瑞氏木霉β-葡萄糖苷酶在纤维素酶诱导表达过程中作用机制的研究
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摘要
木质纤维素来源的生物燃料是一种清洁的可再生能源,能部分替代目前人们对化石能源的需求,然而就目前的技术水平而言,将植物生物质高效转化为可发酵的单糖是生物燃料生产的主要瓶颈。自然界中存在大量能够降解植物生物质的微生物,是我们转化和利用木质纤维素的基础。其中丝状真菌瑞氏木霉(Trichoderma reesei)是纤维素降解微生物的典型代表,它的一个重要生理特性是在不溶性的纤维素、半纤维素以及一些植物生物质组分存在条件下,能够大量分泌纤维素酶,从而有效水解植物生物质。因为这些不溶性的底物无法进入细胞,由它们释放的可溶性寡糖包括纤维二糖以及槐糖等被认为是真正的诱导物,但目前这种假设还缺少足够的实验证据。
除了纤维二糖和槐糖,非植物细胞壁组分的乳糖也可以诱导瑞氏木霉产生纤维素酶,但目前人们对乳糖诱导纤维素酶表达的机制也不甚了解。虽然有实验证据表明乳糖的胞外水解以及随后的半乳糖代谢与纤维素酶诱导表达有一定相关性,但是也有证据表明瑞氏木霉对乳糖的吸收和胞内代谢在纤维素酶的诱导表达过程中起到重要作用。到目前为止在瑞氏木霉中负责胞内水解和转化乳糖的蛋白尚未得到鉴定。
有证据显示瑞氏木霉p-葡萄糖苷酶在诱导物合成以及菌体快速响应纤维素的过程中发挥重要作用,例如胞外主要p-葡萄糖苷酶bgl1/cel3a基因的缺失导致纤维素酶基因诱导表达延迟,而过表达bgl1/cel3a基因的菌株在纤维素和槐糖诱导条件下纤维素酶基因的表达水平提高。基因组信息分析表明,瑞氏木霉一共含有11个p-葡萄糖苷酶编码基因,其中Bgl1/Cel3a是胞外主要的p-葡萄糖苷酶,属于糖苷水解酶第3家族,而Cel1a和Cel1b是胞内p-葡萄糖苷酶,属于糖苷水解酶第1家族。转录组学数据显示,这三个p-葡萄糖苷酶在纤维素和乳糖诱导条件下转录上调最为显著,是瑞氏木霉最主要的p-葡萄糖苷酶,它们在瑞氏木霉纤维素酶基因诱导表达过程中的作用有待于进一步的研究。大多数糖苷水解酶第1家族的p-葡萄糖苷酶同时具有p-半乳糖苷酶活性,是乳糖胞内代谢酶的重要候选蛋白,瑞氏木霉第1家族的p-葡萄糖苷酶Cel1a和Cel1b是否具有乳糖水解活性以及它们在乳糖诱导纤维素酶表达过程中是否发挥作用目前还不清楚。
为了深入阐释瑞氏木霉三个主要的β-葡萄糖苷酶Bgl1/Cel3a、Cel1a和Cel1b在纤维素酶基因诱导表达过程中的作用,我们将这三个β-葡萄糖苷酶的编码基因分别进行了敲除以及组合多敲除。详细研究了β-葡萄糖苷酶基因缺失菌株响应纤维素、纤维二糖、槐糖以及乳糖等碳源表达纤维素酶的能力,对瑞氏木霉β-葡萄糖苷酶影响纤维素酶诱导表达的机制进行了分析。同时研究了瑞氏木霉两个糖苷水解酶第1家族的β-葡萄糖苷酶Cel1a和Cel1b对乳糖的水解和转化作用,并分析了Cel1a和Cel1b对乳糖诱导纤维素酶表达的影响及其发挥作用的机制。本论文的主要创新结果如下：
1.对瑞氏木霉三个主要β-葡萄糖苷酶编码基因bgl1/cel3a, cel1a以及cel1b进行基于同源重组原理的基因敲除,同时构建了多基因缺失菌株△cel1a△cel1lb和△tripG,以研究不同β-葡萄糖苷酶的生物学功能。
为了研究不同β-葡萄糖苷酶的功能,我们通过同源双交换的手段构建了一系列β-葡萄糖苷酶基因缺失菌株△cel1a,△cel1b,△cel1a△cel1b和△tripG(bgll, cel1a和cel1b同时缺失),锚定PCR以及Southern blot验证了基因敲除以及单拷贝整合。我们进一步分析了不同β-葡萄糖苷酶的缺失对瑞氏木霉胞内外β-葡萄糖苷酶总酶活的影响。在纤维素培养条件下,与野生型菌株相比△cel1b菌株胞内β-葡萄糖苷酶酶活只有轻微下降,而△cel1a菌株胞内β-葡萄糖苷酶酶活下降75%,△cel1a△cel1b菌株胞内β-葡萄糖苷酶酶活与△cel1a菌株类似,说明Cella是胞内主要的β-葡萄糖苷酶,Cel1b对胞内总β-葡萄糖苷酶酶活的贡献不大。Bgl1/Ce13a缺失后瑞氏木霉胞外β-葡萄糖苷酶活性仅有10%残留,说明Bgl1/Ce13a是瑞氏木霉胞外最主要的β-葡萄糖苷酶。
2.比较了不同β-葡萄糖苷酶基因缺失菌株在纤维素以及槐糖上的生长和纤维素酶诱导表型,证明在不溶性纤维素诱导条件下纤维素酶基因的快速诱导表达需要β-葡萄糖苷酶的参与。
我们首先研究了瑞氏木霉β-葡萄糖苷酶是否参与不溶性纤维素诱导纤维素酶表达的过程。通过测定不同β-葡萄糖苷酶基因缺失菌株在纤维素培养过程中的生长曲线,我们发现β-葡萄糖苷酶的缺失影响菌株对纤维素的利用。此外,在纤维素诱导条件下β-葡萄糖苷酶基因缺失菌株发酵液中总蛋白量和纤维素酶酶活均比野生型菌株低。我们进一步使用Northern blot和qRT-PCR的方法研究了纤维素诱导条件下不同β-葡萄糖苷酶基因缺失菌株纤维素酶基因cbh1的转录动力学。结果显示瑞氏木霉野生型菌株在纤维素诱导3h之后cbh1转录即可达到较高水平,而△cel1b菌株需要诱导6h,△cel1a菌株需要诱导24h,△cel1a△cel1b菌株需要诱导36h,△triβG菌株则需要诱导48h才能达到与野生型类似的水平。β-葡萄糖苷酶基因缺失菌株发酵液中CBHI的表达也呈现与转录类似的结果。与纤维素不同,槐糖诱导条件下△cel1a△cel1b菌株cbhl转录动力学与野生型一致,说明β-葡萄糖苷酶在槐糖诱导纤维素酶表达过程中并未发挥重要作用。综上所述,我们证明了这三个β-葡萄糖苷酶在瑞氏木霉菌株快速响应纤维素启动纤维素酶表达过程中发挥了重要作用,它们有可能参与了诱导物的合成。
3.通过分析在纤维二糖诱导条件下瑞氏木霉主要β-葡萄糖苷酶基因缺失菌株的纤维素酶表达表型,证明主要β-葡萄糖苷酶的缺失能提高纤维二糖对纤维素酶的诱导能力,瑞氏木霉主要β-葡萄糖苷酶对纤维二糖的转化作用在纤维素酶诱导表达过程中并未发挥重要作用。
作为纤维素的主要降解产物,纤维二糖在低浓度下也可以诱导瑞氏木霉产生纤维素酶,而且有证据显示瑞氏木霉β-葡萄糖苷酶能够在体外将纤维二糖通过转糖基作用转化为槐糖这一强效诱导物。为了分析瑞氏木霉β-葡萄糖苷酶在纤维二糖诱导纤维素酶表达过程中发挥的作用,首先我们摸索了在瑞氏木霉中纤维二糖诱导纤维素酶表达的最佳浓度。实验确定0.25%(w/v)纤维二糖为最佳诱导浓度。其次我们通过Western blot测定了β-葡萄糖苷酶基因缺失菌株发酵液中CBHI表达量,并使用qRT-PCR检测了菌体纤维素酶cbhl基因的转录水平,结果显示在纤维二糖诱导条件下β-葡萄糖苷酶基因缺失菌株中纤维素酶的表达水平明显高于野生型,说明降低瑞氏木霉β-葡萄糖苷酶的活性能提高纤维二糖诱导纤维素酶表达的能力,瑞氏木霉主要的β-葡萄糖苷酶对纤维二糖的转化作用在纤维素酶的诱导表达过程中并未发挥重要作用。最后,我们在纤维素培养过程中外源添加一定比例的纤维二糖,发现β-葡萄糖苷酶基因缺失菌株快速响应纤维素产生纤维素酶的能力得到恢复,表明β-葡萄糖苷酶可能通过调控胞外纤维二糖的水平影响纤维素诱导条件下纤维素酶基因的快速表达。
4.证明瑞氏木霉胞内主要β-葡萄糖苷酶Cel1a和Cel1b在乳糖诱导纤维素酶表达过程中发挥重要作用。Cel1a和Cel1b的缺失影响纤维素酶基因转录调控因子Xyrl的表达,但是组成型回补表达Xyr1并不能恢复β-葡萄糖苷酶基因缺失菌株响应乳糖产生纤维素酶的能力。
瑞氏木霉糖苷水解酶第1家族的β-葡萄糖苷酶是乳糖胞内代谢酶的重要候选蛋白,因此我们分析了β-葡萄糖苷酶Cel1a和Cel1b是否影响菌体对乳糖的利用以及乳糖诱导纤维素酶基因的表达。通过生长曲线的测定,我们发现胞内p-葡萄糖苷酶的缺失影响菌体在乳糖碳源上的生长,但是并不影响菌体对半乳糖的利用。通过Western blot分析乳糖培养条件下β-葡萄糖苷酶基因缺失菌株发酵液中纤维素酶CBHI的表达,我们发现β-葡萄糖苷酶基因cel1a的缺失导致乳糖诱导纤维素酶表达延迟,而在双缺菌株△cel1a△cel1b中纤维素酶的表达完全缺失。进一步通过qRT-PCR分析纤维素酶基因cbh1的转录发现β-葡萄糖苷酶基因缺失菌中纤维素酶表达的滞后或缺失发生在转录层面,说明瑞氏木霉胞内β-葡萄糖苷酶Cel1a和Cel1b对乳糖诱导纤维素酶表达是必需的。Xyr1是调控纤维素酶基因诱导表达的关键转录因子,因此我们分析了β-葡萄糖苷酶基因缺失菌株中xyr1基因的转录水平,实验发现虽然β-葡萄糖苷酶的缺失造成xyr1基因的转录水平降低,但在△cel1a△cel1b菌株中组成型表达xyr1并不能恢复菌株响应乳糖产生纤维素酶的能力。上述结果表明胞内β-葡萄糖苷酶在乳糖诱导纤维素酶表达过程中发挥了重要作用,β-葡萄糖苷酶不仅影响了关键转录因子Xyr1的表达,还通过其他途径影响乳糖诱导纤维素酶表达的过程,β-葡萄糖苷酶发挥作用的机制还需要进一步的分析。
5.通过分析Cel1a和Cel1b重组蛋白的底物特异性,证明瑞氏木霉糖苷水解酶第1家族的β-葡萄糖苷酶具有乳糖水解活性和转化活性。
糖苷水解酶第1家族的β-葡萄糖苷酶往往具有β-半乳糖苷酶活性,瑞氏木霉糖苷水解酶第1家族的β-葡萄糖苷酶Cel1a和Cel1b可能在乳糖胞内代谢过程中发挥作用。因此我们通过大肠杆菌异源表达和纯化了瑞氏木霉β-葡萄糖苷酶Cel1a和Cel1b,并对其底物特异性进行了分析。我们发现Cel1a和Cel1b不仅能够水解纤维二糖及其类似物pNPG,还能够水解乳糖类似物DNPG,证明其具有β-半乳糖苷酶活性。通过HPLC分析β-葡萄糖苷酶Cel1a与乳糖反应的产物,我们发现除了D-葡萄糖和D-半乳糖,还有其他半乳寡糖产生,表明β-葡萄糖苷酶Cel1a可能具有乳糖转糖基活性。瑞氏木霉β-葡萄糖苷酶具有的这种糖苷水解活性在纤维素酶诱导表达过程中的作用还需要进一步的研究。
6.证明了瑞氏木霉主要β-葡萄糖苷酶具有的糖苷水解活性对乳糖诱导纤维素酶表达是必需的。
为了进一步分析瑞氏木霉β-葡萄糖苷酶Cel1a和Cel1b所具有糖苷水解活性对乳糖诱导纤维素酶表达的影响,我们在瑞氏木霉△cel1a菌株中组成型表达了野生型Cel1a蛋白以及水解活性缺失点突变体Cel1a (Q367A)蛋白。在乳糖诱导条件下,组成型回补野生型Cel1a蛋白使得△cel1a菌株cbh1的转录得到恢复,cbh1转录起始时间甚至比野生型提前,cbh1转录量也有所升高,而回补水解活性缺失点突变体Cella (Q367A)蛋白的菌株cbh1转录并未恢复,转录起始时间与野生型相比仍然有明显的滞后,这些结果说明Cel1a的糖苷水解活性对乳糖诱导纤维素酶的表达是必需的。为了进一步分析β-葡萄糖苷酶具有的乳糖水解活性在纤维素酶诱导表达过程中的作用,我们在△cel1a菌株中组成型表达了乳酸克鲁维酵母来源的胞内β-半乳糖苷酶Lac4,结果显示回补Lac4并不能恢复△cel1a菌株纤维素酶基因cbh1的转录,表明仅有乳糖胞内水解活性并不足以启动纤维素酶的诱导表达,Cel1a还通过其他途径对纤维素酶基因的诱导表达产生影响。
Biofuel derived from lignocellulose is a clean, renewable energy that can partially replace our current demand for fossil fuels. However, degrading the insoluble plant biomass into fermentable monosaccharaides represents a main bottleneck for biofuel production. Trichoderma reesei secretes large amounts of (hemi) cellulase that could hydrolyze plant biomass into soluble oligosaccharides, and is now one of the most important industrial strains to produce cellulases and hemicellulases. The mechanism of cellulase induction is still indistinct and the best inducers of the cellulase for T. reesei are preferably insoluble cellulose, hemicellulose, and plant biomasses. Currently it is generally believed that small molecules such as cellobiose and sophorose released from these insoluble substrates are the real inducers.
In addition to cellobiose and sophorose, lactose can also induce the production of cellulase, however currently we still do not understand the mechanism underlying this induction. There are some experimental evidences showing that lactose transport and subsequent intracellular hydrolysis or conversion may play important roles in the induction process. T. reesei lacks typical intracellular β-galactosidase which belongs to glycoside hydrolase family2, and the intracellular protein(s) involved in intracellular lactose hydrolysis and conversion has (have) not yet been identified.
There are lines of evidence that β-glucosidases play a vital role in the process of rapid response to cellulose and the induced synthesis of cellulase in T. reesei. For example, deletion of the major extracellular β-glucosidase encoding gene bgll/cl3a delayed cellulase expression on cellulose, while the bgll/cel3a overexpressing strain had an elevated expression of cellulase not only on cellulose but also on sophorose (a potential inducer of cellulase). T. reesei contains11P-glucosidases, among which Bgll/Cel3a is an extracellular β-glucosidase, belonging to glycoside hydrolase family3, and Cell a and Cellb are intracellular enzymes both belonging to glycoside hydrolase family1. Transcription of these three β-glucosidases on cellulose and lactose is significantly raised in T. reesei. Additionally, in some cellulase-high-yielding strains, transcription of these three β-glucosidases is higher than that of the wild type, suggesting that they may be involved in the process of cellulase induction. It is a common phenomenon that β-glucosidases of glycoside hydrolase family1also possess side β-galactosidase activity, making them important candidates for intracellular conversion of lactose. Whether the two GH1P-glucosidases Cel1a and Cel1b of T. reesei have β-galactosidase activity and their roles in lactose induction need further study.
In order to illustrate the roles of β-glucosidases in cellulase induction of T. reesei, three main β-glucosidase genes bgl1/cel3a, Cel1a and Cel1b were knockout singly and multiply. Cellulase expression capacity of these β-glucosidase-deficient strains was studied on different carbon sources such as cellulose, cellobiose, sophorose and lactose. Besides, the two intracellular β-glucosidases Cel1a and Cellb were heterologously expressed to study their activities on lactose and their roles in cellulase induction by lactose. The main innovative results of this study are as follows:
1. A series of β-glucosidases deletion strains were constructed by gene replacement to study the in vivo function of these proteins in cellulase induction.
In order to study the in vivo function of different β-glucosidases in T. reesei, we constructed a number of β-glucosidase deletion strains by means of homologous recombination, including△cel1a,△cel1b,△cel1a△cel1b and△triβG. Anchored PCR and Southern blot were used to verify gene knockout and single copy integration. During culture on cellulose, intracellular β-glucosidase activity of Acel1b was slightly lower than that of wild type strain, while it fell75%in the Acella strain, the extracellular β-glucosidase activity of Bgll deletion strain dramatically declined. In summary, Cel3a accounts for the major extracellular β-glucosidase activity, while Cel1a is the major intracellular β-glucosidase, Cel1b contributes only a small part to the total intracellular β-glucosidase activity in T. reesei.
2. Deletion of cella, cellb delayed cellulase expression on cellulose, but not on sophorose, suggesting that intracellular Cella and Cellb contribute to the induction of cellulase genes probably through participating in the formation of cellulase inducer.
To further analyze the influence of P-glucosidases on cellulase expression we studied the kinetics of cbhl gene transcription in different β-glucosidase-mutant strains using Northern blot/qRT-PCR. This analysis showed that in comparison to the rapid induction in the WT strain, which occurred as early as3h upon induction by cellulose, productive activation of transcription of cbhl was delayed by about3h and21h in the△cel1b and△cel1a strains, respectively. This lag in gene expression was further extended to36h in the△cel1a△cel1b strain, although the final level of transcription was almost the same. Western blot of CBHI protein in the fermentation broth also showed similar results. However, the induction effects by sophorose were not affected in the P-glucosidase mutant strains. In summary, all these data suggests that β-glucosidases are vital in rapid cellulase induction, and may be involved in inducer synthesis.
3. Deletion of major β-glucosidases in T. reesei resulted in higher expression levels of cellulase on cellobiose suggesting that lowering the degree of cellobiose hydrolysis may facilitate cellulase synthesis.
As the main degradation products of cellulose, cellobiose at a low concentration can induce the expression of cellulase and it has been shown that cellobiose could be transformed into sophorose by β-glucosidases in T. reesei. We therefore analyzed the impact of β-glucosidases on cellulase induction by cellobiose. The cellulase expression on cellobiose were significantly elevated in△cel1a△cel1b and△triβG strains suggesting that lowering the degree of cellobiose hydrolysis may facilitate cellulase synthesis and the conversion of cellobiose by the main intracellular and extracellular β-glucosidases may not account for the rapid cellulase expression on cellulose. By exogenously adding a certain proportion of cellobiose to cellulose medium, we found that the cellulase induction capacity of β-glucosidase-deficient strains on cellulose recovered. These data indicated that the productive induction defect on cellulose as observed in the absence of the major β-glucosidases may to a larger extent be caused by the insufficient cellobiose initially available for triggering the induction cascade.
4. Intracellular β-glucosidases Cell a and Cellb affect expression of cellulase and Xyrl by lactose. Constitutive expression of Xyrl could not restore the efficient induction of β-glucosidase-deficient strains.
We further analyze the effect of β-glucosidases on induction of cellulase by lactose. By analyzing the cellulase expression of△cella,△cel1b and△cel1a△cel1b strains cultured on lactose, we found that single deletion of intracellular β-glucosidase lead to lower cellulase expression on lactose, while double deletion of cel1a and cel1b almost abolished it. Further analysis of cbhl transcription by qRT-PCR revealed that the decrease of the cellulase expression occurred at the transcriptional level. These results show that intracellular β-glucosidases are essential for efficient induction of cellulase by lactose. Xyrl is the key transcription factor regulating cellulase expression, whose absence abolished cellulase expression on cellulose and lactose. We found transcription of xyrl decreased in the△cel1a△cel1b strain cultured on lactose, however constitutively expression of xyrl could not restore its efficient cellulase expression, suggesting that expression of xyrl alone is not sufficient to activate the cellulolytic transcription by lactosein the absence of Cel1a and Cel1b.
5. Intracellular β-glucosidase Cel1a and Cel1b exhibited apparent hydrolytic activity toward ONPG and lactose.
Enzymes involved in intracellular metabolism of lactose have not so far been identified in T. reesei. Most β-glucosidases belonging to GH1also possess β-galactosidase activity. Besides this, our previous data showed that intracellular β-glucosidases played central roles in the progress of cellulase induction by lactose. All these data make us speculate whether Cel1a and Cel1b of T. reesei possess P-galactosidase activity. So we heterologously expressed and purified Cel1a and Cel1b in E. coli and analyzed their substrate specificity. We found that Cel1a and Cel1b can hydrolyze not only cellobiose and pNPG, but also lactose and ONPG, suggesting that they possess β-galactosidase activity. Analyzing the lactose hydrolyzing products by HPLC, we found oligosaccharides in addition to D-glucose and D-galactose were also produced, suggesting that β-glucosidases of T. reesei also possess transglycosylation activity toward lactose.
6. Both Cel1a and its associated hydrolytic activity are responsible for the efficient induction of cellulase genes by lactose.
To gain insight into the possibility that Cel1a-associated glycoside hydrolytic activity is involved in the induction process, we retransformed△cel1a strain with wild-type Cella and a mutant protein Cella (Q367A) which bears no activity towards cellobiose and lactose. While constitutive expression of WT Cel1a resulted in a relatively higher induced expression of cbhl as compared with the WT strain, cells expressing the Cella (Q367A) displayed the same kinetic of cbhl induction as in the△cel1a strain on lactose. These results suggest that hydrolytic activity of Cel1a was required for cellulase expression. To further test Cel1a-associated hydrolytic activity toward lactose is fully responsible for cellulase induction, β-galactosidase LAC4from Kluyveromyces lactis was expressed in the Acel1a strain. Analysis of the induced expression of cbh1revealed no restoration of induced cbhl expression in the△cel1aCElac4strain. Altogether, these data suggest that intracellular processing of lactose mediated by Cella and Cellb beyond sole hydrolysis may account for their important roles in cellulase induction by lactose.
除了纤维二糖和槐糖,非植物细胞壁组分的乳糖也可以诱导瑞氏木霉产生纤维素酶,但目前人们对乳糖诱导纤维素酶表达的机制也不甚了解。虽然有实验证据表明乳糖的胞外水解以及随后的半乳糖代谢与纤维素酶诱导表达有一定相关性,但是也有证据表明瑞氏木霉对乳糖的吸收和胞内代谢在纤维素酶的诱导表达过程中起到重要作用。到目前为止在瑞氏木霉中负责胞内水解和转化乳糖的蛋白尚未得到鉴定。
有证据显示瑞氏木霉p-葡萄糖苷酶在诱导物合成以及菌体快速响应纤维素的过程中发挥重要作用,例如胞外主要p-葡萄糖苷酶bgl1/cel3a基因的缺失导致纤维素酶基因诱导表达延迟,而过表达bgl1/cel3a基因的菌株在纤维素和槐糖诱导条件下纤维素酶基因的表达水平提高。基因组信息分析表明,瑞氏木霉一共含有11个p-葡萄糖苷酶编码基因,其中Bgl1/Cel3a是胞外主要的p-葡萄糖苷酶,属于糖苷水解酶第3家族,而Cel1a和Cel1b是胞内p-葡萄糖苷酶,属于糖苷水解酶第1家族。转录组学数据显示,这三个p-葡萄糖苷酶在纤维素和乳糖诱导条件下转录上调最为显著,是瑞氏木霉最主要的p-葡萄糖苷酶,它们在瑞氏木霉纤维素酶基因诱导表达过程中的作用有待于进一步的研究。大多数糖苷水解酶第1家族的p-葡萄糖苷酶同时具有p-半乳糖苷酶活性,是乳糖胞内代谢酶的重要候选蛋白,瑞氏木霉第1家族的p-葡萄糖苷酶Cel1a和Cel1b是否具有乳糖水解活性以及它们在乳糖诱导纤维素酶表达过程中是否发挥作用目前还不清楚。
为了深入阐释瑞氏木霉三个主要的β-葡萄糖苷酶Bgl1/Cel3a、Cel1a和Cel1b在纤维素酶基因诱导表达过程中的作用,我们将这三个β-葡萄糖苷酶的编码基因分别进行了敲除以及组合多敲除。详细研究了β-葡萄糖苷酶基因缺失菌株响应纤维素、纤维二糖、槐糖以及乳糖等碳源表达纤维素酶的能力,对瑞氏木霉β-葡萄糖苷酶影响纤维素酶诱导表达的机制进行了分析。同时研究了瑞氏木霉两个糖苷水解酶第1家族的β-葡萄糖苷酶Cel1a和Cel1b对乳糖的水解和转化作用,并分析了Cel1a和Cel1b对乳糖诱导纤维素酶表达的影响及其发挥作用的机制。本论文的主要创新结果如下：
1.对瑞氏木霉三个主要β-葡萄糖苷酶编码基因bgl1/cel3a, cel1a以及cel1b进行基于同源重组原理的基因敲除,同时构建了多基因缺失菌株△cel1a△cel1lb和△tripG,以研究不同β-葡萄糖苷酶的生物学功能。
为了研究不同β-葡萄糖苷酶的功能,我们通过同源双交换的手段构建了一系列β-葡萄糖苷酶基因缺失菌株△cel1a,△cel1b,△cel1a△cel1b和△tripG(bgll, cel1a和cel1b同时缺失),锚定PCR以及Southern blot验证了基因敲除以及单拷贝整合。我们进一步分析了不同β-葡萄糖苷酶的缺失对瑞氏木霉胞内外β-葡萄糖苷酶总酶活的影响。在纤维素培养条件下,与野生型菌株相比△cel1b菌株胞内β-葡萄糖苷酶酶活只有轻微下降,而△cel1a菌株胞内β-葡萄糖苷酶酶活下降75%,△cel1a△cel1b菌株胞内β-葡萄糖苷酶酶活与△cel1a菌株类似,说明Cella是胞内主要的β-葡萄糖苷酶,Cel1b对胞内总β-葡萄糖苷酶酶活的贡献不大。Bgl1/Ce13a缺失后瑞氏木霉胞外β-葡萄糖苷酶活性仅有10%残留,说明Bgl1/Ce13a是瑞氏木霉胞外最主要的β-葡萄糖苷酶。
2.比较了不同β-葡萄糖苷酶基因缺失菌株在纤维素以及槐糖上的生长和纤维素酶诱导表型,证明在不溶性纤维素诱导条件下纤维素酶基因的快速诱导表达需要β-葡萄糖苷酶的参与。
我们首先研究了瑞氏木霉β-葡萄糖苷酶是否参与不溶性纤维素诱导纤维素酶表达的过程。通过测定不同β-葡萄糖苷酶基因缺失菌株在纤维素培养过程中的生长曲线,我们发现β-葡萄糖苷酶的缺失影响菌株对纤维素的利用。此外,在纤维素诱导条件下β-葡萄糖苷酶基因缺失菌株发酵液中总蛋白量和纤维素酶酶活均比野生型菌株低。我们进一步使用Northern blot和qRT-PCR的方法研究了纤维素诱导条件下不同β-葡萄糖苷酶基因缺失菌株纤维素酶基因cbh1的转录动力学。结果显示瑞氏木霉野生型菌株在纤维素诱导3h之后cbh1转录即可达到较高水平,而△cel1b菌株需要诱导6h,△cel1a菌株需要诱导24h,△cel1a△cel1b菌株需要诱导36h,△triβG菌株则需要诱导48h才能达到与野生型类似的水平。β-葡萄糖苷酶基因缺失菌株发酵液中CBHI的表达也呈现与转录类似的结果。与纤维素不同,槐糖诱导条件下△cel1a△cel1b菌株cbhl转录动力学与野生型一致,说明β-葡萄糖苷酶在槐糖诱导纤维素酶表达过程中并未发挥重要作用。综上所述,我们证明了这三个β-葡萄糖苷酶在瑞氏木霉菌株快速响应纤维素启动纤维素酶表达过程中发挥了重要作用,它们有可能参与了诱导物的合成。
3.通过分析在纤维二糖诱导条件下瑞氏木霉主要β-葡萄糖苷酶基因缺失菌株的纤维素酶表达表型,证明主要β-葡萄糖苷酶的缺失能提高纤维二糖对纤维素酶的诱导能力,瑞氏木霉主要β-葡萄糖苷酶对纤维二糖的转化作用在纤维素酶诱导表达过程中并未发挥重要作用。
作为纤维素的主要降解产物,纤维二糖在低浓度下也可以诱导瑞氏木霉产生纤维素酶,而且有证据显示瑞氏木霉β-葡萄糖苷酶能够在体外将纤维二糖通过转糖基作用转化为槐糖这一强效诱导物。为了分析瑞氏木霉β-葡萄糖苷酶在纤维二糖诱导纤维素酶表达过程中发挥的作用,首先我们摸索了在瑞氏木霉中纤维二糖诱导纤维素酶表达的最佳浓度。实验确定0.25%(w/v)纤维二糖为最佳诱导浓度。其次我们通过Western blot测定了β-葡萄糖苷酶基因缺失菌株发酵液中CBHI表达量,并使用qRT-PCR检测了菌体纤维素酶cbhl基因的转录水平,结果显示在纤维二糖诱导条件下β-葡萄糖苷酶基因缺失菌株中纤维素酶的表达水平明显高于野生型,说明降低瑞氏木霉β-葡萄糖苷酶的活性能提高纤维二糖诱导纤维素酶表达的能力,瑞氏木霉主要的β-葡萄糖苷酶对纤维二糖的转化作用在纤维素酶的诱导表达过程中并未发挥重要作用。最后,我们在纤维素培养过程中外源添加一定比例的纤维二糖,发现β-葡萄糖苷酶基因缺失菌株快速响应纤维素产生纤维素酶的能力得到恢复,表明β-葡萄糖苷酶可能通过调控胞外纤维二糖的水平影响纤维素诱导条件下纤维素酶基因的快速表达。
4.证明瑞氏木霉胞内主要β-葡萄糖苷酶Cel1a和Cel1b在乳糖诱导纤维素酶表达过程中发挥重要作用。Cel1a和Cel1b的缺失影响纤维素酶基因转录调控因子Xyrl的表达,但是组成型回补表达Xyr1并不能恢复β-葡萄糖苷酶基因缺失菌株响应乳糖产生纤维素酶的能力。
瑞氏木霉糖苷水解酶第1家族的β-葡萄糖苷酶是乳糖胞内代谢酶的重要候选蛋白,因此我们分析了β-葡萄糖苷酶Cel1a和Cel1b是否影响菌体对乳糖的利用以及乳糖诱导纤维素酶基因的表达。通过生长曲线的测定,我们发现胞内p-葡萄糖苷酶的缺失影响菌体在乳糖碳源上的生长,但是并不影响菌体对半乳糖的利用。通过Western blot分析乳糖培养条件下β-葡萄糖苷酶基因缺失菌株发酵液中纤维素酶CBHI的表达,我们发现β-葡萄糖苷酶基因cel1a的缺失导致乳糖诱导纤维素酶表达延迟,而在双缺菌株△cel1a△cel1b中纤维素酶的表达完全缺失。进一步通过qRT-PCR分析纤维素酶基因cbh1的转录发现β-葡萄糖苷酶基因缺失菌中纤维素酶表达的滞后或缺失发生在转录层面,说明瑞氏木霉胞内β-葡萄糖苷酶Cel1a和Cel1b对乳糖诱导纤维素酶表达是必需的。Xyr1是调控纤维素酶基因诱导表达的关键转录因子,因此我们分析了β-葡萄糖苷酶基因缺失菌株中xyr1基因的转录水平,实验发现虽然β-葡萄糖苷酶的缺失造成xyr1基因的转录水平降低,但在△cel1a△cel1b菌株中组成型表达xyr1并不能恢复菌株响应乳糖产生纤维素酶的能力。上述结果表明胞内β-葡萄糖苷酶在乳糖诱导纤维素酶表达过程中发挥了重要作用,β-葡萄糖苷酶不仅影响了关键转录因子Xyr1的表达,还通过其他途径影响乳糖诱导纤维素酶表达的过程,β-葡萄糖苷酶发挥作用的机制还需要进一步的分析。
5.通过分析Cel1a和Cel1b重组蛋白的底物特异性,证明瑞氏木霉糖苷水解酶第1家族的β-葡萄糖苷酶具有乳糖水解活性和转化活性。
糖苷水解酶第1家族的β-葡萄糖苷酶往往具有β-半乳糖苷酶活性,瑞氏木霉糖苷水解酶第1家族的β-葡萄糖苷酶Cel1a和Cel1b可能在乳糖胞内代谢过程中发挥作用。因此我们通过大肠杆菌异源表达和纯化了瑞氏木霉β-葡萄糖苷酶Cel1a和Cel1b,并对其底物特异性进行了分析。我们发现Cel1a和Cel1b不仅能够水解纤维二糖及其类似物pNPG,还能够水解乳糖类似物DNPG,证明其具有β-半乳糖苷酶活性。通过HPLC分析β-葡萄糖苷酶Cel1a与乳糖反应的产物,我们发现除了D-葡萄糖和D-半乳糖,还有其他半乳寡糖产生,表明β-葡萄糖苷酶Cel1a可能具有乳糖转糖基活性。瑞氏木霉β-葡萄糖苷酶具有的这种糖苷水解活性在纤维素酶诱导表达过程中的作用还需要进一步的研究。
6.证明了瑞氏木霉主要β-葡萄糖苷酶具有的糖苷水解活性对乳糖诱导纤维素酶表达是必需的。
为了进一步分析瑞氏木霉β-葡萄糖苷酶Cel1a和Cel1b所具有糖苷水解活性对乳糖诱导纤维素酶表达的影响,我们在瑞氏木霉△cel1a菌株中组成型表达了野生型Cel1a蛋白以及水解活性缺失点突变体Cel1a (Q367A)蛋白。在乳糖诱导条件下,组成型回补野生型Cel1a蛋白使得△cel1a菌株cbh1的转录得到恢复,cbh1转录起始时间甚至比野生型提前,cbh1转录量也有所升高,而回补水解活性缺失点突变体Cella (Q367A)蛋白的菌株cbh1转录并未恢复,转录起始时间与野生型相比仍然有明显的滞后,这些结果说明Cel1a的糖苷水解活性对乳糖诱导纤维素酶的表达是必需的。为了进一步分析β-葡萄糖苷酶具有的乳糖水解活性在纤维素酶诱导表达过程中的作用,我们在△cel1a菌株中组成型表达了乳酸克鲁维酵母来源的胞内β-半乳糖苷酶Lac4,结果显示回补Lac4并不能恢复△cel1a菌株纤维素酶基因cbh1的转录,表明仅有乳糖胞内水解活性并不足以启动纤维素酶的诱导表达,Cel1a还通过其他途径对纤维素酶基因的诱导表达产生影响。
Biofuel derived from lignocellulose is a clean, renewable energy that can partially replace our current demand for fossil fuels. However, degrading the insoluble plant biomass into fermentable monosaccharaides represents a main bottleneck for biofuel production. Trichoderma reesei secretes large amounts of (hemi) cellulase that could hydrolyze plant biomass into soluble oligosaccharides, and is now one of the most important industrial strains to produce cellulases and hemicellulases. The mechanism of cellulase induction is still indistinct and the best inducers of the cellulase for T. reesei are preferably insoluble cellulose, hemicellulose, and plant biomasses. Currently it is generally believed that small molecules such as cellobiose and sophorose released from these insoluble substrates are the real inducers.
In addition to cellobiose and sophorose, lactose can also induce the production of cellulase, however currently we still do not understand the mechanism underlying this induction. There are some experimental evidences showing that lactose transport and subsequent intracellular hydrolysis or conversion may play important roles in the induction process. T. reesei lacks typical intracellular β-galactosidase which belongs to glycoside hydrolase family2, and the intracellular protein(s) involved in intracellular lactose hydrolysis and conversion has (have) not yet been identified.
There are lines of evidence that β-glucosidases play a vital role in the process of rapid response to cellulose and the induced synthesis of cellulase in T. reesei. For example, deletion of the major extracellular β-glucosidase encoding gene bgll/cl3a delayed cellulase expression on cellulose, while the bgll/cel3a overexpressing strain had an elevated expression of cellulase not only on cellulose but also on sophorose (a potential inducer of cellulase). T. reesei contains11P-glucosidases, among which Bgll/Cel3a is an extracellular β-glucosidase, belonging to glycoside hydrolase family3, and Cell a and Cellb are intracellular enzymes both belonging to glycoside hydrolase family1. Transcription of these three β-glucosidases on cellulose and lactose is significantly raised in T. reesei. Additionally, in some cellulase-high-yielding strains, transcription of these three β-glucosidases is higher than that of the wild type, suggesting that they may be involved in the process of cellulase induction. It is a common phenomenon that β-glucosidases of glycoside hydrolase family1also possess side β-galactosidase activity, making them important candidates for intracellular conversion of lactose. Whether the two GH1P-glucosidases Cel1a and Cel1b of T. reesei have β-galactosidase activity and their roles in lactose induction need further study.
In order to illustrate the roles of β-glucosidases in cellulase induction of T. reesei, three main β-glucosidase genes bgl1/cel3a, Cel1a and Cel1b were knockout singly and multiply. Cellulase expression capacity of these β-glucosidase-deficient strains was studied on different carbon sources such as cellulose, cellobiose, sophorose and lactose. Besides, the two intracellular β-glucosidases Cel1a and Cellb were heterologously expressed to study their activities on lactose and their roles in cellulase induction by lactose. The main innovative results of this study are as follows:
1. A series of β-glucosidases deletion strains were constructed by gene replacement to study the in vivo function of these proteins in cellulase induction.
In order to study the in vivo function of different β-glucosidases in T. reesei, we constructed a number of β-glucosidase deletion strains by means of homologous recombination, including△cel1a,△cel1b,△cel1a△cel1b and△triβG. Anchored PCR and Southern blot were used to verify gene knockout and single copy integration. During culture on cellulose, intracellular β-glucosidase activity of Acel1b was slightly lower than that of wild type strain, while it fell75%in the Acella strain, the extracellular β-glucosidase activity of Bgll deletion strain dramatically declined. In summary, Cel3a accounts for the major extracellular β-glucosidase activity, while Cel1a is the major intracellular β-glucosidase, Cel1b contributes only a small part to the total intracellular β-glucosidase activity in T. reesei.
2. Deletion of cella, cellb delayed cellulase expression on cellulose, but not on sophorose, suggesting that intracellular Cella and Cellb contribute to the induction of cellulase genes probably through participating in the formation of cellulase inducer.
To further analyze the influence of P-glucosidases on cellulase expression we studied the kinetics of cbhl gene transcription in different β-glucosidase-mutant strains using Northern blot/qRT-PCR. This analysis showed that in comparison to the rapid induction in the WT strain, which occurred as early as3h upon induction by cellulose, productive activation of transcription of cbhl was delayed by about3h and21h in the△cel1b and△cel1a strains, respectively. This lag in gene expression was further extended to36h in the△cel1a△cel1b strain, although the final level of transcription was almost the same. Western blot of CBHI protein in the fermentation broth also showed similar results. However, the induction effects by sophorose were not affected in the P-glucosidase mutant strains. In summary, all these data suggests that β-glucosidases are vital in rapid cellulase induction, and may be involved in inducer synthesis.
3. Deletion of major β-glucosidases in T. reesei resulted in higher expression levels of cellulase on cellobiose suggesting that lowering the degree of cellobiose hydrolysis may facilitate cellulase synthesis.
As the main degradation products of cellulose, cellobiose at a low concentration can induce the expression of cellulase and it has been shown that cellobiose could be transformed into sophorose by β-glucosidases in T. reesei. We therefore analyzed the impact of β-glucosidases on cellulase induction by cellobiose. The cellulase expression on cellobiose were significantly elevated in△cel1a△cel1b and△triβG strains suggesting that lowering the degree of cellobiose hydrolysis may facilitate cellulase synthesis and the conversion of cellobiose by the main intracellular and extracellular β-glucosidases may not account for the rapid cellulase expression on cellulose. By exogenously adding a certain proportion of cellobiose to cellulose medium, we found that the cellulase induction capacity of β-glucosidase-deficient strains on cellulose recovered. These data indicated that the productive induction defect on cellulose as observed in the absence of the major β-glucosidases may to a larger extent be caused by the insufficient cellobiose initially available for triggering the induction cascade.
4. Intracellular β-glucosidases Cell a and Cellb affect expression of cellulase and Xyrl by lactose. Constitutive expression of Xyrl could not restore the efficient induction of β-glucosidase-deficient strains.
We further analyze the effect of β-glucosidases on induction of cellulase by lactose. By analyzing the cellulase expression of△cella,△cel1b and△cel1a△cel1b strains cultured on lactose, we found that single deletion of intracellular β-glucosidase lead to lower cellulase expression on lactose, while double deletion of cel1a and cel1b almost abolished it. Further analysis of cbhl transcription by qRT-PCR revealed that the decrease of the cellulase expression occurred at the transcriptional level. These results show that intracellular β-glucosidases are essential for efficient induction of cellulase by lactose. Xyrl is the key transcription factor regulating cellulase expression, whose absence abolished cellulase expression on cellulose and lactose. We found transcription of xyrl decreased in the△cel1a△cel1b strain cultured on lactose, however constitutively expression of xyrl could not restore its efficient cellulase expression, suggesting that expression of xyrl alone is not sufficient to activate the cellulolytic transcription by lactosein the absence of Cel1a and Cel1b.
5. Intracellular β-glucosidase Cel1a and Cel1b exhibited apparent hydrolytic activity toward ONPG and lactose.
Enzymes involved in intracellular metabolism of lactose have not so far been identified in T. reesei. Most β-glucosidases belonging to GH1also possess β-galactosidase activity. Besides this, our previous data showed that intracellular β-glucosidases played central roles in the progress of cellulase induction by lactose. All these data make us speculate whether Cel1a and Cel1b of T. reesei possess P-galactosidase activity. So we heterologously expressed and purified Cel1a and Cel1b in E. coli and analyzed their substrate specificity. We found that Cel1a and Cel1b can hydrolyze not only cellobiose and pNPG, but also lactose and ONPG, suggesting that they possess β-galactosidase activity. Analyzing the lactose hydrolyzing products by HPLC, we found oligosaccharides in addition to D-glucose and D-galactose were also produced, suggesting that β-glucosidases of T. reesei also possess transglycosylation activity toward lactose.
6. Both Cel1a and its associated hydrolytic activity are responsible for the efficient induction of cellulase genes by lactose.
To gain insight into the possibility that Cel1a-associated glycoside hydrolytic activity is involved in the induction process, we retransformed△cel1a strain with wild-type Cella and a mutant protein Cella (Q367A) which bears no activity towards cellobiose and lactose. While constitutive expression of WT Cel1a resulted in a relatively higher induced expression of cbhl as compared with the WT strain, cells expressing the Cella (Q367A) displayed the same kinetic of cbhl induction as in the△cel1a strain on lactose. These results suggest that hydrolytic activity of Cel1a was required for cellulase expression. To further test Cel1a-associated hydrolytic activity toward lactose is fully responsible for cellulase induction, β-galactosidase LAC4from Kluyveromyces lactis was expressed in the Acel1a strain. Analysis of the induced expression of cbh1revealed no restoration of induced cbhl expression in the△cel1aCElac4strain. Altogether, these data suggest that intracellular processing of lactose mediated by Cella and Cellb beyond sole hydrolysis may account for their important roles in cellulase induction by lactose.
引文
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Aro, N., A. Saloheimo, et al. (2001). "ACEII, a novel transcriptional activator involved in regulation of cellulase and xylanase genes of Trichoderma reesei." J Biol Chem 276(26):24309-24314.
Bhatia, Y., S. Mishra, et al. (2002). "Microbial β-glucosidases:cloning, properties, and applications." Critical reviews in biotechnology 22(4):375-407.
Bischof, R., L. Fourtis, et al. (2013). "Comparative analysis of the Trichoderma reesei transcriptome during growth on the cellulase inducing substrates wheat straw and lactose." Biotechnology for biofuels 6(1):127.
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Carle-Urioste, J. C., J. Escobar-Vera, et al. (1997). "Cellulase induction in Trichoderma reesei by cellulose requires its own basal expression." J Biol Chem 272(15):10169-10174.
Cziferszky, A., B. Seiboth, et al. (2003). "The Snfl kinase of the filamentous fungus Hypocrea jecorina phosphorylates regulation-relevant serine residues in the yeast carbon catabolite repressor Migl but not in the filamentous fungal counterpart Crel." Fungal Genetics and Biology 40(21:166-175.
Fekete, E., L. Karaffa, et al. (2012). "Identification of a permease gene involved in lactose utilisation in Aspergillus nidulans." Fungal Genetics and Biology 49(6): 415-425.
Fekete, E., B. Seiboth, et al. (2008). "Lack of aldose 1-epimerase in Hypocrea jecorina (anamorph Trichoderma reesei):a key to cellulase gene expression on lactose." Proc Natl Acad Sci U S A 105(20):7141-7146.
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Fritscher, C., R. Messner, et al. (1990). "Cellobiose metabolism and cellobiohydrolase I biosynthesis by Trichoderma reesei." Experimental mycology 14(4):405-415.
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Holden, H. M., I. Rayment, et al. (2003). "Structure and function of enzymes of the Leloir pathway for galactose metabolism." Journal of Biological Chemistry 278(45): 43885-43888.
Ilmen, M., M.-L. Onnela, et al. (1996). "Functional analysis of the cellobiohydrolase I promoter of the filamentous fungus Trichoderma reesei." Molecular and General Genetics MGG 253(3):303-314.
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Aro, N., A. Saloheimo, et al. (2001). "ACEII, a novel transcriptional activator involved in regulation of cellulase and xylanase genes of Trichoderma reesei." J Biol Chem 276(26):24309-24314.
Bhatia, Y., S. Mishra, et al. (2002). "Microbial β-glucosidases:cloning, properties, and applications." Critical reviews in biotechnology 22(4):375-407.
Bischof, R., L. Fourtis, et al. (2013). "Comparative analysis of the Trichoderma reesei transcriptome during growth on the cellulase inducing substrates wheat straw and lactose." Biotechnology for biofuels 6(1):127.
Bouffard, G. G., K. E. Rudd, et al. (1994). "Dependence of Lactose Metabolism upon Mutarotase Encoded in the gal Operon in Escherichia coli." Journal of molecular biology 244(3):269-278.
Carle-Urioste, J. C., J. Escobar-Vera, et al. (1997). "Cellulase induction in Trichoderma reesei by cellulose requires its own basal expression." J Biol Chem 272(15):10169-10174.
Cziferszky, A., B. Seiboth, et al. (2003). "The Snfl kinase of the filamentous fungus Hypocrea jecorina phosphorylates regulation-relevant serine residues in the yeast carbon catabolite repressor Migl but not in the filamentous fungal counterpart Crel." Fungal Genetics and Biology 40(21:166-175.
Fekete, E., L. Karaffa, et al. (2012). "Identification of a permease gene involved in lactose utilisation in Aspergillus nidulans." Fungal Genetics and Biology 49(6): 415-425.
Fekete, E., B. Seiboth, et al. (2008). "Lack of aldose 1-epimerase in Hypocrea jecorina (anamorph Trichoderma reesei):a key to cellulase gene expression on lactose." Proc Natl Acad Sci U S A 105(20):7141-7146.
Fowler, T. and R. D. Brown, Jr. (1992). "The bgll gene encoding extracellular beta-glucosidase from Trichoderma reesei is required for rapid induction of the cellulase complex." Mol Microbiol 6(21):3225-3235.
Fritscher, C., R. Messner, et al. (1990). "Cellobiose metabolism and cellobiohydrolase I biosynthesis by Trichoderma reesei." Experimental mycology 14(4):405-415.
Furukawa, T., Y. Shida, et al. (2009). "Identification of specific binding sites for Xyr1, a transcriptional activator of cellulolytic and xylanolytic genes in Trichoderma reesei." Fungal Genet Biol 46(8):564-574.
Green, M. R. and J. Sambrook (2012). Molecular cloning:a laboratory manual, Cold Spring Harbor Laboratory Press.
Gruber, F., J. Visser, et al. (1990). "The development of a heterologous transformation system for the cellulolytic fungus Trichoderma reesei based on a pyrG-negative mutant strain." Curr Genet 18(1):71-76.
Hakkinen, M., M. Arvas, et al. (2012). "Re-annotation of the CAZy genes of Trichoderma reesei and transcription in the presence of lignocellulosic substrates." Microb Cell Fact 11(1):134.
Hart1, L., C. P. Kubicek, et al. (2007). "Induction of the gal pathway and cellulase genes involves no transcriptional inducer function of the galactokinase in Hypocrea jecorina." J Biol Chem 282(25):18654-18659.
Holden, H. M., I. Rayment, et al. (2003). "Structure and function of enzymes of the Leloir pathway for galactose metabolism." Journal of Biological Chemistry 278(45): 43885-43888.
Ilmen, M., M.-L. Onnela, et al. (1996). "Functional analysis of the cellobiohydrolase I promoter of the filamentous fungus Trichoderma reesei." Molecular and General Genetics MGG 253(3):303-314.
Ilmen, M., M. L. Onnela, et al. (1996). "Functional analysis of the cellobiohydrolase I promoter of the filamentous fungus Trichoderma reesei." Mol Gen Genet 253(3):303-314.
Ilmen, M., A. Saloheimo, et al. (1997). "Regulation of cellulase gene expression in the filamentous fungus Trichoderma reesei." Applied and environmental Microbiology 63(4):1298-1306.
Ilmen, M., C. Thrane, et al. (1996). "The glucose repressor gene crel of Trichoderma:isolation and expression of a full-length and a truncated mutant form." Mol Gen Genet 251(4):451-460.
Ivanova, C., J. A. Baath, et al. (2013). "Systems Analysis of Lactose Metabolism in Trichoderma reesei Identifies a Lactose Permease That Is Essential for Cellulase Induction." PloS one 8(5):e62631.
Iyayi, C., E.-E. Bruchmann, et al. (1989). "Induction of cellulase formation in Trichoderma reesei by cellobiono-1,5-lacton." Archives of microbiology 151(4): 326-330.
Karaffa, L., L. Coulier, et al. (2013). "The intracellular galactoglycome in Trichoderma reesei during growth on lactose." Applied Microbiology and Biotechnology.
Karaffa, L., E. Fekete, et al. (2006). "D-Galactose induces cellulase gene expression in Hypocrea jecorina at low growth rates." Microbiology 152(Pt 5):1507-1514.
Kubicek, C. P., R. Messner, et al. (1993). "Triggering of cellulase biosynthesis by cellulose in Trichoderma reesei. Involvement of a constitutive, sophorose-inducible, glucose-inhibited beta-diglucoside permease." J Biol Chem 268(26):19364-19368.
Kubicek, C. P., M. Mikus, et al. (2009). "Metabolic engineering strategies for the improvement of cellulase production by Hypocrea jecorina." Biotechnol Biofuels 2(1): 19-32.
KUBICEK, C. P., G. MUHLBAUER, et al. (1988). "Properties of a conidial-bound cellulase enzyme system from Trichoderma reesei." Journal of general microbiolog 134(5):1215-1222.
Li, L. and K. A. Borkovich (2006). "GPR-4 is a predicted G-protein-coupled receptor required for carbon source-dependent asexual growth and development in Neurospora crassa." Eukaryotic cell 5(8):1287-1300.
Luo, C., J. J. Loros, et al. (1998). "Nuclear localization is required for function of the essential clock protein FRQ." The EMBO journal 17(5):1228-1235.
Lynd, L. R., M. S. Laser, et al. (2008). "How biotech can transform biofuels." Nature biotechnology 26(2):169-172.
Mach, R. L., M. Schindler, et al. (1994). "Transformation of Trichoderma reesei based on hygromycin B resistance using homologous expression signals." Current genetics 25(6):567-570.
Mach, R. L., B. Seiboth, et al. (1995). "The bgll gene of Trichoderma reesei QM 9414 encodes an extracellular, cellulose-inducible beta-glucosidase involved in cellulase induction by sophorose." Mol Microbiol 16(4):687-697.
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