复合诱变条件优化结合过表达乳酸脱氢酶高效合成苯乳酸

Optimization of Compound Mutagenesis Conditions Combined with Overexpression of Lactate Dehydrogenase for Efficient Synthesis of Phenyllactic Acid

乳酸菌(lactic acid bacteria,LAB)作为新型生物防腐剂苯乳酸(phenyllactic acid,PLA)的天然生产菌,在自然界中普遍存在,但由于其胞壁较厚、机械强度较大,导致外源基因难以导入。为使LAB自身高效合成PLA,该研究以实验室筛选的乳酸片球菌(Pediococcus acidilactici GM)作为出发菌株,采用复合诱变选育出活力更高、产量更大的突变菌株,即先采用常压室温等离子体(atmospheric and room temperature plasma,ARTP)诱变50 s,再采用0.8%硫酸二乙酯(diethyl sulfate,DES)诱变25 min,并在培养基中加入PLA以提高菌株抗性,进行摇瓶发酵验证和遗传稳定性验证。为解决外源基因难导入的问题,使外源DNA得到高效转化,该研究通过单因素试验对氨基酸、缓冲液、溶菌酶、菌体生长状态和电转化参数进行优化,得到最优电转化条件:添加0.75%L-苏氨酸的MRS培养基培养菌体至OD600=0.8,使用洗涤缓冲液Ⅲ(0.5 mol/L蔗糖,7 mmol/L Na_(2)HPO4,7 mmol/L NaH_(2)PO_(4),20 mmol/L MgCl_(2))洗涤菌体,溶菌酶浓度30 mg/mL于37℃处理菌体30 min,电场强度20 kV/cm。为进一步提高LAB合成PLA的产量,本研究利用基因工程手段,将质粒载体pMG36e自带启动子P32(强度低)替换为Pldh(强度高),构建L型乳酸脱氢酶(lactate dehydrogenase,LDH)过表达质粒,并在最优电转化条件下成功转化于P.acidilactici GM中,实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)分析表明LDH基因ldh表达上调,经过5 L罐发酵培养,添加苯丙酮酸(phenylpyruvic acid,PPA)后,PLA产量提高至56.3 g/L,转化率提高至94.8%。

Lactic acid bacteria(LAB),as natural producers of the novel biopreservative phenyllactic acid(PLA),are widely present in the nature.However,due to their thick cell walls and high mechanical strength,it is difficult to introduce exogenous genes into LAB.Compound mutagenesis was carried out for Pediococcus acidilactici GM,a strain screened out in the laboratory,for the efficient synthesis of PLA.The strain was first induced with atmospheric and room temperature plasma(ARTP)for 50 s and then with 0.8%diethyl sulfate(DES)for 25 min,and PLA was added to the culture medium to improve the strain resistance.A mutant strain with high vitality and a high yield of PLA was selected,and then shake flask fermentation and genetic stability validation were conducted.In view of the difficult introduction and transformation of exogenous DNA,the amino acids,buffer,lysozyme,bacterial growth status,and electroporation parameters were optimized by single factor experiments.The optimal electroporation conditions were obtained as follows.The bacterial cells were cultured in the MRS medium with 0.75%L-threonine to reach OD600 of 0.8,washed with washing buffer III(0.5 mol/L sucrose,7 mmol/L Na_(2)HPO4,7 mmol/L NaH_(2)PO_(4),and 20 mmol/L MgCl_(2)),and treated with lysozyme(30 mg/mL)at 37℃for 30 min and then at the electric field intensity of 20 kV/cm.To further increase the production of PLA by LAB,genetic engineering was employed to replace the promoter P32(low intensity)of the plasmid pMG36e with Pldh(high intensity)to construct a lactate dehydrogenase(LDH)overexpression plasmid,which was then transformed into P.acidilactici GM under the optimal electroporation conditions.RTReal‐time quantitative polymerase chain reaction(RT‐qPCR)results showed that the obtained mutant presented upregulated expression of the LDH gene ldh,and the yield of PLA was increased to 56.3 g/L and the conversion rate was increased to 94.8%after fermentation and culture in 5 L tanks with phenylpyruvic acid(PPA).