Modification Of Metabolic Engineering Of Engineering Strains Producing Isoprene And Study On Enzymatic Synthesis

Isoprene as an important platform compounds has a wide range of applications in the rubber industry and the fine chemical industry.At present,isoprene production methods mainly include C5 fraction separation method,chemical synthesis and dehydrogenation method.However,these chemical synthesis processes are highly dependent on petroleum resources and are prone to environmental pollution.Therefore,it is necessary to study alternative bio-isoprene production processes and methods using renewable biomass as raw materials.In this paper,two isoprene biosynthesis processes were further studied: First,the existing engineering Escherichia coli metabolic engineering transformation and fermentation process optimization to improve the fermentation efficiency,the second is to establish and optimize the isoprene in vitro enzymatic synthesis process.In this study,we first carried on the metabolic engineering transformation of the biological isoprene engineering bacteria in the laboratory through the genetic engineering method,constructs an engineering strain E.coli BL21（DE3）::Trc-low△adhE△ackA.The major remodeling strategy was knocked out of the competitive bypass metabolic pathway gene of the alcohol dehydrogenase gene（adhE）with the acetic acid kinase gene（ackA）to reduce the production of acetic acid and ethanol in the fermentation byproducts;At the same time,five enzyme genes of isoprene downstream metabolic pathway were introduced into the genome of engineering bacteria to improve the fermentation stability.The modified strain can produce isoprene with mevalonate as substrate.It has the advantages of good fermentation stability and low by-product content,and can realize the co-fermentation of isoprene and lactic acid（both in this study without metabolic competition）.In this study,the strain was optimized for the fermentation process.The optimal results showed that the best medium was high-density fermentation medium（20 g/L glucose,0.24 g/L MgSO₄,5 g/L beef extract,2.5 g/L KH₂PO₄,1 g/L betaine,3 g/L（NH₄）₂SO₄,1 g/L monohydrate lemon Acid,1 g/L trisodium citrate,1.86 g/L KCl,80 mg/L FeSO₄ · 7H₂O）.The optimum final concentration of the inducer was 0.5 mmol / L,and the pH adjustment was ammonia.The strain was incubated for 36 hours at a shake flask level using an optimized process,the yield of isoprene was 85.8 mg/L,the yield of lactic acid was 16.9 g/L,the yield of by-product ethanol was reduced by about 50%,and the yield of acetic acid was reduced by about 47%.In addition,this study also studied the enzymatic synthesis of isoprene in vitro.The research strategy is to use synthetic biology principles to construct extracellular systems catalyzed by multiple enzymes and their cofactors.In this study,the enzyme used in the enzymatic enzyme in vitro is the five enzymes of the isoprene downstream pathway（which catalyzes the synthesis of mevalonate to isoprene）.First,the purified enzyme was obtained by the following steps: induction of expression,sonication,nickel column purification,ultrafiltration and other steps.And then through the initial establishment of multi-enzyme in vitro catalytic system,we optimized the methylvalerate acid concentration,ATP concentration and the best ratio of five enzymes and so on.The experimental results show that the best in vitro enzyme reaction system for producing isoprene with mevalonate as substrate is:Potassium phosphate buffer 50 mmol/L,KCl 30 mmol/L,MgCl₂ 10 mmol/L,β-mercaptoethanol 4 mmol/L,ATP 10 mmol/L,mevalonate 2.5 mmol/L,enzyme concentration MVK: PMK: MVD: IDI: ISPS = 1: 1: 1: 2: 16.In addition,we compare the in vitro and in vivo isoprene synthesis methods by controlled trials.The results of the controlled trials show that the method of producing isoprene in vitro by enzymatic method has higher productivity than the whole cell production method and is continuous effective production time is longer.Application of in vitro enzymatic process for 40 hours,the yield of isoprene can reach 302 mg/L.