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Production of the versatile cellulase for cellulose bioconversion and cellulase inducer synthesis by genetic improvement of Trichoderma reesei

机译：纤维素生物转化和纤维素酶诱导剂的多功能纤维素酶的生产通过Trichoderma Reesei的遗传改善

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Abstract Background The enzymes for efficient hydrolysis of lignocellulosic biomass are a major factor in the development of an economically feasible cellulose bioconversion process. Up to now, low hydrolysis efficiency and high production cost of cellulases remain the significant hurdles in this process. The aim of the present study was to develop a versatile cellulase system with the enhanced hydrolytic efficiency and the ability to synthesize powerful inducers by genetically engineering Trichoderma reesei. Results In our study, we employed a systematic genetic strategy to construct the carbon catabolite-derepressed strain T. reesei SCB18 to produce the cellulase complex that exhibited a strong cellulolytic capacity for biomass saccharification and an extraordinary high β-glucosidase (BGL) activity for cellulase-inducing disaccharides synthesis. We first identified the hypercellulolytic and uracil auxotrophic strain T. reesei SP4 as carbon catabolite repressed, and then deleted the carbon catabolite repressor gene cre1 in the genome. We found that the deletion of cre1 with the selectable marker pyrG led to a 72.6% increase in total cellulase activity, but a slight reduction in saccharification efficiency. To facilitate the following genetic modification, the marker pyrG was successfully removed by homologous recombination based on resistance to 5-FOA. Furthermore, the Aspergillus niger BGLA-encoding gene bglA was overexpressed, and the generated strain T. reesei SCB18 exhibited a 29.8% increase in total cellulase activity and a 51.3-fold enhancement in BGL activity (up to 103.9 IU/mL). We observed that the cellulase system of SCB18 showed significantly higher saccharification efficiency toward differently pretreated corncob residues than the control strains SDC11 and SP4. Moreover, the crude enzyme preparation from SCB18 with high BGL activity possessed strong transglycosylation ability to synthesize β-disaccharides from glucose. The transglycosylation product was finally utilized as the inducer for cellulase production, which provided a 63.0% increase in total cellulase activity compared to the frequently used soluble inducer, lactose. Conclusions In summary, we constructed a versatile cellulase system in T. reesei for efficient biomass saccharification and powerful cellulase inducer synthesis by combinational genetic manipulation of three distinct types of genes to achieve the customized cellulase production, thus providing a viable strategy for further strain improvement to reduce the cost of biomass-based biofuel production.
机译：摘要背景下木质纤维素生物量有效水解的酶是经济上可行的纤维素生物转化过程的发展的主要因素。截至目前，纤维素酶的低水解效率和高生产成本仍然是该过程中的重大障碍。本研究的目的是开发一种具有增强的水解效率的通用纤维素酶系统，并通过基因工程richoderma Reesei合成强大的诱导症的能力。结果我们的研究中，我们采用了一种系统的遗传策略来构建碳分解粘土毒性菌株T.Reesei SCB18以产生纤维素酶复合物，其表现出对生物质糖化的强纤维素分解能力和纤维素酶的非凡的高β-葡糖苷酶（BGL）活性。 - 减少二糖合成。我们首先将高晶醇溶解和尿嘧啶营养慢性菌株T.ReeSei SP4作为碳分子粘土菌素抑制，然后在基因组中删除碳分解代谢物抑制基因Cre1。我们发现，通过选择标记Pyrg删除Cre1导致总纤维素酶活性的72.6％增加，但糖化效率略有降低。为了促进以下遗传修饰，通过基于抗5-FOA的同源重组成功地除去标记PyrG。此外，曲霉菌尼日尔BGLA编码基因BGLA已经过表达，并且所产生的菌株T.ReeseiSCB18显示出总纤维素酶活性的29.8％，BGL活性的51.3倍的增强（高达103.9 IU / ml）。我们观察到SCB18的纤维素酶系统对不同预处理的玉米菌残基的糖化效率显着更高，而不是对照菌株SDC11和SP4。此外，具有高BGL活性的SCB18的粗酶制剂具有强血糖基化能力，以合成葡萄糖的β-二糖。最终用作纤维素酶生产的诱导剂的转糖基化产物，其与常用可溶性诱导剂，乳糖相比，总纤维素酶活性的增加了63.0％。总结结论，我们通过组合生物量糖化和强大的纤维素酶诱导剂合成，通过组合生物质糖化和强大的纤维素酶诱导型合成，三种不同类型基因来实现定制的纤维素酶生产，从而为进一步应变改善提供可行的策略降低基于生物质的生物燃料生产成本。

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