Construction And Analysis Of Cyanobacteria Chassis With Increased Flux Of Acetyl-CoA Under Photo-Mixotrophic Condition

Photosynthetic microorganisms such as cyanobacteria（blue-green algae）possess the ability to absorb solar radiation and fix carbon dioxide（CO₂）as sole energy and carbon sources,respectively.All these merits make cyanobacteria a promising alternative to produce value-added chemicals.However,the productivity must be improved before its application at industry scale.Synechococcus elongatus UTEX 2973（hereafter Synechococcus 2973）is a newly isolated strain with shorter double time,as well as the distinct advantage in tolerance to high light and high temperature,making it a potential chassis candidate for cyanobacterial cell factory.However,its intracellular acetyl-CoA content is much lower than that of other model cyanobacterial chassis,which might become one of the limiting factors to increase its productivity.Phtoto-mixotrophic cultivation is recently considered as the effective alternative to improve cell growth and chemical production in cyanobacteria,as the cyanobacteria can perform both autotrophic and heterotrophic metabolism of utilizing additional organic carbon simultaneously in photo-mixotrophic condition.However,so far,few researches are on photo-mixotrophic cultivation of Synechococcus 2973.Xylose is a major component of lignocellulose and the second most abundant sugar present in nature after glucose.However,no natural strain of cyanobacteria is known to utilize xylose.To construct photo-mixotrophic cultivation of Synechococcus2973,three xylose utilization genes（xyl EBA）from Escherichia coli were expressed in wild type strain of Synechococcus 2973.It was found that the growth rate and cell dry weight（DCW）of the engineered strain were significantly increased,and the content of acetyl-CoA was also increased by 379.2%with the addition of 6 g/L D-xylose.In addition,metabolomic analysis showed that several key metabolites in central carbon metabolism pathway,including F6P,FBP,R5P,E4P and G3P were significantly increased under photo-mixotrophic condition,especially F6P which was increased by21.5 folds.All these results indicated that the carbon metabolism and the acetyl-CoA centent in Synechococcus 2973 could be improved by photo-mixotrophic cultivation.To further increase the content of acetyl-CoA,the following approaches to redirect central carbon metabolism were taken:i)enhancing carbon flux into acetyl-CoA by expression of phosphoketolase gene（all1483）derived from Anabaena sp.PCC 7120;ii)improving the phosphoketolase substrate（F6P）supply by overexpressing the fructose diphosphatase gene（M744＿04600,fba）and knocking out the fructose phosphokinase gene（M744＿13890,pfk）.The results showed that the content of acetyl-CoA in the two engineered strains increased by 24.6%and 163.1%,respectively.Finally,the 3-hydroxypropionic acid（3-HP）biosynthetic pathway was introduced to evaluate the productivity increase of the new chassis.As expected,under the xylose-based photo-mixotrophic condition,the production of 3-HP in new chassis with rewired central carbon metabolism pathway was increased up to approximately 2 folds than that in the wild type producting strain.In this study,the construction of xylose utilization engineered strain confirmed that photo-mixotrophic cultivation with supplemental organic carbon sources could promote cell growth and productivity in Synechococcus 2973.In addition,the accumulation of acetyl-CoA and 3-HP in Synechococcus 2973 could be improved by rewiring the central carbon metabolism pathway under photo-mixotrophic condition.It worth noting that this strategy could be applied not only for the improvement of intracellular concentration of acetyl-CoA but also for the production of value-added chemicals that require acetyl-CoA as a key precursor in cyanobacterial chassis.