Metabolic Regulation Of CO₂ Fixation Associated With Succinate Biosynthesis

As an important function factor,succinate is widely used as food additives and pharmaceutical intermediates in food and pharmaceutical industries.Because of the high cost and serious environmental pollution,the traditional chemical synthesis based on non renewable petroleum resources inhibits the development of succinate.So non renewable petrochemical resources must be replaced by renewable biological resources.Therefore,production of succinate via a green technology such as microbial fermentation has drawn much attention.Further,CO2,a primary greenhouse gas,can be consumed during the fermentation process,which can be considered as an environmental benefit.As the essential substrate for the biosynthesis of succinic acid,CO2 is regarded as the key factor to determine the efficiency of succinic acid production.The CO2 fixed reaction from phosphoenol pyruvate to oxaloacetic acid,which is catalysed by phosphoenolpyruvate(PEP)carboxykinase(PCK)and carboxylase(PPC),has been proved to be the key step.In this study,in order to establish a balanced and efficient CO2 fixation process for improving succinic acid biosynthesis,the regulation of CO2 fixation genes was investigated at genetic level and transcriptional level,respectively.A two-step process is involved in CO2 integration:CO2 uptake into the cell and CO2 fixation by carboxylation enzymes.A dual expression system was employed,in which genes regulating both CO2 uptake and fixation were co-overexpressed,or overexpressed individually to improve succinate biosynthesis.The succinate production was significantly increased only when a CO2 fixation gene(ppc or pck)and a CO2 transport gene(sbtA or bicA)were co-expressed.Co-expression of pck and sbtA provided the best succinate production among all the strains.The highest succinate production of 73.4 g L-1 was 13.3%,66.4%or 15.0%higher than that obtained with the expression of PCK,SbtA alone,or with empty plasmids,respectively.Thus,a balanced CO2 transport and fixation process was established.The specific cell phenotype is usually regulated by multiple genes and the desired effect is hard to be obtained by simply genetic engineering.For further enhancing succinic acid production,this study was investigated by engineering the E.coli global transcription factor,Cra(catabolite repressor/activator).The amino acids at 57-62 of Cra protein was mutated into the amino acid of the LaCl/GalR family protein.The highest succinic acid production of 85.8 g/L was obtained in Arg60Glu,which was 26.0%greater than that obtained using an empty vector control and overespression of Cra.In order to significantly change the protein structure of Cra,and then affect the regulation function of Cra on several genes,regulatory domain was replaced by the regulatory domain of the family protein while the DNA binding domain and linker was maintaining to construct the chimera Cra.The highest succinic acid production of 90.7 g/L was obtained when the regulatory domain was from RbsR,which was 26.0%greater than that obtained using an empty vector control and overespression of Cra.Metabolic network of succinate biosynthesis in E.coli was constructed,and then metabolic flux of succinate biosynthesis was computed.It was found that the CO2 fixation and glyoxylate pathway was active,ultimately contributing to succinate biosynthesis.In summary,based on the fact that CO2 is responsible for determining the biosynthesis of succinic acid,this work significantly enhanced succinic acid production through combinatorial optimization of CO2 fixation.This work provides useful information for the combinatorial regulation of metabolic pathways to improve succinate production.