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首页> 美国卫生研究院文献>Applied and Environmental Microbiology >Addition of Genes for Cellobiase and Pectinolytic Activity in Escherichia coli for Fuel Ethanol Production from Pectin-Rich Lignocellulosic Biomass
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Addition of Genes for Cellobiase and Pectinolytic Activity in Escherichia coli for Fuel Ethanol Production from Pectin-Rich Lignocellulosic Biomass

机译：富含果胶的木质纤维素生物质生产乙醇中纤维二糖酶和果胶分解活性基因的添加

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Ethanologenic Escherichia coli strain KO11 was sequentially engineered to contain the Klebsiella oxytoca cellobiose phosphotransferase genes (casAB) as well as a pectate lyase (pelE) from Erwinia chrysanthemi, yielding strains LY40A (casAB) and JP07 (casAB pelE), respectively. To obtain an effective secretion of PelE, the Sec-dependent pathway out genes from E. chrysanthemi were provided on a cosmid to strain JP07 to construct strain JP07C. Finally, oligogalacturonide lyase (ogl) from E. chrysanthemi was added to produce strain JP08C. E. coli strains LY40A, JP07, JP07C, and JP08C possessed significant cellobiase activity in cell lysates, while only strains JP07C and JP08C demonstrated extracellular pectate lyase activity. Fermentations conducted by using a mixture of pure sugars representative of the composition of sugar beet pulp (SBP) showed that strains LY40A, JP07, JP07C, and JP08C were able to ferment cellobiose, resulting in increased ethanol production from 15 to 45% in comparison to that of KO11. Fermentations with SBP at very low fungal enzyme loads during saccharification revealed significantly higher levels of ethanol production for LY40A, JP07C, and JP08C than for KO11. JP07C ethanol yields were not considerably higher than those of LY40A; however, oligogalacturonide polymerization studies showed an increased breakdown of biomass to small-chain (degree of polymerization, ≤6) oligogalacturonides. JP08C achieved a further breakdown of polygalacturonate to monomeric sugars, resulting in a 164% increase in ethanol yields compared to those of KO11. The addition of commercial pectin methylesterase (PME) further increased JP08C ethanol production compared to that of LY40A by demethylating the pectin for enzymatic attack by pectin-degrading enzymes.
机译：依次对产乙醇的大肠杆菌菌株KO11进行改造，使其包含产酸克雷伯氏菌纤维二糖磷酸转移酶基因（casAB）和来自欧文氏菊花的果胶酸裂合酶（pelE），分别产生菌株LY40A（casAB）和JP07（casAB pelE）。为了获得有效的PelE分泌，在粘粒中将来自大肠杆菌的Sec依赖性途径的基因提供给菌株JP07，以构建菌株JP07C。最后，加入来自菊花大肠杆菌的寡半乳糖醛酸苷裂解酶（ogl）以产生菌株JP08C。大肠杆菌LY40A，JP07，JP07C和JP08C菌株在细胞裂解物中具有明显的纤维二糖酶活性，而仅菌株JP07C和JP08C表现出细胞外果胶酸裂解酶活性。通过使用代表甜菜果肉（SBP）组成的纯糖混合物进行发酵，发现LY40A，JP07，JP07C和JP08C菌株能够发酵纤维二糖，与之相比，乙醇产量增加了15％至45％ KO11的在糖化过程中，在极低的真菌酶负荷下用SBP进行发酵，与KO11相比，LY40A，JP07C和JP08C的乙醇生产水平显着提高。 JP07C乙醇的收率并不比LY40A的高很多。然而，低聚半乳糖醛酸聚合研究表明，生物质分解为小链（聚合度≤6）的低聚半乳糖醛酸增加了。 JP08C进一步将聚半乳糖醛酸酯分解为单体糖，与KO11相比，乙醇的收率提高了164％。与LY40A相比，商业化果胶甲基酯酶（PME）的添加进一步提高了JP08C乙醇的产量，这是通过将果胶脱甲基以进行果胶降解酶的酶促攻击。

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