Characterization of a series of 5’ truncated promoters of cdna1
As a strong constitutive promoter in T. reesei, Pcdna1 has been successfully utilized to express cellulases using glucose as the carbon source . Here, the 928-bp promoter region upstream of ATG of the gene cdna1 was analyzed as shown in Fig. 1. The promoter Pcdna1 contains several core cis-acting elements, such as TATA box, CCAAT box and GC box, which are essential elements to initiate transcription or enhance the transcriptional level [36–38]. To functionally validate the cdna1 promoter, a promoter library based on Pcdna1 was established by sequence truncation. Five 5’ truncated promoters of cdna1, Pcdna1-1 (928 bp), Pcdna1-2 (794 bp), Pcdna1-3 (640 bp), Pcdna1-4 (490 bp) and Pcdna1-5 (247 bp), were fused to the reporter gene cbh1 (coding cellobiohydrolase 1) and transformed into T. reesei using hph as the selectable marker gene, respectively (Fig. 2a). All these five truncated promoters were capable of directing CBH activity after culturing with glucose (GMM) or Avicel (AMM, Fig. 2b and c). When glucose was used as the sole carbon source, PD1-2 (cbh1 directed by Pcdna1-2) showed the lowest CBH activity, which was about 0.7-fold of PD1-1 (cbh1 directed by Pcdna1-1). However, PD1-3 (cbh1 directed by Pcdna1-3) exhibited the highest CBH activity, which was 1.2-fold higher than that of PD1-1, indicating that the deletion of the 154-bp sequence (-794 to -640 bp) located between Pcdna1-2 and Pcdna1-3 enhanced promoter activity. Similar to that under the glucose condition, when the strains were cultured with Avicel, the greatest improvement of CBH activity was imparted by Pcdna1-3 (1.4-fold higher than Pcdna1-1) while the promoter activity of Pcdna1-2 was relatively weak (0.4-fold lower than Pcdna1-1), indicating that the cis-acting elements facilitating gene expression exist in the 640-bp (-640 to -1 bp) sequence of the promoter (Pcdna1-3). Indeed, three kinds of essential cis-acting elements, CCAAT box, GC box and TATA box, were contained in Pcdna1-3 (Fig. 1a). Thus, Pcdna1-3 could be selected as the high-efficiency core region to initiate gene expression.
Identification of the activation region of the cbh1 promoter
The promoter of cbh1 is known as the strongest inducible promoter in T. reesei, and it contains different types of transcriptional regulator-binding sites [17–21]. It has been reported that several binding sites of transcriptional activators Xyr1, Ace2 and Hap2/3/5 exist in the 248-bp (-869 to -622 bp) regulation region, and three Cre1-binding sites are located in the 48-bp (-725 to -678 bp) transcriptional repression region . Insertion of the 200-bp (-821 to -622 bp) regulation region without Cre1-binding sites results in significant higher activity than the wild-type promoter , demonstrating that the 200-bp (-821 to -622 bp) region of Pcbh1 can serve as an activation region.
Here, the Cre1-binding sites-containing region (-725 to -678 bp) was deleted to construct the modified promoter Pcbh1-dc (821 bp, containing the activation region AR and the core region CR), and the regulation region RR (-869 to -622 bp) was removed from Pcbh1 to construct the core-region promoter Pcbh1-cr (621 bp) as shown in Fig. 3a. To eliminate the interference of native CBH1, the enhanced green fluorescent protein (eGFP)-encoding gene, egfp, was selected as the reporter gene. Then, the promoters Pcbh1-dc and Pcbh1-cr were fused with egfp to construct the corresponding cassettes, respectively (Fig. 3a). The fluorescence intensities of the transformants under the glucose (Fig. 3b) and Avicel (Fig. 3c) conditions were measured, respectively. It was found that the Pcbh1-cr transformant PBC1 showed a barely detectable level of fluorescence intensity, which suggested that the activity of the Pcbh1 core region was relatively weak. On the contrary, the Pcbh1-dc transformant PBD1 displayed remarkable improvement of fluorescence intensity compared to PBC1 under both glucose and Avicel conditions (Fig. 3b and Fig. 3c). Especially, when cultured with Avicel, PBD1 generated a 5.3-fold higher fluorescence intensity than that of PBC1 (Fig. 3c). These results demonstrated that the 200-bp (-821 to -622 bp) region of Pcbh1 could be selected as the high-efficiency activation region to facilitate gene expression.
Construction and assessment of the hybrid promoter P cc
As shown above, Pcdna1-3 possessed the ability to efficiently initiate gene expression, and the 200-bp AR of Pcbh1 was determined to be an enhancer to facilitate the process, which led to the hypothesis that the combination of these two regions may generate a powerful hybrid promoter. In this regard, a novel hybrid promoter Pcc was created by fusing the 200-bp (-821 to -622 bp) activation region of Pcbh1 to the 640-bp (-640 to -1 bp) core region of Pcdna1 (Fig. 4a). To assess the activity of Pcc, the promoter was used to direct the expression of the reporter gene egfp in T. reesei. Meanwhile, the promoters Pcbh1 and Pcdna1 were adopted to direct the expression of egfp as control, respectively. Fluorescence intensity measurement was performed as shown in Fig. 4c. As expected, the transformant PCC (egfp expressed by Pcc) had the highest fluorescence intensity and reached 1.6 and 1.8 times more than the transformants PB1 (egfp expressed by Pcbh1) and PD1 (egfp expressed by Pcdna1) under the Avicel condition, respectively (Fig. 4b). While under the glucose condition, the fluorescence intensity of PCC was comparable to that of PD1 (data not shown). The fluorescence emission detected by fluorescence microscopy showed that the eGFP protein was produced successfully in the transformant PCC under both glucose and Avicel conditions (Fig. 4c). These results indicated that the hybrid promoter Pcc could drive gene expression with high efficiency under both repressive and inducible conditions.
Construction of the T. reesei strains overexpressing BGLA directed by Pcc
The β-glucosidase (BGL) was recognized as one of the bottlenecks of cellulase cocktail in hydrolyzing cellulosic substrates due to its low activity in T. reesei. Thus, the hybrid promoter Pcc was first attempted to direct BGL expression with the aim of optimizing the hydrolysis efficiency of cellulolytic enzyme system. Here, the Aspergillus niger β-glucosidase gene bglA was overexpressed by Pcc in T. reesei using pyrG as the selectable marker gene. Two transformants, QPB55 and QPB74, were selected to be cultured on esculin plates to detect their BGL activity using glucose and sodium carboxymethyl cellulose (CMC-Na) as the sole carbon source, respectively (Fig. 5a). The sizes of dark halos of these transformants were much larger than that of the parental strain QP4 no matter on glucose-esculin plates or CMC-esculin plates, which indicated that the overexpression of bglA driven by Pcc resulted in remarkably improvement of BGL activity. Then, fermentation of these transformants were conducted in GMM and CPM for cellulase production, respectively. SDS-PAGE analysis showed that an approximately 120-kDa band (the expected molecular weight of A. niger BGLA) were clearly observed under two conditions in the fermentation broths of QPB55 and QPB74, which were not present in QP4 (Fig. 5b and c). And the bands were determined to be BGLA by MS analysis (data not shown). The transformants QPB55 and QPB74 exhibited the significant increase of BGL activities (16.6 IU/mL and 15.1 IU/mL, respectively) when using glucose as the carbon source (Fig. 5d), which was in line with the results observed on esculin plates and SDS-PAGE analysis. Besides, when cultured with Avicel, QPB55 and QPB74 provided 43.5- and 25.7-fold higher BGL activities (27.2 IU/mL and 16.0 IU/mL) than that of the parental strain (0.6 IU/mL), which led to 85.2% and 58.6% enhancements of FPA (1.6 IU/mL and 1.4 IU/mL, filter paper activity) in comparison to that of QP4 (0.9 IU/mL), respectively (Fig. 5d and e). These results implied that the overexpression of bglA driven by the hybrid promoter Pcc remarkedly increased the BGL activity, therefore enhancing the total cellulase activity of T. reesei.
Construction of the T. reesei strains overexpressing EG2 directed by Pcc
The T. reesei EG2 has been determined to contribute the most to the endoglucanase activity, suggesting its importance in hydrolysis of cellulosic substrates . Therefore, the endoglucanase gene eg2 was also employed to be overexpressed directed by Pcc in T. reesei using pyrG as the selectable marker gene. Then two transformants, QPE15 and QPE24, were selected and fermented in GMM and CPM, respectively. The SDS-PAGE and MS analysis demonstrated that the EG2 bands in the transformants QPE15 and QPE24 were clearly observed while the band of QP4 was not detected (cultured with glucose) or relatively weak (cultured with Avicel, Fig. 6a and b). Notably, strikingly increased EG activities (0.3 IU/mL) of these transformants were detected under the glucose condition, whereas EG activity of QP4 was considerably weak (0.06 IU/mL, Fig. 6c). While cultured with Avicel, QPE15 and QPE24 showed 19.1% and 15.7% increase of EG activities (6.2 IU/mL and 6.0 IU/mL) compared to QP4 (5.2 IU/mL), leading to 2.5- and 2.0-fold higher FPA (2.1 IU/mL and 1.7 IU/mL) than that of QP4 (0.9 IU/mL), respectively (Fig. 6c and d). These results indicated that overexpression of eg2 driven by Pcc remarkedly increased the EG activity, therefore improving the total cellulase activity of T. reesei. Based on the observation that the total cellulase activity of T. reesei can be improved by overexpressing specific cellulases directed by Pcc, this hybrid promoter was demonstrated to be applied to optimize cellulase system.
Construction of the T. reesei strains co-overexpressing BGLA and EG2 directed by Pcc
Given that the individual overexpression of BGLA and EG2 driven by Pcc could led to improvement of the activities of BGL and EG, respectively, and both of them resulted in the FPA augmentation, a hypothesis was proposed that construction of strains co-overexpressing BGLA and EG2 directed by Pcc could further optimize the T. reesei enzymatic system to improve the cellulolytic ability. To this end, the expression cassette of bglA driven by Pcc (Pcc-bglA-ptrA) was transformed to EG2-overexpression strain QPE15 using ptrA as the selectable marker gene. Afterwards, two transformants QPEB29 and QPEB70 were selected and verified to successfully express bglA directed by Pcc (data not shown). Then, QPEB29 and QPEB 70 were cultured in CPM for 7 days at 30°C for cellulase production. QPEB29 and QPEB70 showed 2.4 IU/mL and 2.2 IU/mL filter paper activities at the end of fermentation, respectively, which were increased by 178.1% and 156.5% in comparison to QP4, and were also higher than that of QPB55 and QPE15 as expected (Fig. 7). These results indicated that the double overexpression of BGLA and EG2 directed by Pcc could generate a more efficient cellulolytic enzyme system than the overexpression of the individual ones in T. reesei.
Saccharification of corncob residues by the T. reesei strains co-overexpressing BGLA and EG2 directed by Pcc
The improved total cellulase activities of QPEB29 and QPEB70 led to the presumption that their extracellular cellulase cocktails might perform better than that of the parental strain in the deconstruction of lignocellulosic biomass. To confirm this, the cellulase cocktails produced by the bglA-eg2 co-overexpression strains QPEB29 and QPEB70 were used in the saccharification of acid-pretreated corncob residue (ACR) and delignified corncob residue (DCR), respectively. Glucose released in saccharification towards ACR by cellulase cocktails of QPEB29 and QPEB70 were 9.9 ~ 10.5 mg/mL (corresponding to 28.5 ~ 30.2% cellulose conversion), which were 2.2- and 2.3-fold higher than that by QP4 (4.5 mg/mL corresponding to 12.9% cellulose conversion, Fig. 8a). When DCR was used as the substrate, the released glucose detected in the saccharification with QPEB29 and QPEB70 cellulase cocktails reached 31.4 ~ 33.2 mg/mL (corresponding to 86.0 ~ 91.0% cellulose conversion), which were 2.2- and 2.4-fold higher than the value of QP4 (14.0 mg/mL, corresponding to 38.4% cellulose conversion, Fig. 8b). These results demonstrated that the double overexpression of BGLA and EG2 directed by Pcc significantly improved the capacity of the T. reesei cellulase system for biomass hydrolysis, implying that this hybrid promoter Pcc has the potential to be utilized as a robust promoter to optimize cellulolytic enzyme system for highly efficient bioconversion of lignocellulosic biomass.