【作者】 杜燕；

【导师】 班睿；

【作者基本信息】 天津大学 ， 生物化工， 2021， 硕士

【摘要】 D-对羟基苯甘氨酸（D-HPG）作为一些重要的β-内酰胺类抗生素的侧链基团,是半合成抗生素的医药中间体。海因酶法是目前最有潜力的D-HPG生产方法,其以D,L-对羟基苯海因为原料,经D-海因酶（DHase）和D-氨甲酰水解酶（DCase）两步催化反应生成D-HPG。DHase和DCase通常存在于野生型细菌或重组菌中,由于DCase活性低于DHase,限制了海因酶法的反应速率,并造成中间物积累。本文研究了重组枯草芽孢杆菌（Bacillus subtilis）表达DCase,以及影响细胞DCase活性和稳定性的一些关键因素。采用人工基因P_AE-adc表达DCase,构建了不同拷贝数整合表达和质粒表达的重组B.subtilis菌株。结果表明,基因拷贝数与细胞DCase活性不成比例关系,双拷贝整合表达菌株DN02的DCase活性最高,为132 U/g DCW。葡萄糖和蔗糖作为碳源,有利于提高细胞DCase活性。但是,PTS碳源所引起的碳分解代谢物阻遏效应导致发酵液酸化,而降低细胞DCase活性与稳定性。缺陷葡萄糖运输蛋白Pts G或抗终止蛋白Glc T,使葡萄糖吸收速率降低57%,能够显著缓解发酵液酸化,衍生菌DN12的DCase活性达到518 U/g DCW。Pts G缺陷与Glc T缺陷等效,表明pts G的表达强烈依赖Glc T的抗终止作用。Pts G能够补偿Glc U或Glc P的单缺陷或双缺陷,但Pts G,Glc P和Glc U共缺陷不再降低细胞的葡萄糖吸收速率,表明B.subtilis还存在未知的葡萄糖运输途径。在以葡萄糖为碳源的条件下,通过修饰cre box,而减弱Ccp A介导的葡萄糖对cit Z表达的抑制。结果使柠檬酸循环增大,乙酸合成量降低24%,发酵液酸化程度减轻,衍生菌株DN16p2的DCase活性达到615 U/g DCW。通过修饰调控元件而减弱Cod Y对cit B表达的抑制,结果使柠檬酸循环进一步增大及乙酸合成减少,衍生菌株DN17p3的DCase活性达到641 U/g DCW。由此过分增大柠檬酸循环通量,在供氧不变的条件下,将会诱导乳酸合成而加重发酵液酸化,对细胞DCase活性产生不利影响。通过Cit B的R741E点突变,消除Cit B对cit Z m RNA的去稳定作用,导致柠檬酸循环增大,乙酸产量降低71%,羟基丁酮产量降低16%。但是,进一步降低乙酸产量对提高细胞DCase活性仅有轻微作用。在B.subtilis中共表达DCase和DHase,细胞DCase活性低于对应的DCase单表达菌株,表明DCase的表达受到多拷贝质粒p UBS存在的抑制。更多还原

【Abstract】 D-p-hydroxyphenylglycine（D-HPG）is a side-chain group of some importantβ-lactam antibiotics and a pharmaceutical intermediate for semi-synthetic antibiotics.The hydantoinase method is currently the most promising D-HPG production method.It uses D,L-p-hydroxybenzene as raw material to produce D-HPG through two-step catalytic reaction of D-hydantoinase（DHase）and D-carbamoylase（DCase）.DHase and DCase are usually present in wild-type bacteria or recombinant bacteria.Since the activity of DCase is lower than DHase,the reaction rate of the hydantoinase method is limited,and intermediates are accumulated.This paper studies the expression of DCase by recombinant B.subtilis and some key factors that affect the activity and stability of DCase in cells.Using artificial gene P_AE-adc to express DCase,recombinant B.subtilis strains with different copy number integration expression and plasmid expression were constructed.The results showed that there was a disproportionate relationship between gene copy number and cell DCase activity.The double-copy integrated expression strain DN02 had the highest DCase activity of 132 U/g DCW.Glucose and sucrose as a carbon source can help to increase the DCase activity of cells.However,the carbon catabolism repression（CCR）effect caused by the PTS carbon source leads to acidification of the fermentation broth,which reduces the DCase activity and stability of the cell.Defective glucose transporter Pts G or anti-termination protein Glc T can reduce glucose absorption rate by 57%,which can significantly alleviate the acidification of fermentation broth,and the DCase activity of derivative strain DN12 reaches 518 U/g DCW.The defective Pts G is equivalent to the defective Glc T,indicating that the expression of pts G strongly depends on the anti-termination effect of Glc T.Pts G can compensate the single defect or double defect of Glc U or Glc P,but the co-defect of Pts G,Glc P and Glc U no longer reduces the glucose absorption rate of cells,indicating that B.subtilis also has unknown glucose transporter.Under the condition of using glucose as the carbon source,by modifying the cre box,Ccp A-mediated glucose inhibition of cit Z expression was reduced.As a result,the citrate cycle was increased,the amount of acetate synthesis decreased by 24%,the degree of acidification of the fermentation broth was reduced,and the DCase activity of derivative strain DN16p2 reached 615 U/g DCW.By modifying the regulatory elements,Cod Y’s inhibition of cit B expression is weakened.As a result,the citrate cycle was further increased and acetic acid synthesis was decreased,and the DCase activity of the derivative strain DN17p3 reached 641 U/g DCW.If the citrate circulation flux is excessively increased in this way,under the condition of constant oxygen supply,it will induce lactate synthesis and aggravate the acidification of the fermentation broth,which will adversely affect the cell DCase activity.Through the R741E point mutation of Cit B,the destabilizing effect of Cit B on cit Z m RNA was eliminated,resulting in an increase in the citate cycle,a 71%decrease in acetate production,and a 16%decrease in acetate production.However,further reduction of acetate production has only a slight effect on increasing cell DCase activity.Coexpression of DCase and DHase in B.subtilis showed that the DCase activity of the cells was lower than that of the corresponding DCase single expression strain,indicating that the expression of DCase was inhibited by the presence of the multicopy plasmid p UBS.更多还原