Synthesis Of Alginate Oligosaccharide And PHA_MCL By Pseudomonas Mendocina NK-01and Its Metabolic Pathway Modification

In this study, a strain was isolated from farmland soil which cansynthesize medium-chain-length polyhydroxyalkanoate (PHA_MCL) andalginate oligosaccharides （AO） simultaneously from glucose. It wasidentified to belong to Pseudomonas mendocina species using BIOLOGsystem and16S rDNA sequence identification and denominated asPseudomonas mendocina NK-01. Structure identification and physicalpropersities characterization of the two products were carried out. ThePHA_MCL biosynthesis pathway was blockaded via gene knock out. Theyield of AO was improved and the relationship of AO and PHA_MCL in themetabolic system was investigated. Finally, the substrate specificity ofPHA_MCL synthases PhaC1and PhaC2was investigated.When Pseudomonas mendocina NK-01was cultivated in a200Lfermentor using glucose as carbon source,0.316g L-1m PHA_MCL and 0.57gL-1AO were obtained at the end of the process. GC/MS was usedto characterize the PHA_MCL, which was found to be a polymer mainlyconsisting of3HO （3-hydroxyoctanoate） and3HD （3-hydroxydecanoate）.Tmand Tgvalues for the PHA_MCL were51.03°C and-41.21°C,respectively, by DSC. Its decomposition temperature was about300°C.The elongation at break was700%under12MPa stress. MS and GPCwere also carried out to characterize the AO which had weight averagemolecular weights of1546and1029Da, respectively, for the two maincomponents at the end of the fermentation process. MS analysisrevealed that the AO were consisted of β-D-mannuronic acid and/orα-L-guluronic acid, and the β-D-mannuronic acid and/or α-L-guluronicacid residues were partially acetylated at position C2or C3. It wasrevealed that the weight average molecule weight （Mw） of the twocomponents of AO were1546and1029Da respectively using Gelpermeation chromatography （GPC）. Pseudomonas mendocina NK-01can simultaneously synthesizePHA_MCL and AO from glucose under conditions of limited nitrogen.Therefore, it was proposed that AO production would be promoted if thePHA_MCL synthesis pathway was blockaded. In this study, the PHA_MCL synthase （type II） operon was cloned via polymerase chain reaction（PCR）. The operon （4.5kb） was composed with two PHA_MCL synthasegenes （about1.7kb each） and one PHA_MCL depolymerase gene （0.85kb）.The three genes, phaC1, phaZ and phaC2, were deposited on GenBank（NCBI） with accession numbers of DQ316602.1, HM585027.1andEU580408.1. The PHA_MCL synthesis pathway was blocked by a deletionof approximately57%of the sequence of PHA synthase operon mediatedby the suicide plasmid, pEX18TcC1ZC2Amp. Deletion of the PHAsynthase operon in P. mendocina NK-01was confirmed by PCR andantibiotic resistance assays to form the gene knockout mutant, P.mendocina C7. Shake-flask and30L fermentor cultures of P. mendocina C7showed a2.21-fold and2.64-fold accumulation of AO from glucose,respectively, compared with the wild-type strain. Mass spectrometry （MS）and GPC characterization revealed that P. mendocina C7and P.mendocina NK-01produced AO were identical in terms of monomercomposition and MW. Thus, the mutant P. mendocina C7has potential usein large scale fermentation of AO. Furthermore, it was demonstrated thatthe PHA_MCL and AO synthesis pathways compete for the use of carbonsources in P. mendocina NK-01. Although some report claimed thatPHA_MCL and Alginate （not AO） synthesis pathways compete for the useof carbon sources in Pseudomonas aeruginosa, it was different form ourconclusion that one of the product in our study was AO but not alginate.Due to this, it was presumed that there may be some alginate lyase in theculture broth which could degradate the secreted alginate into AO.Due to P. mendocina C7was a PHA production negative strain that itcould be used in the function investigation of PHA_MCL synthase PhaC1 and PhaC2. PHA_MCL produced by strain NK-01was found to be apolymer mainly consisting of3HO （3-hydroxyoctanoate） and3HD（3-hydroxydecanoate）. Thus, it is of importance to investigate whichsynthase works in the incorporating of monomers into PHA_MCL and theirsubstrate spe specificity. The PHA_MCL synthase genes phaC1and phaC2of Pseudomonas mendocina NK-01were cloned into expression plasmidpBBR1MCS-2to form pBBR1MCS-C1and pBBR1MCS-C2which wereexpressed respectively in the PHA_MCL-negative strain Pseudomonasmendocina C7whose PHA_MCL synthesis operon was defined knock out.The PHA_MCL produced from synthase PhaC1and PhaC2were comparedin the aspect of production rate, monomer composition, molecular weightand physical properties such as thermal properties and the degree ofcrystallinity using fermentation, GC/MS, GPC, DSC, TGA and XRD.The results demonstrated that the PHA_MCL from PhaC1had higherproduction rate to that from PhaC2but lower molecular weight. GC/MS analysis revealed that the two PHA_MCL had high similarity in monomercomposition with3HD as the favorite i.e. PhaC1and PhaC2had the samesubstrate specificity. DSC, TGA and XRD also revealed that the twoPHA_MCL had the same physical properties. P. mendocina NK-01was thefirst reported strain whose PHA_MCL synthase PhaC1and PhaC2had thesame substrate specificity. This conclusion has not been reported inpresent studies yet.