| D-danshensu(Salvianolic acid)is a phenolic acid compound and mainly obtained from the root of Salvia miltiorrhiza(a traditional Chinese medicinal plant).D-danshensu can be used in the treatment of cardio-cerebrovascular related diseases,also shows other pharmacological functions,such as anti-oxidant,anti-tumor,anti-inflammatory and anti-liver fibrosis.D-danshensu can also be used as a substrate for the synthesis of other medicinal compounds,such as IDHP and DBZ,which have shown to enhance the treatment of cardiovascular and cerebrovascular diseases.At present,D-danshensu is mainly extracted from the root of Salvia miltiorrhiza.However,the content of D-danshensu in Salvia miltiorrhiza is low,and the yield of Salvia miltiorrhiza is easily affected by weather,which makes the yield of D-danshensu unstable and difficult to meet the market demand.Therefore,it is particularly urgent to apply modern biological technology to develop sustainable methods for D-danshensu production.Firstly,the production pathway of D-danshensu was designed according to the catalytic characteristics of enzyme.Secondly,the strain for D-danshensu production was constructed and different copy numbers of plasmids were used to balance the expression of pathway genes.Then,to improve the supply capacity of NAD+and the production of D-danshensu,the NAD+synthesis pathway of Escherichia coli was modified by modularization strategy.Finally,the culture conditions of the strain,the catalysis conditions of whole cells and the separation and purification conditions of D-danshensu were optimized.The main research contents of this paper are as follows:(1)Design of D-danshensu synthesis pathwayThree production pathways of D-danshensu were designed using catechol or L-DOPA as substrates.The first pathway was composed of L-amino acid oxidase(aadL),D-Lactate dehydrogenase(ldhD)and glucose dehydrogenase(gdh).The second pathway was composed of phenol lyase(TPL),aadL,ldhD and gdh.The third pathway consists of aminotransferase(tyrB),D-aromatic lactate dehydrogenase(csldhD)and L-glutamate dehydrogenase(LGDH).The pathway strains were constructed and compared under the same catalytic conditions.After 1 h of transformation reaction,the yield of D-danshensu from pathway one reached 0.35 g·L-1.After 6 h of transformation reaction,the yield of D-danshensu from pathway one was the highest and reached 1.67 g·L-1,which was 5.6 times of pathway two and 2.7 times of pathway three.(2)Optimization of the synthesis pathway of D-danshensuIn order to prevent the accumulation of intermediate products and maximize the production of D-danshensu,it is necessary to balance the expression of pathway genes.In this study,we used different copy numbers(high copy of pRSFDuet-1,medium copy of pETDuet-1 and low copy of pCDFDuet-1)plasmids to regulate the expression of aadL,ldhD and gdh.A total of 7 engineered strains were constructed.Then the activities of ldhD,gdh and production of D-danshensu were measured.The E.coli ALG7 expressing gdh with pCDFDuet-1 and aadL,ldhD with pRSFDuet-1 can produce the most D-danshensu.After 1 h of transformation reaction,the yield of D-danshensu reached 1.25 g·L-1,which was 3.6 times of that of E.coli ALG2(pRSF-aadL,pET-ldhD-gdh).(3)Modification of NAD+synthesis pathway in E.coliThere are many genes involved in the synthesis of NAD+from E.coli.In order to simplify the complexity,a modular metabolic engineering strategy was introduced to enhance the NAD+synthesis pathway.The NAD+synthesis pathway from E.coli was divided into three modules.Module one was the de novo synthesis pathway with L-aspartic acid as the substrate,module two was the rescue pathway with niacin as the substrate,and module three was the synthesis pathway common to both the de novo synthesis pathway and the rescue pathway.Firstly,Module one was optimized by single or combined overexpression of nadB,nadA and nadC.Secondly,module two was optimized by single or combined overexpression of niaP,pncB and nadV.Then,module three optimization was carried out by single or combined overexpression of nadD,nadE and nadM.Finally,niaP,pncB,nadD,nadE and nadM were overexpressed individually or in combination for module combination optimization.E.coli A5 produced the highest NAD+/NADH and D-danshensu yield,which were 36.48 ?mol·g-1 DCW and 2.31 g·L-1,2.6 and 1.85 times higher than those of the original strain.(4)Optimization of strain culture conditionsFirstly,the composition of the medium was optimized,including carbon source,nitrogen source,inorganic salt and vitamin.The optimal medium composition was obtained as follows:25 g·L-1 glucose,20 g·L-1 yeast powder,6 g·L-1 disodium hydrogen phosphate,2 g·L-1 potassium dihydrogen phosphate,3 g·L-1 magnesium sulfate,lmg·L-1 VB1,1.5 mg·L-1 VB2 and 1.5 mg·L-1 VB6.Then,the culture conditions of the strain were optimized,including pH,inoculation amount,dissolved oxygen amount,induction condition and feeding method.The optimal culture conditions were obtained as follows:pH 7,inoculation amount of 5%,dissolved oxygen of 20%,inducer concentration of 0.6 mM,initial cell volume(OD600)of 15.92,induction duration of 14 h,glucose flow of 3.42 g·L-1·h-1.The OD600 reached 47.45 after cultured for 22 h under the optimal culture conditions.The amplification experiment was carried out in a 200 L fermenter.After 22 h culture,the OD600 reached 48.72.(5)Optimization of whole cell transformation conditionsFirstly,the single factor condition was optimized,including pH,temperature,biomass,stirring speed and glucose content;Then orthogonal experiments were carried out to optimize pH,the conversion temperature and stirring speed.The optimal conversion conditions were as follows:pH 7.5,35?,bacteria volume of 50 g·L-1,rotating speed of 400 rpm,and the mass ratio between L-DOPA and glucose of 1:1.2.Under the optimal transformation conditions,the yield of D-danshensu reached 117.05 g·L-1 and 6.5 g·L-1·h-1 after 18 h.Enlargement experiment of 200 L bioreactor was carried out.Finally,120 g·L-1 L-DOPA was transformed into 117.85 g·L-1 D-danshensu within 12 h,with yield of 97.71%and a production intensity of 8.42 g·L-1·h-1.(6)Optimization of purification conditions of D-danshensuThe static adsorption and desorption experiments of 20 macroporous resins were carried out.Among them,LX-17 had the highest adsorption capacity for D-danshensu,and was easy to be desorbed by ethanol solution.Then,the loading conditions in the process of separation and purification were optimized,and the optimal loading conditions were obtained as follows:the concentration of loading solution was 20.45 g·L-1,the pH of loading solution was 3,and the flow rate of loading solution was 2 mL·min-1.Finally,the elution conditions were optimized,50%ethanol was selected as the elution agent,and the elution flow rate was 2 mL·min-1.The optimized separation and purification conditions were used to purify D-danshensu from the conversion solution.The purified solution was concentrated under reduced pressure and freeze-dried to obtain milky white D-danshensu powder with 99.63%chemical purity. |
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