Novel Engineering Strategies To Improve The Cofactors Imbalance In Cyanobacteria

In recent years,great progress have been made in chemicals production from carbon dioxide in cyanobacteria.So far,more than 30 kinds of chemicals have been synthesized in cyanobacteria,but the yield of the target chemicals is basically at the level of mg/L(mmol/L),and the fermentation period is up to 30 days for some chemicals production.The productivity of the same chemicals in cyanobacteria is nearly 100 times lower than that of microbial cell factories such as E.coli and yeast.The syntheses of most chemicals require the participation of oxidoreductases.Because the reaction catalyzed by oxidoreductases usually utilize NAD(P)H as the cofactors,the insufficient supply of NAD(P)H is often the rate-limiting step of chemicals procdution in bacteria.As the metabolic reactions and mechanism of metabolic regulation between heterotrophic microorganisms and autotrophic microorganisms are different,the strategies of metabolic engineering used in heterotrophic microorganisms are not totally applicable in autotrophic microorganisms such as cyanobacteria.In heterotrophic microorganisms,the content of NADH is far more abun dant than NADPH.NADH-dependent dehydrogenases functions well in these cells.However,in most autotrophic microorganisms,the content of NADPH is far more abundant than NADH,NADH-dependent dehydrogenases are faced with the problem of insufficient supply of NADH in these cells.Cyanobacteria are photoautotrophic microorganisms,and the content of NADPH in cells is far more abundant than NADH.However,most of the dehydrogenases in nature are NADH-dependent dehydrogenases.The physiological environment with a low ratio of NADH/NADPH limits the application of NADH dependent dehydrogenase in cyanobacteria.In previous researchs,researchers tried different strategies to solve the problem of cofactors supply,such as screening a NADPH-dependent dehydrogenase in order to directly utilize the NADPH in cells or expressing a soluble transhydrogenase that capable of converting NAPDH into NADH.However,the enzymatic activities of NADPH-dependent dehydrogenases obtained by mutagenesis are low.Moreover,both of these two methods require the consumption of NADPH in cyanobacteria.NADPH is mainly used to fix CO2 in calvin cycle.Overconsumption of NADPH will affect the fixation of CO2 and the growth rate of cells.Therefore,in this work,in order to meet the demand of cofactor NADH in the process of cyanobacterial chemical synthesis without affecting photosynthetic carbon fixation,cyanobacterium,Synechococcus elongatus UTEX 2973(Syn2973)is used as the research object,and the biosynthesis of D-lactic acid consuming NADH is uesd as the model.We develops two strategies to solve the problem of insufficient supply of NADH in the synthesis of chemicals in cyanobacteria.The first strategy is to design and construct a NADPH dependent D-lactate dehydrogenase,which can efficiently utilize both NADH and NADPH through rational design.The second strategy is to express an electron transport protein OmcS,which capable of channeling the electrons from the photosynthetic electron transport chain into the respiratory electron transport chain to support the synthesis of ATP,so as to save the consumption of NADH in the respiratory chain and finally increase the content of intracellular NADH.Photosynthesis is consist of light reaction and dark reaction.It is generally believed that the dark reaction in cyanobacteria is the rate-limiting step of photosynthesis.Under the condition of high light intensity,extra photosynthetic electrons produced by the photosynthetic systems will be consumed by some photoprotective mechanisms,such as thermal dissipation and photorespiration to protect the photosynthetic systems from photo damage and photoinhibition.It is calculated that only about 30%of the absorbed energy can be captured by algae and stored in the forms of ATP and NADPH,and nearly 70%of the energy is lost in the ways of light reflection,heat and fluorescence etc.If part of these lost light energy can be reused to drive the synthesis of ATP in the respiratory chain,it will help save the consumption of NADH in the respiratory electron transport chain,and improve the efficiency of light energy utilization.Therefore,we expressed the electron transport protein(OmcS)from the Geobacter sulfurreducens in Syn2973 to realize that channeling the photosynthetic electrons from the photosynthetic electron transport chain into the respiratory electron transport chain.The final results showed that the constructed NADPH-dependent D-lactate dehydrogenase(D-LDH*)was an enzyme that can efficiently utilize both NADH and NADPH as cofactors.Compaired with the control D-LDH,the specific enzymatic activity of D-LDH*to NADPH increased 62-fold,and it's worth noting that the specific enzymatic activity of D-LDH*to NADH did not decrease,but increase by 28%.In the D-lactic acid fermentation,the production of D-lactic acid in the mutant strain DH5?(D-LDH*)was 38.5%higher than that of the control strain DH5?(D-LDH).Moreover,D-LDH*will function better in NADPH rich cells for that the specific enzymatic activity of D-LDH*to NADPH is twice as that of D-LDH*to NADH.The second strategy is to express the electron transfer protein OmcS in Syn2973.Comparied with the control strain Syn2973-Ldh?GlcD1 and Syn2973-Ldh?Nbla,the rate of oxygen evolution,relative electron transfer rate(rETR)and actual quantum efficiency(Y)of the mutant strains Syn2973-OmcSLdh and Syn2973-OmcS carring the gene omcS were significantly enhanced.Besides,the production of D-lactic acid increased by 58.1%in the strain Syn2973-OmcSLdh,and the intracellular contents of NADH and ATP in Syn2973-OmcSLdh were much higher than that of Syn2973-?GlcD1Ldh.These results clearly showed that the introduction of OmcS can not only provided more NADH for the production of D-lactic acid,but also improved the photosynthesis efficiency in Syn2973.To solve the problem of insufficient supply of cofactor NADH in cyanobacteria,two novel strategies were developed.The strategy of introducing extra photosynthetic electrons into the respiratory chain not only benefits to maintaining the NADH balance in the process of cyanobacterial chemical synthesis,but also contributes to photosynthetic carbon fixation.These strategies provide new ideas for the synthesis of NADH-dependent chemicals and the research of photosynthesis in the photoautotrophic microorganisms and higher plants.