一体化生物加工过程进行大肠杆菌的半纤维素琥珀酸生产
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摘要
复杂的结构及解聚所需的高成本是阻碍半纤维素作为底物进行生物燃料和生物化学品生产的主要障碍。一体化生物加工过程（Consolidated Bioprocessing,CBP）通过利用一种微生物同时行使水解酶的生产，生物质原料的降解以及生物产品的转化这三大功能，能够显著降低木质纤维素原料包括半纤维素利用过程的成本。在本文中，通过借鉴代谢工程与合成生物学的基本原理与思想，改造大肠杆菌使其表达分泌三种半纤维素酶，实现了直接以山毛榉木聚糖为底物进行琥珀酸生产的生物转化过程。
木聚糖酶的胞外分泌是通过与OsmY蛋白的融合实现的。将十二个不同来源的并与OsmY融合的木聚糖内切酶-木糖苷酶组合进行酶学表征，选择出一组具有最优酶学特性的木聚糖酶组合，即来自于Fibrobacter succinogenes S85的木聚糖内切酶XynC-A以及来自于Fusarium graminearum的木糖苷酶XyloA。在该菌株中，木聚糖酶诱导表达8h时，即可向胞外分泌0.18±0.01g/l的蛋白质，木聚糖内切酶活性为12.14±0.34U/mg，木糖苷酶活性为92±3mU/mg。
随后通过一系列措施来进一步优化木聚糖酶的表达分泌，以提高其水解效率，更好的满足CBP生产的需要，包括敲除大肠杆菌Braun’s lipoprotein的编码基因lpp；过表达催化二硫键形成的氧化还原酶编码基因dsbA；优化木聚糖酶表达载体的表达水平；通过Blc蛋白于外膜表面展示木糖苷酶；过表达来自于枯草芽孢杆菌168的阿拉伯糖苷酶编码基因abf2等。
将半纤维素水解能力与一株琥珀酸高产菌进行整合，实现了在没有提供外部木聚糖水解酶及任何有机组分的情况下直接以木聚糖为底物进行琥珀酸的生产。由此构建的大肠杆菌菌株Z6373在厌氧条件下以木聚糖为底物，可以得到0.37g/g的琥珀酸，转化率达到了以木聚糖酸水解液为底物时的76%。
本文通过表达和分泌三种半纤维素酶成功的建立起一个大肠杆菌以木聚糖为底物进行琥珀酸生产的CBP系统。本文结果表明了通过CBP进行半纤维素到琥珀酸转化的可行性，为其进一步改良及木质纤维素材料向其他化学品的转化上奠定了一定基础。
The recalcitrant nature of hemicellulosic materials and the high cost indepolymerization are the primary obstacles preventing the use of xylan as feedstockfor fuel and chemical production. Consolidated bioprocessing (CBP), incorporatingenzyme-generating, biomass-degrading and bioproduct-producing capabilities into asingle microorganism, could potentially avoid the cost of the dedicated enzymegeneration in the process of xylan utilization. In this study, drawing on the basicprinciples of metabolic engineering and synthetic biology, we engineered Escherichiacoli strains capable of exporting three hemicellulases to the broth for the succinateproduction directly from beechwood xylan.
Xylanases were extracellular environment-directed by fusing with OsmY.Subsequently, twelve variant OsmY fused endoxylanase-xylosidase combinationswere characterized and tested. The combination of XynC-A from Fibrobactersuccinogenes S85and XyloA from Fusarium graminearum which appeared to haveoptimal enzymatic properties was identified as the best choice for xylan hydrolysis(0.18±0.01g/l protein in the broth with endoxylanase activity of12.14±0.34U/mgprotein and xylosidase activity of92±3mU/mg protein at8h after induction).
To improve the hydrolysis efficiency and for better meet the needs of the CBPproduction, further improvements of hemicellulases secretion were investigated bylpp deletion, dsbA overexpression, blc surface displaying and expression leveloptimization. With co-expression of α-arabinofuranosidase, the engineered E. colicould hydrolyze beechwood xylan to pentose monosaccharides.
The hemicellulolytic capacity was further integrated with a succinate-producing strainto demonstrate the production of succinate directly from xylan without externallysupplied hydrolases and any other organic nutrient. The resulting E. coli Z6373wasable to produce0.37g/g succinate from xylan anaerobically equivalent to76%of thatfrom xylan acid hydrolysates.
This report represents a promising step towards the goal of hemicellulosic chemicalproduction. This engineered E. coli expressing and secreting three hemicellulasesdemonstrated a considerable succinate production on the released monosaccharidesfrom xylan. The ability to use lower-cost crude feedstock will make biological succinate production more economically attractive.
木聚糖酶的胞外分泌是通过与OsmY蛋白的融合实现的。将十二个不同来源的并与OsmY融合的木聚糖内切酶-木糖苷酶组合进行酶学表征，选择出一组具有最优酶学特性的木聚糖酶组合，即来自于Fibrobacter succinogenes S85的木聚糖内切酶XynC-A以及来自于Fusarium graminearum的木糖苷酶XyloA。在该菌株中，木聚糖酶诱导表达8h时，即可向胞外分泌0.18±0.01g/l的蛋白质，木聚糖内切酶活性为12.14±0.34U/mg，木糖苷酶活性为92±3mU/mg。
随后通过一系列措施来进一步优化木聚糖酶的表达分泌，以提高其水解效率，更好的满足CBP生产的需要，包括敲除大肠杆菌Braun’s lipoprotein的编码基因lpp；过表达催化二硫键形成的氧化还原酶编码基因dsbA；优化木聚糖酶表达载体的表达水平；通过Blc蛋白于外膜表面展示木糖苷酶；过表达来自于枯草芽孢杆菌168的阿拉伯糖苷酶编码基因abf2等。
将半纤维素水解能力与一株琥珀酸高产菌进行整合，实现了在没有提供外部木聚糖水解酶及任何有机组分的情况下直接以木聚糖为底物进行琥珀酸的生产。由此构建的大肠杆菌菌株Z6373在厌氧条件下以木聚糖为底物，可以得到0.37g/g的琥珀酸，转化率达到了以木聚糖酸水解液为底物时的76%。
本文通过表达和分泌三种半纤维素酶成功的建立起一个大肠杆菌以木聚糖为底物进行琥珀酸生产的CBP系统。本文结果表明了通过CBP进行半纤维素到琥珀酸转化的可行性，为其进一步改良及木质纤维素材料向其他化学品的转化上奠定了一定基础。
The recalcitrant nature of hemicellulosic materials and the high cost indepolymerization are the primary obstacles preventing the use of xylan as feedstockfor fuel and chemical production. Consolidated bioprocessing (CBP), incorporatingenzyme-generating, biomass-degrading and bioproduct-producing capabilities into asingle microorganism, could potentially avoid the cost of the dedicated enzymegeneration in the process of xylan utilization. In this study, drawing on the basicprinciples of metabolic engineering and synthetic biology, we engineered Escherichiacoli strains capable of exporting three hemicellulases to the broth for the succinateproduction directly from beechwood xylan.
Xylanases were extracellular environment-directed by fusing with OsmY.Subsequently, twelve variant OsmY fused endoxylanase-xylosidase combinationswere characterized and tested. The combination of XynC-A from Fibrobactersuccinogenes S85and XyloA from Fusarium graminearum which appeared to haveoptimal enzymatic properties was identified as the best choice for xylan hydrolysis(0.18±0.01g/l protein in the broth with endoxylanase activity of12.14±0.34U/mgprotein and xylosidase activity of92±3mU/mg protein at8h after induction).
To improve the hydrolysis efficiency and for better meet the needs of the CBPproduction, further improvements of hemicellulases secretion were investigated bylpp deletion, dsbA overexpression, blc surface displaying and expression leveloptimization. With co-expression of α-arabinofuranosidase, the engineered E. colicould hydrolyze beechwood xylan to pentose monosaccharides.
The hemicellulolytic capacity was further integrated with a succinate-producing strainto demonstrate the production of succinate directly from xylan without externallysupplied hydrolases and any other organic nutrient. The resulting E. coli Z6373wasable to produce0.37g/g succinate from xylan anaerobically equivalent to76%of thatfrom xylan acid hydrolysates.
This report represents a promising step towards the goal of hemicellulosic chemicalproduction. This engineered E. coli expressing and secreting three hemicellulasesdemonstrated a considerable succinate production on the released monosaccharidesfrom xylan. The ability to use lower-cost crude feedstock will make biological succinate production more economically attractive.
引文
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