Research On Improving The Production Performance Of Biodegradable Materials Of PHB And Levan Based On Metabolic Engineering Strategies

PHB is a green alternative for petrochemical-based plastics,a biodegradable 3-hydroxyalkanoate,and different degrees of PHB polymerization have different application properties.Levan,a natural fructose polymer,is not only an important prebiotic polysaccharide,but also has biological activities such as antitumor,diabetes,and immune stimulation.However,the current low production performance limits the commercial scale production of PHB and Levan.It is necessary to construct a finely regulated cell factory to reduce the production cost and increase their yields,and the utilization of by-products in the production process also deserves attention.Metabolic engineering could control the yield and production intensity of synthetic pathways by controlling the flow of metabolic pathways,the flow direction of carbon pathways,and the flow rate.In addition,enzyme engineering provides theoretical and technical support for mining new metabolic pathways.Therefore,Escherichia coli was taken as the chassis microorganism in this study,and heterologous expression of PHB synthesis pathway was constructed based on a high-efficiency expression system.Then,the strategies including co-production with Levan based on metabolic engineering,global metabolic regulation based on pressure-responsive promoter Rpp and s RNA,and dynamic regulation based on Cre recombinase have been developed,so as to achieve the efficient PHB synthesis pathway,reduce production cost,and balance the production of target compounds and cell growth.At the same time,the new levansucrases were characterized,and the yield of Levan was improved by biomimetic mineralization technology,which also laid the foundation for the subsequent preparation of Levan and the realization of more efficient co-production with PHB.On this basis,the production performance of E.coli factory has been optimized in vitro and in vivo.The main research results are as follows:1)A novel dual expression vector of E.coli was constructed,and the efficient preparation of PHB was realized.E.coli BL21（DE3）was used as the starting strain to construct an in vitro metabolic synthesis pathway of PHB.Based on the consideration of the adaptability of promoters for the expression of different target genes,a dual expression vector p ANY4 containing different promoters in the complementary DNA strands was constructed.Therefore,recombinant plasmids for either isopropyl-β-D-thiogalactoside（IPTG）-induced or temperature-induced protein expression could be simultaneously constructed in a single molecular cloning process,which can be used for convenient expression and protein purification for parallel comparison.In addition,on the basis of splitting the dual promoter p R/p L,this study revealed for the first time that there is no significant cumulative effect of the activation efficiency between the single temperature-sensitive promoters p R and p L.A library of promoter-optimized site-saturated mutants was constructed by degenerate PCR,which increased the level of promoter activation by 2 times,reaching or even exceeding the expression level of T7 promoter.On this basis,PHB expression plasmids of p ANY4-T7-PHB and p ANY4-p L18-PHB containing different promoters were constructed,and the yield of PHB reached up to 3.21 g/L.Related research also provides a foundation for the follow-up researches.2)The efficient preparation of PHB by using different carbon sources and the effective co-production of levan and PHB were achieved.Firstly,the carbon source utilization repression mechanism（CCR）which preferentially utilizes glucose was lifted in E.coli by utilizing CRISPR/Cas9 andλ-Red recombination technologies to knock out the pts G gene.On this basis,the effect of E.coli BL21（DE3）（Δpts G）on utilization of various carbon sources was evaluated,and it was found that xylose and fructose exhibited better PHB synthesis ability than glucose.Then,the engineered strain was used to prepare PHB by utilizing glucose+xylose and glucose+fructose as sugar sources.The results showed that the knockout of pts G gene increased the yield of PHB by about 15-25%with different carbon sources,indicating that the knockout of pts G was beneficial for the synthesis of PHB.In addition,novel levansucrases derived from anaerobic bacteria were heterologously expressed and characterized in this study.The results showed that the conversion rate of sucrose was as high as 96.1%,while the yield of levan was as high as 52.2%.E.coli containing the above two metabolic pathways finally co-produced with sucrose as a substrate to reach the final PHB yield of 4.8 g/L and the Levan yield of 47.9%.3)PHB production was enhanced based on Rpp promoter and small RNA global metabolic regulation.Based on the bidirectionality promoter of p ANY4-p L18-ccd B,a stress-responsive promoter Rpp system was developed to express metabolic synthesis pathway of PHB in the forward direction in responding to E.coli cell density.And the global regulators of Ryh B and Fsr A were expressed in the reverse direction,and then cofactors for down-regulating succinate dehydrogenase were accumulated,which increased the expression level to 72.1%,and finally reached the accumulation of PHB to 4.92 g/L.4)PHB production was further improved based on the dynamic regulation strategy of E.coli.A dynamic switch was designed to achieve the dynamic balance of cell growth and PHB metabolic synthesis pathway.Firstly,the double knockout system was used to knock out the pox B and pta genes of the acetate metabolism branch to reduce the waste of carbon source.Then,based on the Cre recombinase,the Rpp stress-responsive promoter,and the thermosensitive promoter that upregulates gene expression in response to different temperatures,the metabolic lifespan of E.coli was divided into three modules:cell growth,gene excision within the Lox P site at 37°C,and PHB synthesis at 40°C,respectively.Then,the stringency of the switch was improved by optimizing the Cre recombinase start codon,and the fluorescence signal and growth curve were detected to demonstrate the controlability and portability of the dynamic switch.As a result,the final PHB yield reached 5.62 g/L,which was 1.7 times higher than the control.5)The highly active expression and simple purification of Sac B and other proteins can be realized based on the multifunctional His-ELP-intein tag.A multifunctional fusion protein expression system of His-ELP-intein tag is constructed by combining Ni-NTA-based strategy and ELP-based strategy with a specially designed purification protocol,which has the purification ability of both His-tag and ELP-intein tag.In this way,proteins should be purified and dissolved in buffers free of imidazole and high concentration salt ions without losing enzyme activity,which facilitates the efficient preparation of extracellular Levan.6)A new strategy for non-column protein purification and CaHPO₄-based enzymatic biomimetic mineralization was established to further improve the extracellular Levan yield.Considering the commercial value of fructan Levan,levansucrase Sac B was used to prepare hydrangea-like nanoflowers with mesoporous structure CaHPO₄-HEIS-HH using the biomimetic mineralization technology based on CaHPO₄.The resulting CaHPO₄-HEIS-HH showed good reusability,excellent storage stability,and up to 254.3%improved relative Levan yield.Additionally,the CaHPO₄-HEIS-HH could achieve 96%substrate sucrose conversion and 58%Levan yield,the and up to 75%enzymatic activity could remain after reused for ten times.In addition,CaHPO₄-HEIS-HH could catalyze 75%of the sucrose in different juices,so it should have application potential in the functional juice industry.