CRISPR-Microfluidics Screen For Cellular Determinants Affecting The Production And Secretion Of Recombinant Proteins

The microbial protein cell factory mainly adopts the construction process combining expression element optimization and chassis strain engineering.However,due to the multi-layer network structure and complexity of biological systems,especially the lack of systematic understanding of the mechanism of unknown loci in the genome affecting the synthesis and secretion of recombinant proteins,the chassis strains engineering is still mainly dependent on"trial and error"methods,resulting in long development cycle,high cost and low production efficiency.Therefore,how to quickly and efficiently mine unknown associated genes within the genome is an important direction to improve the efficiency of designing microbial protein cell factories.To address this,our study proposed the"CRISPR-microfluidic screen"strategy.We combined CRISPRi for trackable genetic perturbations with a droplet microfluidics system for high-throughput phenotypic screening to systematically identify cellular determinants that limit the production of secreted proteins from the entirety of the C.glutamicum genome.The following are the particular research contents:（1）Based on the FlAsH-tetracysteine assay,we created an enzyme-independent approach for assessing the yield of secreted proteins in picoliter droplets.And the high-throughput screening platform of"Generation-offline culture-substrate injection-offline incubation-droplet sorting"was designed,with a throughput of>10⁵ single-cell/day.（2）The all-in-one CRISPRi system was built and used to screen for genes linked with the activity of the ethanol-induced promoter P_A256,indicating that transcriptional activation of this promoter is caused by ethanol metabolism entering the TCA cycle.The first genome-wide CRISPRi library of Corynebacterium glutamate was designed and constructed,containing46,549 sg RNAs to perturb 99.71%protein-coding genes and 85.36%non-coding genes.More than 0.5 million single cells in the library were screened by droplet microfluidic platform with the nanobody VHH as the model secretory protein,and the strains with significantly increased VHH production were successfully enriched.（3）Genotype-phenotype mapping of protein secretion in Corynebacterium glutamate was generated by NGS analysis of sorting libraries,revealing a large number of beneficial targets with functions in various biological processes,including redox regulation,amino acid metabolism and ABC transporters;among them,24 effective gene targets were demonstrated by reconstruction experiments and phenotypic performance evaluation,in which transcriptional inhibition of 8 amino acid metabolism-related genes increased VHH yield by 8.8%～48.1%,and that of 8 ABC transporter related genes increased VHH yield by 4.3～48.1%;redox-transcription factors were proved to be important factors affecting protein production;through engineering on CosR and RshA,we constructed a chassis strain with 2.78-fold increase in VHH production,and it was used to efficiently secrete collagen and scFV.