利用五碳糖产高纯度L-乳酸的大肠杆菌基因工程菌的构建

Production of L-lactic acid from pentose by a genetically engineered Escherichia coli

摘要:

摘要:【目的】本研究以已敲除多个产杂酸酶基因的大肠杆菌(Escherichia coli)乙醇工程菌SZ470 (ΔfrdBC ΔldhA ΔackA ΔfocA-pB ΔpdhR::pBp6-pBrbs-ceEF-lpd)为起始菌株，进一步敲除其乙醇脱氢酶(alcohol dehydrogenase，ADH)基因，同时插入带有自身启动子的乳酸片球菌(Pediococcus acidilactici)的L-乳酸脱氢 酶(L-lactate dehydrogenase，LLDH)基因，构建可利用五碳糖同型发酵L-乳酸重组大肠杆菌。【方法】利用λ噬菌体Red重组系统构建乙醇脱氢酶基因(adhE)缺失菌株Escherichia coli JH01，并克隆P．acidilactici的ldhL基因，利用染色体插入技术将其整合到JH01基因组，构建产L-乳酸大肠杆菌基因工程菌Escherichia coli JH12，利用无氧发酵15 L发酵罐测定重组菌株L-乳酸产量。【结果】工程菌JH12 在15 L发酵罐中以6%的葡萄糖为碳源进行发酵，发酵到36 h的过程中葡萄糖的消耗速率为1. 46 g/(L·h)，乳酸生产强度为1.14 g/(L·h)，乳酸的产量达到41.13 g/L。发酵产物中未检测到琥珀酸、甲酸的生成，仅有少量乙酸生成，L-乳酸纯度达95.69%(L-乳酸在总发酵产物的比率)。工程菌JH12以6%的木糖为碳源进行发酵，发酵到36 h的过程中葡萄糖的消耗速率为0.88 g/(L·h)，乳酸生产强度为0.60 g/(L·h)，乳酸的产量达到34.73 g/L。发酵产物中杂酸少，乳酸的纯度高达98%。【结论】本研究通过基因敲除、染色体插入及无氧进化筛选获得一株产L-乳酸的大肠杆菌工程菌JH12，该菌株不需利用外源质粒，稳定性好，可利用五碳糖进行发酵，发酵产物中杂酸少，L-乳酸的纯度高。本研究为L-乳酸大肠杆菌工程菌的构建提供一定的技术支持，同时也为大肠 杆菌L-乳酸的工业化生产提供了参考依据。

Abstract:

Abstract:［Objective］In this study，we constructed a recombinant Escherichia coli strain for the production of high-purity L-lactic acid，using a homoethanol fermenting mutant E．coli SZ470 (ΔfrdBC ΔldhA ΔackA ΔfocA-pB ΔpdhR::pBp6-pBrbs-aceEF-lpd) as the starting strain． ［Methods］By using homologous recombination，we deleted the adhE gene from SZ470 to obtain a mutant Escherichia coli JH01，which could not grow under anaerobic conditions． Then we cloned the Llactate dehydrogenase gene (ldhL) of Pediococcus acidilactici and inserted it into the chromosome of JH01 via electroporation to obtain a recombinant strain Escherichia coli JH12． We evaluated the L-lactic acid production of the recombinant strain in a 15 L fermenter．［Results］In 10 L LB medium supplemented with 6% glucose，JH12 maintained maximal cell growth and an efficient L-lactic acid production rate for 36 h． Glucose consumption rate achieved was 1.46 g/(L·h) and L-lactic acid production rate was 1. 14 g/(L·h)．The results also show that 41.13 g/L lactic acid was produced，achieving a purity of 95.69% (based on total fermentation products) ．Xylose consumption rate was 0.88 g/(L·h) and L-lactic acid production rate was 0.60 g/(L·h)．The production of lactic acid was 34.73 g/L， achieving a purity of 98%．There were no succinic acid and formic acid detected and only little amount of acetic acid generated during the fermentation．［Conclusion］ We constructed a homolactic acid fermentation strain E． coli JH12，which could efficiently convert glucose and xylose into high-purity L-lactic acid． JH12 could have great potential in industrial fermentation for L-lactic acid production．