Development And Optimization Of Cell Factory For Photosynthetic Synthesis Of Ethanol And Lactic Acid By Cyanobacteria

With the rapid growth of global energy demand and the increasingly serious greenhouse gas emission crisis,the development of green and sustainable energy,materials and chemical production routes has been paid more and more attention.Using photosynthetic microorganisms as a platform,photosynthetic bio-manufacturing technology can directly convert carbon dioxide and water into target chemicals by using solar energy,which can play a role in carbon sequestration and emission reduction while carrying out green synthesis.Therefore,it has become a very potential route for the synthesis of biofuel and bio-based chemicals.Cyanobacteria is the most potential microbial photosynthetic platform,with the advantages of fast growth rate,high photosynthetic efficiency,convenient genetic operation,etc,has been widely used in the research of photosynthetic synthetic biotechnology and the development of photosynthetic cell factories.Ethanol and D-lactic acid are two typical biofuels and bio-based chemicals.It is of great scientific and technical significance to synthesize ethanol and D-lactic acid by solar energy drives fixed carbon on the platform of cyanobacteria.The main content of this thesis is to study the construction and optimization strategy of cyanobacteria ethanol and D-lactic acid photosynthetic cell factory.In the first part of the work,the effects of high temperature and high light stress resistance of cyanobacteria chassis cells on the growth and product synthesis ability of photosynthetic cells were explored using ethanol synthesis as a model.Previous studies have found that the mutation of cysteine 252 on the alpha subunit of FoF1-ATP synthase in PCC7942 to one of the four conjugated amino acids,tyrosine,phenylalanine,histidine and tryptophane,can significantly improve the high temperature and high light tolerance and growth rate of cells.In this paper,the ethanol synthesis pathway composed of pyruvate decarboxylase and alcohol dehydrogenase was firstly introduced into the genome of Synechococcus PCC 7942,which realized the ethanol synthesis of this engineered cyanobacteria strains,and then the site-directed mutation(C252F)of 252 amino acids was introduced into the coding gene of F0F1-ATP synthase alpha subunit of this cyanobacteria strain,While the high temperature and high light tolerance and growth rate of the engineered cyanobacteria strain were effectively improved,the ethanol yield was also significantly increased,indicating that the optimization of physiological robustness of the cyanobacteria photosynthetic cell factory was helpful to improve the overall efficiency of the photosynthetic synthesis.In the second part of the work,the importance of matching heterogenous metabolic pathways with the photosynthetic physiological background of cyanobacteria chassis strains was explored using lactic acid synthesis as a model.Previously,the development of cyanobacteria lactic acid photosynthetic cell factory was mainly realized by introducing exogenous NADH-dependent lactate dehydrogenase.However,the characteristics of photosynthetic autotrophic metabolism of cyanobacteria determined that NADPH was the main form of reducing power in its metabolic environment,which meant that there was a risk of incompatibility between heterologous pathway and endogenous background.On the basis of chassis optimization,this study further explored the effects of introducing different cofactor-dependent(NADH/NADPH)lactate dehydrogenases on the lactic acid synthesis ability of photosynthetic cell factories.The results showed that D-lactate dehydrogenase with NADPH preference was constructed by rational design,which showed significant advantages in lactic acid synthesis compared with the starting gene for cofactor preference optimization.The research results of this paper show that,in addition to the basic strategies such as pathway introduction and expression regulation,optimizing the photosynthetic physiological background and pathway-chassis adaptability of cyanobacteria is also of great significance to the development of high-efficiency photosynthetic cell factory,which is helpful to improve the overall efficiency of the photosynthetic biological manufacturing process of cyanobacteria.