Disruption of lactate dehydrogenase through homologous recombination to improve bioethanol production in Thermoanaerobacterium aotearoense
Disruption of lactate dehydrogenase through homologous recombination to improve bioethanol production in Thermoanaerobacterium aotearoense
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通过同源重组破坏乳酸脱氢酶以提高奥特罗热厌氧杆菌的生物乙醇产量
DOI：
10.1016/j.enzmictec.2010.10.006
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发表时间：
2011-02-08
影响因子：
3.4
通讯作者：
Wang, Jufang
中科院分区：
文献类型：
--
作者：
Cai, Youhua;Lai, Chaofeng;Wang, Jufang
为了提高嗜热厌氧杆菌（Thermoanaerobacterium aotearoense）的乙醇产量，通过同源重组成功地破坏了乳酸脱氢酶（10）基因，该基因负责乳酸生产的关键分支途径。基于JW/SL-YS 485-AY 278026设计并扩增ldh-up和ldh-down，随后将它们作为插入红霉素抗性基因的同源片段用于构建基于pBLUESCRIPT II SK（+）的靶向载体。Southern杂交和PCR检测证实了Deltaldh突变体中的ldh基因被红霉素抗性基因的插入所破坏。与野生型相比，Delta ldh突变体在葡萄糖和木糖培养下的细胞产量分别增加了31.0%和31.4%，这可能是因为敲除ldh基因导致乙酸和ATP水平增加。在葡萄糖和木糖培养下，乳酸脱氢酶的敲除分别使乙醇的产率（摩尔/摩尔底物）增加2.37倍和2.1倍。此外，在Delta ldh突变体发酵混合物中没有检测到乳酸（HPLC的检测限：0.5mM），但是对于野生型菌株在葡萄糖和木糖上的生长，容易检测到乳酸，最终浓度分别高达59.24mM和56.06mM。这一过程的成功彻底证明了在热厌氧杆菌中通过同源重组进行基因敲除的方法学可能性。(C)2010年爱思唯尔公司All rights reserved.
To enhance ethanol production in Thermoanaerobacterium aotearoense, the lactate dehydrogenase (10) gene, which is responsible for lactic acid production in a key branch pathway, was successfully disrupted via homologous recombination. ldh-up and ldh-down were designed and amplified based on JW/SL-YS485-AY 278026, and they were subsequently used as homologous fragments with an inserted erythromycin resistance gene to construct the targeted vector based on pBLUESCRIPT II SK(+). Southern hybridization and PCR-based assay definitely confirmed that the ldh gene in the Delta ldh mutant was disrupted by the insertion of the erythromycin resistance gene. Compared with the wild type, the Delta ldh mutant exhibited increases of 31.0% and 31.4% in cell yield under glucose and xylose cultivation, respectively, probably because knocking out the ldh gene results in increased acetate and ATP levels. Knockout of lactate dehydrogenase produced 2.37- and 2.1-fold increases in the yield of ethanol (mole/mole substrate) under glucose and xylose cultivation, respectively. Moreover, no lactic acid was detected in Delta ldh mutant fermentation mixtures (detection limit of HPLC: 0.5 mM), but lactic acid was readily detected for growth of the wild-type strain on both glucose and xylose, with final concentrations up to 59.24 mM and 56.06 mM, respectively. The success of this process thoroughly demonstrates the methodological possibility of gene knockout through homologous recombination in Thermoanaerobacterium. (C) 2010 Elsevier Inc. All rights reserved.