Biological Production Of 1,3-Propanediol And 2-Phenylethanol By Metabolic Engineering

Alcohols are a group of organic compounds,which are composed of fatty alcohols,alicyclic alcohols and aromatic alcohols.They have a wide range of appealing applications in food,medical,energy,chemical and material industries.Due to the increasing consumption of non-renewable fossil resources and the environmental concerns,the development and utilization of renewable resources for sustainable production have drawn more and more attention.In addition,generating biofuels and chemicals from some inexpensive materials such as crude glycerol,lignin,or CO₂ have drawn much more attention with the advancements of metabolic engineering and synthetic biology.This thesis focused on the microbiological synthesis of 1,3-propanediol and 2-phenylethanol,and the following studies were carried out.Firstly,a more efficient cell factory was constructed by means of systematic metabolic engineering to improve the biological production of 1,3-propanediol.Klebsiella is a gram-negative bacterium,belonging to the enterobacteriaceae,which is widely used in industrial production.However,there are still some problems in the existing 1,3-propanediol biosynthetic methods of Klebsiella,such as low product concentration and low substrate utilization efficiency.Based on the problems mentioned above,a K.oxytoca strain PDL-0 was metabolically engineered by applying a systematic approach to driving the carbon flux of oxidative pathway of glycerol to1,3-propanediol synthesis.The biosynthetic pathways were blocked progressively.Combined with cofactor engineering and cellular respiratory rate improvement,the 1,3-propanediol concentration reached 74.9 g L^-1,which was approximately 107.5%higher than that of wide-type strain;and the total concentration of by-products was approximately 42.8%lower than that of wide-type strain.Secondly,we proposed a strategy for solving the incompatibility in synthetic biology by using the natural compartmentalization.Cyanobacteria have received more and more attention because they can directly convert solar energy and carbon dioxide into chemicals.However,the incompatibility between the introduced heterogeneous cassettes and cyanobacteria hosts limited the production of the target chemical,such as oxygen-sensitivity,cofactor balance and metabolites tolerance.1,3-Propanediol is an important chemical which was widely used for the synthesis of novel polyesters?such as polytrimethylene terephthalate,PTT?,cosmetics,lubricants,and drugs.However,in the study of 1,3-propanediol photosynthetic production,the key enzyme of the biological synthetic pathway?glycerol dehydrase,GDHt?is sensitive to oxygen,and the incompatibility of oxygen-sensitivity limits the photosynthetic production of 1,3-propanediol.To bypass the obstacle of oxygen-sensitivity in cyanobacteria,we selected1,3-propanediol as the target compound,using the spatial separation of natural cell differentiation?heterocysts?formed by Anabaena sp.PCC7120 to solve the incompatibility of oxygen-sensitivity of 1.3-propanediol photoproduction.We constructed two1,3-propanediol production cassettes,cassette KP containing genes from facultative anaerobe Klebsiella pneumoniae and cassette CB containing genes from strictly anaerobic bacterium Clostridium butyricum.Then the synthetic cassettes were integrated into the chromosome of Anabaena sp.strain PCC7120 by homologous recombination,respectively.Thereafter we investigated the 1,3-propanediol production characteristics of all the engineered strians?P11,P12,P13,and P14?with or without heterocysts.As expected,when heterocysts were present,the amount of 1,3-propanediol accumulation by the engineered Anabaena sp.PCC7120 strain P11 was 46mg L^-1,approximately 1.7-fold higher than that without the presence of heterocysts.Moreover,as for the strains?P12,P13,and P14?containing cassette CB,the product1,3-propanediol could only be detected when heterocysts were present.These results demonstrate that using the spatial separation of Anabaena sp.PCC7120 heterocysts has realized the functional assembling of the oxygen-sensitive cassettes of 1,3-propanediol production and 1,3-propanediol photosynthesis.The oxygen-free microenvironment formed by heterocysts is effective for keeping the activity of the oxygen-sensitive 1,3-propanediol synthetic cassette.The strategy that using natural spatial separation to overcome the incompatibility of oxygen-sensitivity in photosynthesis may have the potential of serving as a universal strategy to this challenge.Thirdly,this study is the light-driven photosynthesis of 2-phenylethanol based on the plasticity of metabolic pathways and photosynthetic chain.Due to the limited photosynthetic efficiency and metabolic rigidity,most target products select the central metabolites close to carbon fixation as the starting points for synthesis.Recently,a few attempts have been made to redirect carbon flux to low-flux pathways for the synthesis of target chemicals directly from CO₂.However,there are still many other low-flux pathways involved in the synthesis of numerous valuable chemicals synthesis which remain largely unexploited,such as shikimate pathway.Shikimate pathway is a metabolic bridge between central carbon metabolism and aromatic amino acids?AAAs?network in plants and microbes.Thus,a model aromatic compound derived from shikimate pathway,2-phenylethanol,was chosen as the target product to investigate the plasticity of shikimate pathway in cyanobacteria.We constructed two artificial biosynthetic pathways of 2-phenylethanol which were respectively introduced into cyanobacteria model strain Synechococcus elongatus PCC7942.The engineered strains realize the production of 2-phenylethanol from CO₂ and solar energy.Then,a feedback-inhibition-resistant cassette?FBR cassette?was constructed and introduced into the engineered strains.As a result,the final engineered strain P120 have been demonstrated to accumulate 2-PE to 119.5 mg L^-1.Compared with the wild-type Synechococcus elongatus PCC7942,the specific rate of carbon fixation and O₂ evolution were respectively increased by 68.7%and 29.9%,and more than 30%of photosynthetically fixed CO₂ was channeled towards aromatic compounds synthsis in strain P120.Assays of transcriptional levels demonstrated that the introduction of FBR cassette led to the redirection of metabolic flux and remodeling of photosynthetic chain.This discovery provides a new technique for the production of aromatic compounds and guidance for the improvement of plant photosynthetic efficiency and the design targets of artificial photosynthetic system.1,3-Propanediol,2-phenylethanol and their derivatives are widely used in food,medical,energy and material industries,which play important physiological role in microbial metabolism.This thesis focused on the utilization of CO₂ or glycerol for bio-production by metabolic engineering of microorganisms.We selected 1,3-propanediol and 2-phenylethanol as the target chemicals to study the incompatibility and the plasticity of target pathways in engineered strains.In addition,the results and strategies used in this thesis will provide guidance for the bio-production of alcohols by metabolic engineering and synthetic biology.