Enhancing Malic Acid Yield In Escherichia Coli MG1655 Using A Synthetic Scaffold Protein Complex

Malic acid,a kind of tetra-dicarboxylic acids,is widely used in food,chemical,pharmaceutical and agricultural fields.As an intermediate metabolite of TCA cycle,L-malic acid is one of the most promising renewable platform compounds.Compared with chemical synthesis and enzyme catalysis,microbial fermentation can not only obtain single L-malic acid,but also has the characteristics of mild reaction conditions,economic and environment friendly.High yield genetic engineering strains of L-malic acid constructed by gene editing or overexpression of multiple genes often face the problems of rapid diffusion and degradation of intermediate metabolites and low substrate utilization.Therefore,,a protein scaffold RIAD-RIDD was used in this study to achieve co-locating enzymes of L-malic acid metabolism pathway and form substrate channels in multi-enzyme complexes,so as to improve the conversion efficiency of the key enzymes and the yield of target products.The specific research contents are as follows:(1)Functional verification of RIAD-RIDD protein scaffolding: The fluorescent gene reporting systems RIAD-sf GFP and RIDD-Mcherry containing protein scaffoldings were constructed respectively.With bimolecular fluorescence complementary technology,the fused expression RIAD-sfGFP and RIDD-Mcherry were separated and purified and co-incubated,and the binding of target proteins was determined by observing the fluorescence color.Then,the separate RIAD-sf GFP and RIDD-Mcherry proteins were used as the control groups,and the protein size after mixed incubation was detected by Native-PAGE to further determine the binding status of the target protein.Fluorescence detection results showed that RIAD-sf GFP and RIDD-Mcherry proteins were green and red,respectively,and orange after co-incubation.Native-PAGE electrophoresis results showed that RIAD-sf GFP and RIDD-Mcherry proteins could be anchored together with the help of protein scaffolds,suggesting that RIAD-RIDD protein scaffolds could achieve self-assembly of target proteins in vitro.(2)The assembly of key metabolic node enzymes with protein scaffolds and optimization of induced expression conditions: RIAD-RIDD protein scaffolding was used to assemble pyruvate kinase(Pyk F)and malate enzyme(mae B)in the one-step synthesis of L-malic acid pathway to construct PyKF-RIAD and RIDD-mae B.In Escherichia coli,mae B can catalyze the bidirectional reaction between malic acid and pyruvate,but limited by enzyme kinetics,mae B catalyzes the conversion of malic acid to pyruvate much more than the reverse reaction.Co-localization of the two enzymes to form an enzyme complex is conducive to the accumulation of high concentration of metabolic intermediate pyruvate,thus achieving the reverse catalysis of malate(mae B),and thus improving the yield of L-malate in E.coli MG1655.Combined with the protein expression and fermentation conditions,the induction temperature and IPTG concentration of the fusion proteins Py KF-RIAD and RIDD-mae B were determined to be 30℃ and 0.2 m M,respectively.The expression of scaffold protein was purified by nickel column,and the binding of scaffold protein was detected by Native-PAGE.The results showed that Pykf and mae B could achieve intracellular co-localization by scaffold RIAD and RIDD.(3)Analysis of acid production by fermentation of engineering strain: After optimization of culture temperature and IPTG concentration,the recombinant strain MG7(Py KF-RIAD-RIDD-mae B)accumulated 2.4012 g malic acid after 48 h of aerobic fermentation.The control recombinant strain MG6(PyKF-RIAD+RIDD-mae B),which co-expressed Pykf and mae B free,only accumulated 0.4886 g malic acid,while MG1 in the blank control group barely accumulated malic acid,indicating that the co-localization of Pykf and mae B into enzyme complex through protein scaffold could prevent the diffusion of pyruvate.Thus,malate enzyme mainly catalyzed pyruvate to synthesize malic acid.At the same time,the growth rate and bacterial density of strains containing recombinant scaffold protein were higher than those of the control group,and scaffold tag proteins RIAD and RIDD had no effect on the activity of target protein.(4)Protein scaffold system involving transmembrane transporters: Both synthesis and transmembrane transport of malic acid affect the yield of malic acid.Therefore,after realizing the co-localization of cytoplasmic protein Pykf and mae B with protein scaffold,another metabolic pathway involving transmembrane transporter was explored.Fumaridase(Fumc)in the cytoplasm and tetracarboxylic acid transporter(Dc Uc)in the cell membrane were assembled to transport the intracellular L-malic acid to extracellular and remove the product feedback inhibition effect.The experimental results showed that: When fum C-RIAD-RIDD-Dc Uc was constructed in wild-type E.coli MG1655,no significant increase in malic acid production was detected.When Co-located bidirectional catalysis of Fum C and transport protein Dc Uc(fumaric to malic)was no significant effect on malic acid production because there was no accumulation of malic acid in wild-type E.coli MG1655.In this study,protein scaffold RIAD-RIDD was used to co-locate key enzymes in the L-malic acid metabolic pathway,so as to improve the yield of L-malic acid,which has certain reference significance for the construction of high-yield L-malic acid genetic engineering strains through metabolic engineering.