Development And Application Of Novel Cofactor Systems In Microorganisms

Microbial manufacturing utilizes renewable resources as raw materials and produces high-value chemicals in an environmentally friendly and sustainable manner.Currently,microbial manufacturing is widely applied in various sectors such as food,pharmaceuticals,materials,and energy,playing a significant role in achieving sustainable development.The essence of microbial production lies in the electron-driven assembly of elements,enabling material synthesis,decomposition,and energy exchange.Cofactors are important carriers of electron flow and energy transfer in cells and play an important role in microbial manufacturing.In this paper,with cofactors as the core research content,novel,and efficient microbial cofactor systems were developed using metabolic engineering and protein engineering to achieve stable and controllable synthesis of intracellular cofactors and effectively improve microbial production performance.The specific results are as follows:1.Constructing a novel 3’-phosphoadenosine-5’-phosphosulfate（PAPS）cofactor synthesis system:The compound PAPS serves as a sulfate group donor in the production of valuable sulfated compounds.However,elevated costs and low conversion efficiency limit the industrial applicability of PAPS.The novel PAPS synthesis system consists of a main enzyme module and an auxiliary module,in which the main enzyme module converts substrate ATP to PAPS using ATP sulfurylase and APS kinase.Addressing issues such as byproduct inhibition and low substrate theoretical conversion rates in the main module,an auxiliary module was developed to degrade byproducts and promote ATP regeneration,increasing the theoretical conversion rate of PAPS from 50%to 100%.APS kinase was identified as the limiting step in this catalytic system by in vitro and in vivo catalysis experiments.The key residues that restrict the activity of APS kinase were identified through the analysis of catalytic mechanism and molecular dynamics simulation.The key residues were modified to broaden the product release channel of the enzyme and increase the flexibility of the"lid structure"of the APS kinase.This resulted in a 46.39-fold increase in APS kinase catalytic activity and achieved a 98.1%conversion rate in the PAPS system.Finally,utilizing this PAPS system to provide sulfate groups,chondroitin sulfate A was catalytically produced from chondroitin,achieving a sulfation efficiency of98.71%.2.Developing a novel non-natural cofactor system:Natural redox cofactors participate in numerous complex reactions within metabolic networks,reducing predictability and controllability of metabolic regulation,thus impacting microbial manufacturing efficiency significantly.In order to reduce the interference of metabolic network to cofactor system,the non-natural cofactor nicotinamide uracil dinucleotide（NUD/NUDH）was developed using uridine triphosphate（UTP）and nicotinamide mononucleotide（NMN）as precursors.Subsequently,the NUD synthetase Hs Nmnat*was selected through the binding pocket sieve of reprogrammed nicotinamide transferase,which could efficiently synthesize NUD using UTP and NMN as substrates,and its activity reached 110.25 U/mg.The substrate specificity of phosphite dehydrogenase was further changed to obtain Ps PTDH*,to catalyze NUD to form NUDH with phosphite as electron donor,and the activity reached 102.69 U/mg.Finally,Hs Nmnat*and Ps PTDH*were introduced into E.coli,and the NUD（H）synthetic strains were constructed through metabolic engineering to enhance the supply of precursors UTP and NMN.The NUDH concentration and NUDH/NUD ratio were 7.61 m M and 51.2,respectively,which were much higher than the natural redox cofactors.3.Adaptation of non-natural cofactor systems to carbon fixation pathways:Microbial fixation and resource utilization of CO₂ is critical to achieving carbon-negative biofabrication.Based on literature mining,a simple CO₂fixation pathway was constructed and named FGPM pathway.This pathway was made up of formate dehydrogenase（FDH）,formaldehyde dehydrogenase（FADH）,glycolaldehyde synthase（GALS）,acetyl-phosphate synthase（ACPS）,phosphate acetyltransferase（PTA）,pyruvate:ferredoxin oxidoreductase（r PFOR）,and the malic enzyme（ME）,which was demonstrated to be feasible in E.coli.However,the results showed that the FGPM pathway was difficult to be driven by the natural intracellular cofactor system,and the CO₂ fixation efficiency of E.coli was only 21.63 mg/g DCW/h.To improve the preference of pathway enzymes for NUDH,we proposed a universal strategy（the IESE strategy）involving four steps to modify the steric hindrance of the NUDH-binding pocket.Step I:Identification of mutational hotspot residues.Step II:Conserved residues are excluded.Step III:Screening of mutants to reshape the steric hindrance of the NUDH-binding pocket.Step IV:Evaluate the kinetic parameters and NUDH preference of the mutants.This strategy was used to reshape the steric hindrance of the binding pockets of FDH,FADH,and ME.Finally,the mutants Ts FDH*,Bm FADH*,and Pc ME*were screened,and their NUDH preferences reached 106.62,192.20,and 87.75,respectively,which were 1.26×10⁴,2.6×10⁴ and 7.5×10³folds higher than those of the wild type,respectively.The non-natural cofactor NUD（H）system is effectively adapted to the FGPM CO₂fixation pathway.4.Metabolic application and regulation of non-natural cofactor system:The NUD（H）cofactor system was assembled with the FGPM pathway in E.coli.Then,the rate-limiting steps of FGPM pathway were optimized by protein scaffold engineering and knockout of negative regulatory proteins,so that the CO₂ fixation efficiency of E.coli reached 54.67 mg/g DCW/h.To address competition between excess NUD（H）cofactor synthesis and cell growth metabolism,a NUDH-responsive sensor Rex3**with a response range of 0-1.5 m M was developed based on the oxygen-responsive transcriptional repressor Rex.Subsequently,the sensor Rex3**was used to control the recombination or division of Tet repressor proteins to construct negative feedback gene circuits in response to NUDH.By changing the Tet O1 region of the Tet operon（ACTCTATCATTGATAGAGT）,a gene circuit with different response thresholds was developed to achieve stable and controllable synthesis of intracellular NUDH,and the biomass was increased by 34.11%compared with that before regulation.Finally,using glucose and simulated industrial exhaust gas containing 30%CO₂ as co-substrates for fermentation,the engineered strain synthesized 323.96 m M malate and fixed 379.1 m M CO₂,achieving significant fixation of CO₂ into commercially valuable malate.