstly found to be one-pot synthesized from 1,3-propanediol and its maximum conversion and selectivity can be reached to 92.0% and 40.2% respectively by Au/CeO 2 rods ({111} and {100}) catalysts. The Au/CeO 2 cubes ({100}) catalysts with limited weak acidic and basic sites exhibit high selectivity towards methyl 3-hydroxypropionate and dimethyl malonate. Au/CeO 2 cube catalyst can be reused for three cycles without significant deactivation. To increase 1,3-propanediol conversion and dimethyl malonate selectivity, the influence factors such as calcination temperature of CeO 2 and Au/CeO 2 , the pressure of oxygen, reaction time and the Au loading were studied in detail. The preparation conditions were optimized and the results were related to the catalytic performance. Compared with the single catalyst, the Pd/CeO 2 and Au/CeO 2 samples can together catalyze this one-pot reaction with higher dimethyl malonate selectivity: dimethyl malonate selectivity reached 58.4% when 1,3-propanediol conversion reached 99.6%. This was the highest reported dimethyl malonate yield by the 1,3-propanediol oxidative transformation. In addition to the construction of high-yield strains for HFAs, we studied the mechanisms of 1,3-propanediol oxidation transformation, These results would be helpful to lay the foundation of these two platform chemicals.