Screening Of Lignin-degrading Bacteria And Modification Of Metabolic Pathways Enhances The Accumulation Of PHA

Lignin has complex structure and abundant content in nature.Lignin degrading bacteria can synthesize a variety of high value-added products with lignin as substrate.However,the ability of bacteria to metabolize lignin is limited,metabolic pathways are complex,and the production of synthetic high value-added products is low.In view of the above problems,this paper has carried out the follow ing three aspects of research:1.Screening and characterization analysis of lignin degrading bacteria.40 strains of bacteria were isolated from the soil of the straw field by using]ignin as the sole carbon source.Two strains of J2 and J5 with lignin degradation ability were obtained by sieving 40 strains with Azure B and RBBR and KL-MSM plates.The lignin degradation rate of J2 and J5 within 7 days was 6.9%and 8.1%,respectively,determined by UV spectrophotometry.DMP was used as a substrate to detect the activity of different kinds of lignin degrading enzymes.The results showed that both strains produced laccase and peroxidase activity,the highest enzyme activity of two strains reached 4.4 mU/mL with the presence of H2O2 and Mn2+.The strains were identified as Pseudomonas sp.J2 and Klebsiella sp.J5 by 16S rRNA gene sequence analysis.In addition,according to the genomic information of strains that are closely related,some of the lignin degradation-related genes Lac,Glu,and MnO were cloned from J2 and J5 strains,which further demonstrated the lignin degradation ability of these strains.2.The metabolic engineering method was used to modify the lignin degradation and PHA biosynthetic pathways of P.putida KT2440 and improve the efficiency of biosynthesis of PHA using lignin.In order to facilitate genetic manipulation,the hsdR gene on KT2440 genome was knocked out by double exchange of homologous recombination,and the hsdR gene deletion mutant P.putida QSR1 was obtained.Using QSR1 as the starting strain,a PHA degrading gene phaZ deletion mutant P.putida QSRZ6 was constructed,and lignin-degrading enzymes derived from MnSOD1,MnSOD2 from DyPA,DyPB and Sphingobacterium sp.T2 of Rhodococcus jostii RHA1 were heterologous expressed in QSRZ6.Compared with the strain QSR1,the growth and PHA accumulation of QSRZ6 increased by 29%and 80%,respectively;The PHA yield of engineered strain QSRZ61(heterologously expressed MnSOD1)reached 156 mg/L,which was increased by 11%and 255%compared with QSRZ6 and QSR1;The PHA yield of engineered strain QSRZ62(heterologously expressed MnSOD2)reached 158 mg/L.which was 32%and 259%higher than QSRZ6 and QSR1.3.Construction of stable genetically engineered strains.The SOD operon,which can simultaneously express MnSOD1 and MnSOD2.was designed and integrated into the 16S rDNA site of the QSRZ6 genome,and the metabolic engineering strain P.putida QSRZ6S was obtained.QSRZ6S was cultured in 1 X M9 lignin fermentation medium containing 1.4mM ammonium sulfate that accepted 220 mg/L PHA in 48 h,which was 77%and 400%higher than that of QSRZ6 and QSR1.The results of this paper show that the rational transformation of the lignin degradation system and product accumulation system is conducive to the biotransformation of lignin and the accumulation of effective products and metabolic engineering is an effective way to control lignin transformation and PHA accumulation...