Enhanced Itaconic Acid Production By Multi-enzyme Self-assembly And Metabolic Engineering In Escherichia Coli

Itaconic acid is a C5-dicarboxylic acid,which has broad application as an important building block for the production of plastics,polyacrylic acid,resins etc.In 2004,itaconic acid was chosen as a top 12 value-added chemical from biomass by the Department of Energy.Currently,the most commonly used method to produce itaconic acid is the fermention of polysaccharide by Aspergillus terreus.However,many disadvantages exsit in the A.terreus,including the difficulties of genetic engineering,slow growth during the fermentation and the excess oxygen demand for the itaconic acid.Thus,many researchers have focused on searching other host cells to produce itaconic acid,such as saccharomyces cerevisiae,corynebacterium glutamicum and so on.Here,the study aims to manufacture a directional and sequential self-assembly system to form prominently efficient multi-enzymatic nanoreactors involving bio-catalytically multi-enzymes and using a CRISPR-Cas9 mediated metabolic engineering strategy in E.coli to construct genetically manipulated strains to enhance IA production.The main works are listed as follows:1.Construction and self-assembly of the two-enzyme pathway for itaconic acid biosynthesisTwo enzymes of itaconic acid biosynthesis pathway in A.terreus,including aconitase(ACN)and cis-aconitate decarboxylase(CAD),were co-expressed into E.coli rosetta competent(DE3)cell with genetic engineering.Fusing a PDZ domain and its corresponding ligand(PDZlig)to the C-termini of enzymes ACN and CAD yield ACN-PDZ(APd)and CAD-PDZlig(CP1),respectively.The molecular masses of enzymes APd and CPI are determined by gel filtration chromatography(HPLC)and the results are consistent with one subunit of APd(monomer)and two subunits of CPI(dimer).The enzyme-fusion doesn't significantly disrupt the enzymatic conformations and activities.The multi-enzyme complex formed in vitro were characterized by DLS test,showing that the best assembly conditions are mixing after 30 min in the APd to CPI molar ratio of 2:1.Compared with the unassembled strain uCA,self-assembled strain sPP shows a higher catalytic efficiency in the conditions of 0.5 M substrate at pH 6.5 for 30 h.Finally,the strain sPP results in a titer of 8.7 g/L itaconic acid,which is 1.7-fold higher than that of strain uCA.These results suggest that the high local concentration of intermediate cis-aconitic acid in sPP is converted into itaconic acid more efficiently when compared to uCA,which suggest the substrate channeling in multi-enzyme complex.Notably,itaconic acid production by sPP diminished with the increasing reaction,which may be due to the inhibition of enzyme CAD by itaconic acid.2.Self-assembly and characterization of three-enzymes of itaconic acid pathway in vivo and in vitroThe cadA gene from A.terreus and gltA and acnA genes from Corynebacterium glutamicum are reconstruted with codon optimization for increasing the innitial enzymatic activities.First of all,the linear self-assembly system(LA)for identical biosynthesis enzymes is modified by fusing sole interaction proteins(PDZ domain and PDZlig),yielding CAD-PDZlig(CP1),ACN-PDZ(AP),and GA-PDZ(GP).Then the designed assembly system(DA)is construcconpated by fusing two pairs of protein-peptide interaction domains(mouse SH3 and PSD95/DlgA/Zo-1 domains)and ligands(SH3 ligand and PDZ ligand).Varying the constructive sequence of the SH3 and PDZ domains to the C-terminus or N-terminus of the GA enzyme provided diversified scaffolds for the co-localization of the multi-enzyme,which logically generated four different types of enzymes.Four assembled strains are fermented for the production of itaconic acid in MM media at 30°C and the highest titer shows a 3.84-fold increasement compared with the unassembled strain uaCGA.The assembly of three enzymes is performed by mixing modified proteins in vitro or co-expressing in vivo.The multi-enzyme nanoreactors formed in vitro is characterized by DLS,field-emission scanning electron(SEM and FE-SEM)and atomic force microscopy(AFM).The results of LA showed many nanoscale particle-like structures with sizes ranging from 30 nm to greater than 200 nm and the multi-enzyme bioreactors of DA self-assembled as highly ordered nano-scale particle-like structures ranging from 80 nm to 120 nm.The IrisFP-based three-fragment fluorescence complementation(TFFC)system is used for the visualizing the formation of the self-assembled multi-enzyme nanoreactors with PPIs in vivo.The result show that the fluorescence intensities of the fragment-fused self-assembly strain increased rapidly with time,wherein the strain expressing protein mlrisFP was taken as the control,and reached its peak values(red and green)after about 20 h,thereby indicating that the self-assembly was completed along with the protein expression in vivo.The strains LA and DA are used for the fermentation of itaconic acid in opMM media for 100 h,which result in a 1.6-fold(LA)and 3.5-fold(DA)increasment compared with uaCGA.DA performs higher catalytic efficiency than that of LA.3.Construction of genetically engineered strain with CRISPR-Cas9 based metabolic engineeringGiven that the IA biosynthesis pathway was integrated into the downstream of cis-aconitate,various genes exhibited impacts on the metabolic fluxes towards IA production,including the glyoxylate shunt pathway(aceA),TCA cycle(icd),and byproduct pathways(poxB,pflB and ldhA).The metabolic engineering method was used to further increase the productivity using a CRISPR-Cas9 system-based genomic modification tool.The fermentation results of the strains expressing LA and DA show that elevated metabolic flux towards the desired IA biosynthesis strongly enhance the IA production up to 3.06 g/L in the strain DA-A6 and significantly decreasing the concentrations of the byproducts.The highest itaconic acid yield from glucose is 0.43 mol/mol,which reveals an 612%increasement compared with the uaCGA strain.In conclusion,through various metabolic engineering,gene optimization and multi-enzyme self-assembly strategies,this study successfully constructed an engineering strain for the high production of itaconic acid.A strategy is developed to directionally control the three-enzyme self-assembly and generate highly ordered nano-bioreactors.The study provides a novel and simple method for the assembly of many important multi-enzyme cascades,especially for more than three enzymes to enhance the catalytic efficiencies with the relevant substrate channeling.