重组大肠杆菌利用醋酸及乳酸为碳源合成PHB  

Synthesis of PHB by Recombinant E.coli Using Acetic Acid and Lactic Acid as Carbon Sources

作　　者：肖萌 蒋莹 崔逸璇 Sadaf Riaz Maurycy Daroch XIAO Meng;JIANG Ying;CUI Yixuan;Sadaf Riaz;Maurycy Daroch(School of Environment and Energy,Peking University Shenzhen Graduate School,Shenzhen 518055)

机构地区：[1]北京大学深圳研究生院环境与能源学院,深圳518055

出　　处：《北京大学学报（自然科学版）》2023年第1期115-124,共10页Acta Scientiarum Naturalium Universitatis Pekinensis

基　　金：深圳市基础研究项目(GXWD20201231165807007-20200806170221001)资助。

摘　　要：以廉价的工业副产物醋酸及乳酸为碳源,对产PHB重组大肠杆菌进行培养,考察醋酸及乳酸的添加对重组大肠杆菌生长及PHB产量的影响。将来自Cupriavidus necator的PHB合成操纵子phaCAB基因簇克隆至pBAD载体,得到产PHB菌株BL21_pBAD_phaCAB,以阿拉伯糖为诱导剂,在大肠杆菌中进行重组表达。分别使用LB及M9培养基,对重组菌株BL21_pBAD_phaCAB进行培养,研究其生长速度及PHB产量,探索产PHB重组大肠杆菌最适培养基。以添加0.04 g/L乳酸、1.2 g/L乳酸、0.02 g/L醋酸、0.6 g/L醋酸、0.04 g/L乳酸+0.02 g/L醋酸、1.2 g/L乳酸+0.4 g/L醋酸的M9培养基(均含2 g/L葡萄糖)为实验组,以M9培养基(含2 g/L葡萄糖)为对照组,考察醋酸及乳酸的添加对重组大肠杆菌生长及PHB产量的影响。分别取第6,12,24和36小时的培养液,分析其葡萄糖、醋酸及乳酸含量的变化。结果表明,低氮型M9培养基更适合产PHB重组大肠杆菌在低糖培养环境中生长。在葡萄糖消耗殆尽后,大肠杆菌能够以醋酸及乳酸为碳源进行代谢,因此在培养基中添加一定浓度的醋酸及乳酸能够有效地提高重组菌株BL21_pBAD_phaCAB产PHB能力,在乳酸添加量为1.2 g/L时,PHB产量达到最高(1.43 g/L),比对照组提高78%。Two cheap byproducts lactic and acetic acid were directly used as the carbon sources of polyhydroxybutyrate(PHB)-producing recombinant Escherichia coli to investigate the effects of acetic acid and lactic acid on the growth and PHB yield of recombinant E.coli.The PHB synthetic operon phaCAB gene cluster constructed on the basis of Cupriavidus necator genes was cloned into pBAD vector to obtain PHB-producing recombinant strain BL21_pBAD_phaCAB,which was expressed in E.coli by using arabinose as an inducer.The recombinant strain was cultured using LB and M9 medium respectively,and the growth rate and PHB yield were studied to lead the exploration of optimum medium for PHB-producing recombinant E.coli.To explore the effect of lactic acid and acetic acid on the growth and PHB yield of the recombinant strain,0.04 g/L lactic acid,1.2 g/L lactic acid,0.02 g/L acetic acid,0.6 g/L acetic acid,0.04 g/L lactic acid+0.02 g/L acetic acid,1.2 g/L lactic acid+0.4 g/L acetic acid were added into M9 medium(containing 2 g/L glucose)as the experimental group where M9 medium(containing 2 g/L glucose)was the control group.The culture media of the 6,12,24,and 36 hours were taken respectively to explore the concentration changes in glucose,acetic acid,and lactic acid.The results show that the low nitrogen M9 medium is more suitable for the growth of PHB-producing recombinant E.coli in a low sugar culture environment.After the consumption of glucose,E.coli can use acetic acid and lactic acid as carbon sources for metabolism,and by adding a proper amount of acetic acid and lactic acid,the PHB yield of the recombinant strain can get improved.When 1.2 g/L lactic acid is added,an up to 1.43 g/L PHB yield can be achieved which is 78% higher than the control.

关 键 词：PHB 乳酸 醋酸 大肠杆菌 

分 类 号：TQ317[化学工程—高聚物工业]