Metabolic Engineering For Poly(3-mercaptopropionate) Homopolymer Production In Recombinant Advenella Mimigardefordensis

PTE (polythioester) is a kind of polymer containing sulfur in its backbone which are artificially transformed. Poly(3-mercaptopropionate)(PMP) is one kind of PTE. It has similar structure with its analogues, polyoxoesters. The only difference is the substitution of oxygen with sulfur in the chain backbone. Because of this alteration, this kind of polymer shows valuable characters, such as higher melting points, slower solubility and increased heat stability. Whereas the most attracting property is its non-biodegradability. This endows PTE plenty of potentials. In the foreseeable future, as the nonrenewable fossil energy consuming, more and more polymer products should be replaced by proper substitutes. But not all non-biodegardable polymers from fossil oil could be replaced by the biodegradable substitutes from renewable sources as we normally thought. Modern construction of houses, automobiles and other major developments would be unthinkable and impractical without the availability of persistent polymers. In considering of these industrial demands, the production of PTE from renewable feedstock by biotechnical method makes sense.The chemical synthesis of PTE has already been reported in1951. A lot of other chemical methods to synthesize PTE have also been reported ever since. But all these methods have not been technically produced and commercialized due to difficult preparations, high costs and low yields from laborious synthesis. The key point is that its synthesis still needs petroleum base feedstock which is still in the influence of fossil oil. One recombinant E. coli strain which harbors the non-natural BPEC pathway was successfully used to produce PMP. It contains the following recombining enzymes:butyrate kinase (Buk) and phosphotransbutyrylase (Ptb) from Clostridium acetobutylicum in addition to the PHA synthase from Thiocapsa pfennigii, PhaEC which is recognized as BPEC pathway. This PMP material has sole monomer and it has higher degree of polymerization. But this strain could not use low toxic TDP or DTDP to produce PMP which makes this method hard to be industrially promoted.To find one strain which could use the low toxic and more stable precursor DTDP, our team screened plenty of strains in large scale. Finally, some strains that could use DTDP as the sole carbon source to grow were shown. One new identified strain A. mimigardefordensis DPN7T attracted our interests. After Tn5mutagenesis screening and relevant enzymes assay, a clear DTDP degradation pathway was figured out. The whole genome sequence has been got and the detailed annotation was processed. The first step of this degradation pathway is at the help of LpdA to form3MP which is just the start precursor for PMP production.Basing on these facts, the first option is to use DTDP degradation strain A. mimigardfordensis DPN7T to produce PMP. First, we find a proper vector pBBR1MCS5which could be autonomously replicated in this strain. The entire BPEC pathway is inserted into the vector pBBR1MCS5and mobilized into the strain. However, the recombinant vector could not stably replicate in this strain SHX1. Finally, the plasmid structural instability is found to be related with the buk-ptb operon which could lead to growth repression of this strain. One gene deletion or gene exchange method is established basing on the suicide vector by using of allelic exchange. The integration of buk-ptb operon into the genome relieve the repression affection and stable the plasmid structure in the cell. Mdo is the second enzyme in the DTDP degradation pathway which could catalyze3MP into3SP and decrease the3MP amount in the cell, so it is thought as one key factor to affect PMP accumulation in the cells. Unfortunately, it is not possible to use mdo::Tn5mutant to carry out this strategy due to the transposition events of Tn5transposon in A. mimigardefordensis strains. In some cases, the plasmid size in our strain SHX1is observed in bigger size due to this affection. So the mdo gene is deleted instead of mdo::Tn5mutation in the same method. The PMP production is increased further after the mdo deletion procedure. Gluconate is one common used carbon source for cultivation of A. mimigardefordensis strains. But our results proves that it is not suitable for PMP production, so succinate is chosen instead. It improved the cell growth and the PMP content in the cells synchronously. After all these metabolic engineering modifications, the strain SHX5could produce PMP in5%-6%repeatedly and stably. It ultimately verified that the method to modified A. mimigardefordesis DPN7T for PMP production is feasible.Although we have achieved so much achievements, the production ability of this strain which was around5%is still low and the utilization of costly succinate as carbon source is also limited. To further improve the PMP production and decrease the cost, more different carbon sources, the cultivation styles and the changes of different components along the time course in the medium were all tested. Although cells in glycerol grew slower, it could help the strains to accumulate similar biomass with double PMP content comparing with succinate. It was finally chosen as one cheaper alternative carbon source for PMP production. The addition of propionate increased the polymer production to around18%and recovered the growth of cells. Whereas few3HB and3HV monomers were also incorporated into this polymer (less than10%). The analysis of this polymer composition according to time scale indicated that the appearance of three different kinds of monomers were almost at the same time. It was hard to get rid of the3HB and3HV monomers by changing the cultivation methods.The supernatant analysis demonstrated the increasing of3MP in the medium. It expelled it from one limited factor. The PMP production pathway itself was locked as the critical point for further PMP production. The previous data also implied that the strain itself contained one inherent PMP production pathway which is independent of the BPEC pathway. After genome blast analysis by using a phaC database, its own phaC gene which encodes PhaC Am as the isozyme of PhaEC was revealed. After the comparison of these two different genes encoding PHA synthase, the PHA synthase encoded by its own phaC was proved as the only functional enzyme. PhaEC was proved to be deficient although their transcripts were positive by reverse-transcript PCR assay. The buk-ptb operon was proved to improve the PMP production in the same experiment. Three different origins of PHA synthase were compared afterward and its own PhaCAm was proved as the most efficient. The affection of lac promoter was excluded at the same time. PHA synthase encoded by phaCl from R. eutropha H16also showed its activity to some extent.The improved expression of phaC in the cells increased the PMP production further. And the optimal phaC expression amount was defined. The best PMP production strain SHX22was got finally. To further increase the PMP content, the components in the medium were analyzed according to time scale,'relative surplus'of3MP was thought as one key factor. More experiments proved that the3MP molecule could only be exported and could not be imported by the A. mimigardefordensis strains. It was thought to be the next obstacle for further PMP production. Three different solutions were proposed afterwards. The decrease of DTDP concentration was proved not applicable at last. The second strategy was to find a proper enzyme that could accelerate the catalysis speed from3MP to3MP-CoA. For that purpose, a candidate enzyme was proved to be active for PMP accumulation in E. coli strain. Further attempts are arranged at this moment.Through the modification of PMP purification method, one method was established to purify PMP polymer from this strain. After GC/MS analysis, the PMP was confirmed as homo-polymer. One proper native PMP granules purification method was developed and its protein components were also analyzed by SDS-PAGE which could do great benefit for further research.PMP materials is one new valuable artificial polymer which could be produced through biotechnical method. However its production is limited to the high toxic precursor3MP. Thus the PMP production through lower toxic DTDP is more practical. Through the construction and optimization of BPEC pathway in a DTDP degradation strain A. mimigardedfordensis DPN7T and a serial of fermentation condition optimizations, the native strain which could not accumulate any PMP could accumulated up to10%PMP in its cells after these modifications. Soon after, its own native PMP production pathway was revealed and the key enzyme phaCAm was identified and its expression was optimized. The PMP production in the cells could arrived to around20%with the highest amount to25%. The purification and identification of this PMP material was studied basing on this system. This work achieved the purpose that the PMP was accumulated by using of low toxic DTDP. A new PMP production pathway was constructed in a new developed strain A. mimigardefordensis. It offers a new research thought and potentials of industrialization for biotechnical PMP production.