Construction And Application Of A Self-Assembled Protein Scaffold System On Corynebacterium Glutamicum Cell Surface

Biocatalytic technology with enzymes as the core is a key field of green biological manufacturing,widely used in the synthesis of various fine chemical products.Using cheap biomass resources as starting materials to synthesize high value-added products is an important way to achieve green,efficient,and sustainable development.However,traditional biocatalytic methods cannot simultaneously meet the requirements of biocompatibility and bioregeneration of biocatalytic processes in diverse biomass raw material applications.In recent years,a novel biocatalytic method based on self-assembled protein scaffold systems on cell surfaces have received widespread attention and shown good application prospects.Therefore,selecting microorganisms with both food safety and industrial robustness as chassis cells for conducting research on surface self-assembled protein scaffold systems has important application value.This study aims to establish a self-assembled protein scaffold system on C.glutamicum ATCC13032 cells surface,combining a cross strategy of computational biology and synthetic biology,13 novel self-assembled protein scaffold sysytems were constructed on the surface of C.glutamicum cells using two pairs of genetically encoded orthogonal Spy Catcher/Spy Tag and Snoop Catcher/Snoop Tag as the main scaffold elements.Finally,their operability and application ability in different biological transformation systems were further verified.The main findings of this study are as follows:（1）Based on the mycotic acid layer of the outer membrane of C.glutamicum,a self-assembled protein scaffold mediated system composed of Spy Catcher domains（Spy Catcher domains）was first developed using Spy Catcher/Spy Tag pair that can undergo orthogonal covalent assembly.The effects of five different anchoring motifs（Por B,Por C,por H,Ncgl1337,Pgs A）on the construction of protein scaffolds containing a single Spy Catcher domain were identified through the adaptation screening of anchoring motifs,and immunocytochemistry（IF）,laser confocal microscopy（CLSM）,flow cytometry（FACS）and polyacrylamide gel electrophoresis（SDS-PAGE）were comprehensively applied.Three anchoring motifs（Por B,Por C,Ncgl1337）were obtained,which have the ability to display the Spy Catcher domain on the outer membrane of cells.The green fluorescence probe（egfp-Spy Tag）was used as a model enzyme to investigate the assembly efficiency differences between different scaffolds on the cell surface.The results showed that the unit cell fluorescence intensity using Por B as the anchoring motif was 1.2 and 1.3 times higher than that of Por C and Ncgl1337,respectively.On this basis,we investigated the impact of anchor site flux changes of the three endogenous mycoacid proteins on the self-assembly efficiency of the construction system,seven engineering strains with endogenous mycoacid layer protein deletion in different knockout combinations were constructed.The results showed that the growth and cell morphology of the seven engineering strains were slightly affected,but gene knockout would cause a decrease in the assembly efficiency of the Spy Catcher domain protein scaffold by more than 10%.Therefore,through systematic anchor protein screening and cell membrane engineering,a self-assembled protein scaffold system composed of three different Spy Catcher domains has been successfully developed.（2）In order to realize the controllable assembly of the two enzyme catalytic system on the cell surface,a chemical reaction pair Snoop Catcher/Snoop Tag was introduced based on based on Spy Catcher/Spy Tag,a multivalent co-display protein scaffold composed of Spy Catcher and Snoop Catcher domains was developed on the surface of C.glutamicum cells.Firstly,a chimeric co-display scaffold with a 1:1 ratio of Spy Catcher and Snoop Catcher domains was constructed based on the（GGGGS）₃-connector sequence,and the co-display protein scaffolds was constructed again through the adaptation screening of the anchored motif.It was found that the anchor motifs Por B and Por C were capable of displaying the chimeric scaffolds,but the function of the Spy Catcher domain in the chimeric scaffold was normal,while the function of the Snoop Catcher domain was deactivated.Subsequently,the protein prediction tool Apha Fold2-monomer was used to simulate the structure of chimeric protein scaffolds,revealing that the junction sequence（GGGGS）₃ is the key reason for the inactivation of the co-display function of chimeric protein scaffolds.Then,the connector sequence was replaced withɑ-Helix through rational design,combined with Alpha Fold2-multipler protein complex simulation technology and experimental validation,two chimeric protein scaffolds with different display capabilities were obtained.On this basis,by adjusting the proportion and sequence of the Spy Catcher and Snoop Catcher domains,a co-display protein scaffold with controllable stoichiometry was designed.The assembly ratio of the two functional enzymes on the co-display protein scaffold can reach 1:1 to 1:3.（3）In order to achieve high loading assembly of a single enzyme catalytic system on the cell surface,using synthetic biology techniques,long tandem repeat sequences containing different numbers of（1 to 6）Spy Catcher domains was designed to develop a high loading protein scaffold for directional assembly of single.The results showed that the protein scaffold containing four repetitive Spy Catcher domains had the highest efficiency in a dose-response relationship,and the binding amount of green fluorescent probes per unit cell was 1.8 times that of a single Spy Catcher domain.Subsequently,based on the site-specific recombinant enzyme Bxb1,a"single input-two state"dual-functional logic gate circuit was constructed,which realized the independent operation of two expression modes of State A and State B circuits,respectively.In order to further solve the problem of Bxb1 leakage expression and finely regulate the gene expression circuit,the Ssr A degradation tag and ribosome binding site（RBS）sequence were optimized,achieving a 29.7-times fold change for the State A circuit and a 20.3-times fold change for the State B circuit.（4）Based on different types of protein scaffolds and combining two different biomass resources as substrate materials,two scenarios of self-assembled protein scaffold systems with different functional sugars were designed to achieve stable production from maltodextrin to trehalose,and low-cost and efficient recycling preparation from molasses to isomaltulose,respectively.Firstly,based on the co-displayed protein scaffold,a dual enzyme method was designed to produce trehalose.After optimizing the conversion conditions,the conversion rate was stable at about 69.9%-71.0%in 10 dependent batches,which significantly improves the production stability compared to free enzyme catalysis.In addition,the yield of trehalose reached 215.4 g/Lwith 71.8%conversion rate in a 5 L fermentor.Subsequently,for the production of isomaltuose,the thermalstability of sucrose isomerase was enhanced through enzyme engineering,a combination mutant Pd SIV280L/S499L with improved stability was obtained.The T_m value of the mutant Pd SIV280L/S499L increased by 3.6℃.Finally,the mutant was used in a high loading protein scaffold self-assembly catalytic system,and using50%（v/v）molasses as the starting material,178.5 g/L of isomaltulose could be obtained in a single batch of catalysis,with a conversion rate of 87.3%,and stable production of 10 batches could be achieved simultaneously.