Study On The Improvement Of Cellulose Poly 3-hydroxybutyrate(PHB)of The Engineered Corynebacterium Glutamicum Strain

Poly 3-hydroxybutyrate(PHB)is a class of polymeric polyesters synthesized by microbial cell factories.Not only PHB has the physical properties and thermal processing properties similar to petroleum-based plastics,but also is biodegradable from renewable resources.PHB can be produced by fermentation from biomass resources,but its expensive production cost is the biggest obstacle to industrial large-scale production of PHB.Most of the PHB production uses starch and sugar substances as raw materials,the cost of which accounts for more than 30%-40%of the total production cost,so it is important to reduce the raw material cost of PHB.Lignocellulose is the most promising raw material for future industrial production of PHB because it is widely available,inexpensive and renewable,and there is no "competition for food".The previous work used the highly resistant Corynebacterium glutamicum S9114 as a starting strain and integrated PHB synthesis-related genes and xylose metabolism and transportrelated genes into the genome to obtain a C.glutamicum JH02 strain capable of stable PHB synthesis using glucose and xylose.The objectives of this paper were to further improve the ability of recombinant C.glutamicum to synthesize PHB by molecular biology modification and exploring the optimum NH4+ addition for PHB synthesis by C.glutamicum JH02,and to test the ability of the obtained recombinant strain to produce PHB in straw hydrolysate.The specific research includes:(1)Cellular morphological engineering modifications to increase cell volume and thus provide potential for intracellular accumulation of PHB;(2)membrane localized expression of PHA synthase to improve the stability of PHA synthase and shorten the spatial distance of PHA synthase involved in polymerization reaction;(3)introduction of phaP gene to reduce the particle size of PHB particles to protect the bacterium;(4)increase the metabolic flux of Acetyl-CoA into the PHB synthesis pathway;(5)investigation of the effect of ammonia nitrogen levels(NH4’)on PHB synthesis by C.glutamicum;(6)adaptive evolution to improve the ability of the strain to utilize xylose.The first part was to improve the ability of C.glutamicum JH02 to synthesize PHB through molecular biology modification.To increase cell volume and provide more space for PHB accumulation,cell growth of C.glutamicum cells was promoted by overexpressing the cell morphology-related pknB and WhcD genes,which increased cell dry weight by 1.0-fold and 1.1-fold,respectively,and the final PHB production was increased by 177.0%and 163.0%,respectively.In order to improve the stability of PHA synthase and shorten the spatial distance of PHA synthase involved in the polymerization reaction,PHA synthase was localized on the cell membrane,and the two recombinant strains JH02-Peftu-(Ncgl)phaC and JH02-Peftu(porB)phaC obtained could significantly increase the PHB content by 70.1%and 140.6%,and the cell dry weight increase about 1-fold.The final PHB titer of the two recombinant strains were increased by 195.0%and 310.6%,respectively.The second part explored the effect of ammonia nitrogen on the synthesis of PHB by C.glutamicum.It was determined by concentration gradient experiments that C.glutamicum JH02 could obtain the highest PHB production when NH4+was added at a concentration of 45 g/L.RT-qPCR experiments showed that the transcript levels of genes related to the EMP pathway,TCA cycle and PHB synthesis pathway were significantly up-regulated when appropriate NH4+concentrations were added,which demonstrated that the addition of ammonia nitrogen not only promoted cell growth,but more importantly,effectively promoted the expression of genes related to PHB synthesis,and therefore promoted the accumulation of intracellular PHB.The third part was to examine the ability of the recombinant strain to synthesize PHB in straw hydrolysate and to improve the rate of utilization of xylose from lignocellulose by C.glutamicum through adaptive evolution.Recombinant strain JH02-Peftu-(Ncgl)phaC obtained by membrane-localized expression of PHA synthase was fermented in a 30%solids content straw hydrolysate by fed-batch fermentation to obtain a PHB production of 16.2 g/L.Adaptive evolution experiments of the xylose utilization capacity of C.glutamicum showed that for nonnatural PHB-producing strains,adaptive evolution negatively affected the intracellular synthesis of PHB.The recombinant strain C.glutamicum JH02-Peftu-(Ncgl)phaC obtained in this paper has a significantly higher ability to synthesize PHB from lignocellulose at suitable NH4+concentrations,and the intracellular PHB content can reach up to 39.3 wt%and PHB titer can reach up to 16.2 g/L.Compared with the control strain C.glutamicum JH02,the intracellular PHB content was increased by 134.8%and the PHB titer was increased by 57.3%.The engineered strain obtained in this paper achieved the highest index of PHB production using real lignocellulosic system,providing a promising candidate strain for the next industrial application of PHB production from lignocellulose.