Increased cellulase production by genetically engineering of T. asperellumVel1 gene
The strong promoter TrpC was used to improve the expression of Vel1. The over expression cassette containing TrpC promoter, Vel1 ORF and TrpC terminator was cloned into pCAMBIA1300 and transferred to T. asperellum using A. tumefaciens-mediated transformation. The recombinant vector pCAMBIA1300 Vel1 OE and transformants were shown in Fig. 7. There were 126 T. asperellum recombinant strains were selected for the cellulase production, among them 5 T. asperellum recombinants showed the cellulase activity higher than the wild type strain. The growth of T. asperellum recombinants on the cellulose containing medium was displayed in table S1. The fastest growing T. asperellum recombinants were selected among 5 transformants with the maximum cellulase activities.
Influence of cellulase production by different methods
The four methods (Fig. S1) such as the axenic culture of T. asperellum, co- culture of T. asperellum and B. amyloliquefaciens, the axenic culture of the TA OE-Vel1, and the co-culture of TA OE-Vel1 and B. amyloliquefaciens were analyzed to know the best method to produce lignocellulolytic enzyme for the degradation of lignocellulolytic biomass. The genetically engineered T. asperellum (TA OE-Vel1) showed higher activities of FPAase, CMCase, PNPCase, PNPGase, xylanase I and xylanase II (Fig.1) compare to the T. asperellum. The axenic culture of Bacillus amyloliquefaciens failed to produce the FPAase, CMCase, PNPCase, PNPGase, xylanase I and xylanase II activities. The FPAase, CMCase, PNPCase, PNPGase, xylanase I and xylanase II activity of the TA OE-Vel1 axenic culture was 12.45 ± 0.05 FPIU, 62.14 ± 0.34 IU/mL, 4.95 ± 0.23 IU/mL, 3.26 ± 0.32 IU/mL, 73.67 ± 0.37 IU/mL, and 67.8 ± 0.36 IU/mL after 6 days of fermentation, respectively. This revealed that the Vel1 gene improved the activity of enzymes related to cellulose and hemicellulose hydrolysis. In-addition to that the co-culture of T. asperellum and B. amyloliquefaciens improved the production of enzymes than the axenic culture of T. asperellum, which proved that B. amyloliquefaciens act as a potential inducer of lignocellulose hydrolyzing enzymes producing genes [11]. The enzyme activities including FPAase, CMCase, PNPCase, PNPGase, xylanase I and xylanase II were enriched by the TA + BA. But, it was not as higher than the axenic culture of TA OE-Vel1. This perception might be owing to the different substrates. After 6 days of fermentation, the FPAase, CMCase, PNPCase, PNPGase, xylanase I and xylanase II activity of the co-culture of TA OE-Vel1 and B. amyloliquefaciens were 7.92 ± 0.04 FPIU, 54.16 ± 0.46 IU/mL, 3.24 ± 0.32 IU/mL, 2.56 ± 0.25 IU/mL, 63.23 ± 0.37 IU/mL, and 61.57 ± 0.43 IU/mL respectively. It was identified that the co-cultivation of T. asperellum and B. amyloliquefaciens was a fantastic combination to obtain the higher activity of FPAase, CMCase, PNPCase, PNPGase, xylanase I and xylanase II activity. For the first time, in this investigation, the co-culture of the genetically engineered T. asperellum and B. amyloliquefaciens was attempted to synthesize the highest enzyme production by linking the recombination technology and co-cultivation. As shown in Fig. 1, the FPAase, CMCase, PNPCase, PNPGase, xylanase I and xylanase II activity of the co-culture of TA OE-Vel1 and B. amyloliquefaciens were 15.91 ± 0.14 FPIU, 73.04 ± 0.16 IU/mL, 6.32 ± 0.39 IU/mL, 4.45 ± 0.32 IU/mL, 83.56 ± 0.43 IU/mL, and 78.45 ± 0.38 IU/mL respectively. These enzyme activities were considerably increased than the TA OE-Vel1. Also, the enzyme activities were increased than the co-culture of T. asperellum and B. amyloliquefaciens. The results showed that this method upgraded the synthesis of cellulase. Further, the results recommend that this kind of modified co-cultivation is more valuable than that of recombination technology and co-cultivation.
Influence of the transcription regulating genes, cellulase and xylanase encoding gene expression by different methods
The expression pattern of cbh1, cbh2, egl1, egl2, bgl1, xyn1 and xyn2 were compared to know the regulatory level of cellobiohydrolases, endoglucanases, β-glucosidase and xylanase under different approaches. The expressions of these genes were strongly upregulated in the order of TA OE-Vel1+BA>TA OE-Vel1>TA+BA relative to the axenic culture of T. asperellum (Fig. 2). The expression of the major cellulase gene, including cellobiohydrolases (cbh1 and cbh2) endoglucanases (egl1 and egl2), and β-glucosidase (bgl1) were perfectly supported the enzyme activity of TA+BA, TA OE-Vel1, and TA OE-Vel1+BA. TA OE-Vel1+BA showed the maximum expression of xylanase coding gene xyn1 and xyn2 (Fig. 2a). The cellulase and xylanase encoding genes were coordinated by the group of transcription factors (TFs), including both inducer and inhibitors. The expression of cellulase regulatory genes by the Vel1 has been explored by studying the expression of nine positive regulators and three repressor genes. The stimulation of cellulase was initially verified by the transcription analysis of xyr1, ace II, and ace III, which are the most important inducers of cellulase and xylanase production. As shown in Fig. 2b, the relative quantification of the xyr1, ace II, and ace III gene were upregulated by the over-expression of the Vel1 gene. During co-cultivation of TA OE-Vel1 and B. amyloliquefaciens, the expression of xyr1, ace II and ace III increased to 8.56, 7.98 and 7.14 fold respectively, then the axenic culture of T. asperellum. Meanwhile, the relative transcription folds of these genes were only 4.4, 3.7 and 3.7 in TA+BA. In addition, the transcription factors BglR and Hap2/3/5 complex also positively regulated the cellulase and xylanase. The transcription level of the BglR and the Hap2/3/5 complex was also upregulated in co-ordination with other genes. Among the negatively transcription regulating factors, cre-1 is the carbon catabolite repressor, which completely inhibits the expression of the cellulase and xylanase genes. Relatively, ace1 inhibits the C2H2 zinc finger and negatively regulate the genes encoding cellulase and xylanase. Also, the rce1 is the negative regulator by provoking Xyr1. To detect the influence of ace I, rce 1, and cre 1, the expression level of these genes was quantified. The results showed that these genes were downregulated with the overexpression of the Vel1 gene and TA OE-Vel1+BA. The downregulation of ace I, rce 1, and cre 1 might be involved in the upregulation of cbh1, cbh2, egl1, egl2, and bgl1 through the overexpression of Vel1 gene.
Hydrolysis of cellulosic biomass by the differently sourced cellulases
The pretreated corn stover was hydrolyzed using crude enzymes obtained from different methods were shown in Fig. 3. The enzymes obtained from the TA OE-Vel1+BA showed maximum hydrolysis. This may because of TA OE-Vel1+BA produced a mixture of enzymes in high quantityto hydrolyze the pretreated corn stover. The over-expression of the Vel1 gene in T. asperellum enriched the cellulase production. At 72h, TA OE-Vel1+BA produced the hydrolysis yield of 89.56 ± 0.61%, which was greater than the co-culture of T. asperellum and B. amyloliquefaciens and the axenic culture of genetically engineered T. asperellum. The hydrolysis yield generated by the TA OE-Vel1 + BA and TA + BA was higher than the axenic culture of T. asperellum. However, TA OE-Vel1+BA showed a better hydrolysis yield than TA + BA. This might be due to the reason of the activation of transcription factors and enzyme coding genes by the over expression of the Vel1 gene and by the co-cultivation with B. amyloliquefaciens as an inducer. Consequently, the enzyme production and hydrolysis yield were higher in the TA OE-Vel1+BA method.
Synergistic effect of corn stover amendments and microbial inoculation on lignocellulose degradation, plant growth and defense response
Throughout pot experiment, maize plants were grown healthy without any toxic symptoms. The axenic and co-culture were used to enrich plant growth in soil with and without the amendments of corn stover (Fig. S2). The growth parameters of plants grown in soil samples amended with corn stover differed significantly (P≤0.05) from the plants grown in non-amended soil as assessed by Duncan’s new multiple range test. TA OE-Vel1 + BA and TA + BA co-culture exhibited a remarkable effect on both plant growth and lignocellulolytic degradation. However, the plant growth in the untreated corn stover amended soil was reduced (Table 1). Overall, biodegradation of corn stover amended soil with TA OE-Vel1 + BA co-culture (T13) increased the shoot height and root height of the maize plants when compared to non-amended soil and other treatments (Table 1). Shoot height and root height of maize plants grown in corn stover amended soil treated with co-culture (T12) were 1.68 and 1.31 fold higher, respectively than control (T1). likewise, the shoot height and root height of maize plants grown in non-amended soil treated with co-culture (T5) and TA OE-Vel1 + BA co-culture (T6) were also higher than control (T1). The fresh and dry biomass of shoot and root was also influenced by corn stover amendments treated with TA OE-Vel1 + BA (T13) and TA + BA (T12). The influence of TA OE-Vel1 + BA and TA + BA on the corn stover amendments improved the plant height and biomass of maize than all other treatment. The influence of TA OE-Vel1 + BA and TA + BA co-culture on disease index against Fusarium verticillioides and Cohilohorus herostrophus was also observed in both amended and non-amended soil compared to the control (T7 and T14). On the other hand, the disease index of T12 and T13 were 7 times higher than that of control (T14).
To further understand the plant response to the corn stover amended soil inoculated with co-culture, we studied the induction of defense-related gene expression using semi-quantitative reverse transcriptase (RT)-PCR (Fig. 4). The actin gene has been used as an internal control. Fourteen genes related to different plant defense pathways were selected: allene oxide synthase (AOS) allene oxide cyclase (AOC) (jasmonic acid), 1-aminocyclopropane-1-carboxylic acid synthase (ACS) (ethylene), pathogenesis-related protein 1 (PR1) and pathogenesis-related protein 10 (PR10) (systemic acquired resistance), phenylalanine ammonia-lyase (PAL) and (PAL1) (salicylic acid), hydroperoxide lyase (HPL), lectin, lipase, multiflux efflux synthase (MFS), cystatin ii proteinase inhibitor (Cyst2), peroxidase (PX5), cystatin proteinase inhibitor (Cyst) and thiolase (other defense-related genes). The regulation of these genes by axenic, TA OE-Vel1 + BA and TA + BA co-culture were examined locally at the root and systematically at leaves. The defense gene expression against Fusarium verticillioides and Cohilohorus herostrophus on maize roots and leaves are shown in figure 4. The AOS and AOC gene was upregulated in both roots and leaves of plants infected with Fusarium verticillioides and Cohilohorus herostrophus, respectively, but it was gradually reduced in the plants treated with TA OE-Vel1 + BA (T6 and T13), TA + BA (T5 and T12) co-culture, and axenic culture (T2, T3, T4, T9, T10 and T11) in both amended and non-amended soil. The upregulation of AOS and AOC revealed that the plants were highly infected by the Fusarium verticillioides and Cohilohorus herostrophus in T7 and T14. Based on the expression profiles, the ACS genes was highly induced by TA OE-Vel1 + BA application in the root of T6 and T13 (Fig. 4a and c). Followed by the co-culture of TA+BA induced the ACS genes of maize plants. Interestingly, the expression of these genes was downregulated in T7 and T14. TA OE-Vel1 + BA and TA + BA inoculated maize plants expressed the defense genes locally on the plant root and systematically in leaves as a response of Cohilohorus herostrophus (Fig. 4b and d). The expression of systemic acquired resistance pathway-related genes such as PR1 and PR10 in roots and leaves of maize plants inoculated with TA OE-Vel1 + BA and TA + BA co-culture were upregulated than other treatments of both amended and non-amended soil. The PAL and PAL1 were upregulated in the following order T13>T12>T11>T9>T10 and T6>T5>T4>T2>T3 in both corn stover amended and non-amended soil, respectively. The upregulation of the genes such as HPL, lectin, lipase, MFS, Cyst2, PX5, Cyst, and thiolase was also enhanced by the TA OE-Vel1 + BA and TA + BA co-culture compared to the control.
SOM, TOC, TN and C/N of each treatment had shown in Table 2. There were no notable changes in the content of soil SOM, TOC, TN and C/N in T1 to T7, which was not amended with the corn stover. Amendment of corn stover increased the soil SOM and TOC in the treatment T8, T14, and T10, which has not been treated with the Trichoderma. Biodegradation of corn stover amendment treated with TA OE-Vel1 + BA and TA + BA co-culture reduced the SOM and TOC content of the soil. At the end of the experiments, the C/N ratio of the TA OE-Vel1 + BA and TA + BA treated corn stover amendment soil was rapidly decreased and it was closer to the standard value compared soil treated with axenic culture and control. In comparison with all other treatments, T13 and T12 treatment showed better degradation. In connection to the improvement of the C/N ratio, the cellulose content of the T13 and T12 was completely reduced by the TA OE-Vel1 + BA and TA + BA co-culture (Fig. 5a). Similarly, the lignin content was also reduced in T13 and T12 compared to other treatments and before treatment (Fig. 5b). The cellulase and xylanase content of the T13 and T12 was increased compared to other treatments (Fig. 6a and b).