An Efficient Protein Degradation System In Saccharopolyspora And Saccharomonospora Based On Degradomics

Proteases(Peptidases)are specific enzymes that catalyze the hydrolysis of peptide bonds in proteins or polypeptides.Due to the huge protein sequence space,the enzymes required for hydrolysis system are complex.Therefore,Carlos Lopez-Otín put forward the concept of protease research as a unique subset in proteomics and named it "Degradomics".Relevant research focuses on the substrate-specific recognition and hydrolysis of proteases,such as the synergistic degradation of site-specific proteases like pepsin,trypsin,and chymotrypsin in the digestive system and the precise degradation of intracellular complex substrates by the ubiquitin-proteasome system.How do the limited proteases recognize and hydrolyze unlimited substrate sequences has become a significant research topic.The proteases secreted by Actinomycetes have preeminent stability and catalytic activity and play an irreplaceable role in the efficient degradation of protein biomass waste.Saccharopolyspora and Saccharomonospora appear in large numbers in the natural habitat of antibiotic residue fermentation,which have efficient protein degradation ability.Hence,this research selects the most representative strains Saccharopolyspora erythraea NRRL 2338 and Saccharomonospora viridis DSM 43017 to analyze the timing of expression of functional enzymes and the synergy action between protease components in the process of protein degradation systematically and comprehensively.Based on this research,efficient degradation enzymes can be precisely customized for specific substrates and a theoretical basis for improving the conversion efficiency and green sustainable utilization of protein biomass can be provided.In this thesis,S.erythraea and S.viridis were used as the research objects,through the degradomics technologies and structural proteomics methods such as comparative genomics,transcriptional quantitative analysis and proteomics.The degradation preference of the strains and the efficient degradation system of proteases were preliminarily clarified.The main signs of progress are as follows:1.Comparative genomic analysis was carried out on S.erythraea and S.viridis,and the types of key family proteases and their substrate specific recognition methods were analyzed.The genome of S.erythraea encodes 172 proteases belonging to 44 families and 50 subfamilies,accounting for 2.39%of the protein coding sequences of the strain.S.viridis encodes 91 proteases classified into 35 families and 39 subfamilies,with a complete range of endopeptidases and exopeptidases,which have high degradation potential for protein substrates.The proteases that can be secreted extracellular are mainly composed of serine endopeptidases of the S1-A and S8-A families,metalloendopeptidases of the M23 family,aminopeptidases of M1,M28-A and S15 families and carboxypeptidases of M14-A、M14-B and M15-A family,their genes carry signal peptides and constitute the key functional enzyme systems in the efficient protease degradation system.The substrate specificity of different families and subfamilies included in MEROPS was illuminated and the substrate sequence spectrums were drawn.Based on the difference in total entropy and conservation of substrate sequence at P4-P4’ site,the site-specific recognition pattern of each family and subfamily protease was elucidated.For example,S1-A family trypsin specifically recognizes Arg or Lys at P1;M23 family metalloendopeptidases prefer to degrade the peptide bond after Gly at P1;M28-A family specifically releases Leu,Phe and other amino acid residues at the P1 position of the N-terminal;S15 family proline dipeptidases specifically recognize Pro at P1 position;M14-B family metallocarboxypeptidases specifically hydrolyze Arg or Lys at P1’ site.Furthermore,S8-A family serine endopeptidases and M1 family aminopeptidases show a non-specific recognition pattern,and extensively hydrolyze the peptide bonds formed by hydrophobic amino acids at the P1-P1’ site.The analysis of the substrate-specific recognition patterns of proteases in different families and subfamilies provides a new idea for constructing efficient protein degradation systems.2.The sequential expression of S.erythraea proteases the specific recognition patterns of different(sub)families were clarified based on degradomic analysis;the efficient degradation and metabolism system of Saccharopolyspora proteases was elucidated.Yeast extract,maize protein powder,casein hydrolysate and gelatin,four nitrogen sources with different amino acid compositions,were selected as substrates to culture S.erythraea in shake flasks.The composition and expression timing of key protease systems were determined by quantitative transcription analysis and proteomics.It was found that the same microbial strain with different substrates has the dynamic expression rules of extracellular protease systems:Serine endopeptidases,mainly S1-A and S8-A,are firstly secreted to hydrolyze the peptide bonds formed by Arg,Lys at the P1 site of the hydrophobic amino acids at P1-P1’ sites,degrading the protein with complex sequence space into peptide segments.Secondly,the massively secreted M28-A family aminopeptidases specifically recognize amino acids such as Leu and Phe at the N-terminal of the polypeptide chain to form a smaller peptide segment and the S15 family aminopeptidases that specifically recognize Pro or Ala at the P1 position are secreted to degrade small peptides containing Pro finally.Aminopeptidases and carboxypeptidases appeared in large numbers in the later period,while M14-B family metallocarboxypeptidases specifically recognized peptides with Arg or Lys at the C-end after being cleaved by S1-A family serine endopeptidases.In addition,M7,M23 family metalloendopeptidases,M24-A family aminopeptidases and M15-A family carboxypeptidases specifically recognize Gly,Ala and other sites in the peptide chain,together with M1 family non-specific aminopeptidases,and hydrolyze the polypeptide chain into oligopeptides and amino acids.The determination of a large number of peptide transporters confirmed that S.erythraea could absorb relevant peptides and rapidly degrade and transform them through the efficient synergy of intracellular S9-A,S9-B and other family aminopeptidases,S10,S13 and other family carboxypeptidases for bacterial absorption and utilization.The proteases secreted by S.viridis mainly include S8-A,S1-A,S45,M23,C40 family endopeptidases and M1,M17,M28-A,S15,S9-C and other family exopeptidases analyzed by degradomics technology.The composition and synergistic degradation mode of the key enzyme system are consistent with the efficient proteases Idegradation system of S.erythraea.When maize protein powder rich in Gly was used as the substrate,a large amount of extracellular S45 family encoding genes were induced.The S45 family serine endopeptidases specifically recognize Gly at P1,and degrade the Gly in the peptide chain together with M23 family metalloendopeptidase,which enables the water-soluble corn gluten powder to become the most suitable substrate for the strain.Combined with Alphafold2 to model key family proteases,analyzing the size,shape and surface potential of the active architecture,the results showed that S1-A,S8-A and other family isozymes had different surface properties.The diversity of surface potentials at the active frameworks indicated that the substrates they prefer to bind were diverse and differentiated,laying a structural basis for the efficient synergistic protease degradation system.Through.comparing and analyzing the proteome data of S.rectivirgula,it preliminarily determined the generality of the Saccharopolyspora protease efficient degradation system for substrates such as globulin.3.The composition of the degradation group of various microorganisms was analyzed,and the common laws such as the composition of key families and the cooperative degradation mode in the efficient degradation system of microbial proteases were preliminarily explained.Unlike most glycoside hydrolases,which are specifically induced by different kinds of substrates,the induced expression profile of microbial extracellular proteases is almost the same.The extracellular protease secretion sequences of Bacillus sp.CN2,Streptomyces sp.F-3,S.erythraea NRRL 2338,S.rectivirgula and S.viridis DSM 43017 were compared and analyzed.The results showed that the degradation of complex globular protein substrates by microorganisms generally began with the secretion of S1-A and S8-A family serine endopeptidases,and was followed by M4 family metalloendopeptidases,M28-A family aminopeptidases that specifically recognized amino acid residues such as Leu in the polypeptide chain.S15 family proline dipeptidases were secreted in the later stage and specifically recognized Pro which was difficult to be degraded in the peptide chain.S.erythraea and is had complex protease systems that could completely degrade globular proteins.Bacillus extracellular functional enzyme system was only composed of S1-A、S8-A and M4 family members,without S15 family and other related enzymes.And a large number of peptides were still generated after degradation.In addition,M14 family metallocarboxypeptidases,M23 family metalloproteases and Ml family nonspecific aminopeptidases often appeared in actinomycetes such as S.erythraea,further synergistically degrading polypeptide fragments to form oligopeptides and amino acids.