Biosynthesis Of 3-hydroxypropionic Acid And 3-hydroxybutyric Acid Copolymers Produced By Ralstonia Eutropha

Polyhydroxyalkanoate is a natural biopolyester that has a wide range of applications in the agricultural,pharmaceutical,food and materials industries due to its good biocompatibility and biodegradability.Poly-3-hydroxybutyrate is a biodegradable biopolyester that was discovered earlier,but lower ductility limitaed its applications.Add 3-HP in poly-3-hydroxybutyrate can improve the material properties of the copolymer,but in the early studies,the biosynthesis of 3-hydroxypropionic acid and 3-hydroxypropionic acid copolymers relied on 3-hydroxypropionic acid or its structure precursors,such as acrylic acid,1,3-propanediol etc,they are expensive and cause environmental pollution.In this study,we use Ralstonia eutropha H16 as starting strain.Because the strain has two important characteristics: it can secrete an extracellular esterase to degrade the oil in the surrounding environment to make it into glycerol and free fatty acid;it have two oil degradation(beta oxidation pathway)gene clusters facilitate the transfer and degradation of free fatty acids,allowing them to grow and metabolize using the oil as the sole carbon source.In this study,the exogenous 3-hydroxypropionate synthesis pathway was integrated into the original strain by chromosomal recombination technology,and R.eutropha H16 engineering strain could use oils as sole carbon source for the synthesis of 3-HP and 3-HB copolymer P(3HB-co-3HP).In this experiment,Soxhlet extraction and ice ethanol precipitation were combined to achieve separation and purification,the structure and monomer composition of the copolymer were analyzed by NMR.We also measure the molecular weight distribution by GPC.Finally make sure the target product is P(3HB-co-3HP).In this study,we use three key enzymes: acyl-CoA carboxylase(accADBC),malonyl-CoA reductase(mcr)and propionyl-CoA transferase(pct).The genes of three key enzymes were introduced into the target working plasmid pBBR-pct-mcr-accADBC;and use conjugative transfer to constuct the engineering bacteria,it can produce P(3HB-co-3HP).The engineered strains were fermented with soybean oil as sole carbon source at a level of 5 L fermenter to obtain DCW(cell dry weight)of 6.08 g/L,and copolymer yield of 3.11 g/L(accounting for 51.15% of dry cell weight).The ratio of trihydroxypropionic acid to trihydroxybutyric acid monomer in the copolymer is about 1:2,and the molecular weight reaches 200,000.In this study,we have proved that the arabinose concentration had a great influence on the production of P(3HB-co-3HP).Setting other fermentation conditions as control variables,add the arabinose to 0.25 mol/L,0.5 mol/L,1 mol/L,2 mol/L in the logarithmic growth phase.DCW was 1.52 g/L,2.11 g/L,1.56 g/L and 1.21 g/L respectively.P(3HB-co-3HP)accounts for 57.33%,62.42%,56.76%,52.41% of the dry weight of the cells respectively.Further analysis by NMR showed that when the final concentration of arabinose was 0.25 mol/L and 0.5 mol/L,the product was P(3HB-co-3HP),and was 1 mol/L and 2 mol/L,the product is P3 HB.We also measured the melting point is decreases as the 3HP content increases.Finally,this study also constructed an engineered R.eutropha H16 that can efficiently utilize glycerol.Compared with the unmodified R.eutropha H16,the engineering bacteria expressing glpk(glycerol kinase)greatly improved the utilization efficiency of glycerol.When glycerolas sole carbon source,the growth condition of the engineered strain was significantly better than the control strain,DCW reached 2.16 g/L,and the PHB yield was 68.21% of the dry weight of the cells.On this basis,this study further constructed the plasmid pBBR-pct-mcr-accADBC-glpk,and explored the effect of the introduction of glpk gene on the engineering R.eutropha producing copolymer P(3HB-co-3HP),this part of the study failed.