Agrivida, Inc., Medford, Massachusetts, USA.
Nat Biotechnol. 2012 Nov;30(11):1131-6. doi: 10.1038/nbt.2402. Epub 2012 Oct 21.
Plant cellulosic biomass is an abundant, low-cost feedstock for producing biofuels and chemicals. Expressing cell wall-degrading (CWD) enzymes (e.g. xylanases) in plant feedstocks could reduce the amount of enzymes required for feedstock pretreatment and hydrolysis during bioprocessing to release soluble sugars. However, in planta expression of xylanases can reduce biomass yield and plant fertility. To overcome this problem, we engineered a thermostable xylanase (XynB) with a thermostable self-splicing bacterial intein to control the xylanase activity. Intein-modified XynB (iXynB) variants were selected that have <10% wild-type enzymatic activity but recover >60% enzymatic activity upon intein self-splicing at temperatures >59 °C. Greenhouse-grown xynB maize expressing XynB has shriveled seeds and low fertility, but ixynB maize had normal seeds and fertility. Processing dried ixynB maize stover by temperature-regulated xylanase activation and hydrolysis in a cocktail of commercial CWD enzymes produced >90% theoretical glucose and >63% theoretical xylose yields.
植物纤维素生物质是生产生物燃料和化学品的丰富、低成本原料。在植物饲料中表达细胞壁降解（CWD）酶（如木聚糖酶）可以减少生物加工过程中用于饲料预处理和水解所需的酶量，以释放可溶性糖。然而，木聚糖酶在植物体内的表达会降低生物质产量和植物的繁殖力。为了克服这个问题，我们设计了一种具有热稳定自剪接细菌内含肽的热稳定木聚糖酶（XynB）来控制木聚糖酶的活性。选择了具有<10%野生型酶活性的内含肽修饰的 XynB（iXynB）变体，但在>59°C 时，内含肽自剪接后，酶活性恢复>60%。在温室中种植表达 XynB 的 xynB 玉米，其种子干瘪，繁殖力低，但 ixynB 玉米的种子和繁殖力正常。用商业 CWD 酶混合物通过温度调节的木聚糖酶激活和水解处理干燥的 ixynB 玉米秸秆，可产生>90%理论葡萄糖和>63%理论木糖收率。
