Overexpression of bacterial ethylene-forming enzyme gene in Trichoderma reesei enhanced the production of ethylene

Abstract

In order to efficiently utilize natural cellulose materials to produce ethylene, three expression vectors containing the ethylene-forming enzyme (efe) gene from Pseudomonas syringae pv. glycinea were constructed. The target gene was respectively controlled by different promoters: cbh I promoter from Trichoderma reesei cellobiohydrolases I gene, gpd promoter from Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase gene and pgk I promoter from T. reesei 3-phosphoglycerate kinase I gene. After transforming into T. reesei QM9414, 43 stable transformants were obtained by PCR amplification and ethylene determination. Southern blot analysis of 14 transformants demonstrated that the efe gene was integrated into chromosomal DNA with copy numbers from 1 to 4. Reverse transcription polymerase chain reaction (RT-PCR) analysis of 6 transformants showed that the heterologous gene was transcribed. By using wheat straw as a carbon source, the ethylene production rates of aforementioned 14 transformants were measured. Transformant C30-3 with pgk I promoter had the highest ethylene production (4,012 nl h(-1) l(-1)). This indicates that agricultural wastes could be used to produce ethylene in recombinant filamentous fungus T. reesei.

Keywords: Trichoderma reesei; ethylene-forming enzyme; overexpression.; promoter; wheat straw.

Figure 1

Southern blot analysis of genomic DNA isolated from 14 transformants. (a) Lane 1: DNA molecular weight marker (λDNA/HindⅢ+EcoRⅠ); 2: transformant A10-6 (pPcbh1-efe-hph); 3: transformant A33-2 (pPcbh1-efe-hph); 4: transformant A41-1 (pPcbh1-efe-hph); 5: transformant A55-5 (pPcbh1-efe-hph); 6: transformant A55-9 (pPcbh1-efe-hph); 7: transformant B2-6 (pPgpd-efe-hph); 8: transformant B2-7 (pPgpd-efe-hph); 9: transformant B12-3 (pPgpd-efe-hph); (b) Lane 1: DNA molecular weight marker (λDNA/HindⅢ+EcoRⅠ); 2: transformant B38-2 (pPgpd-efe-hph); 3: transformant B38-4 (pPgpd-efe-hph); 4: transformant C1-3 (pPpgk1-efe-hph); 5: transformant C2-5 (pPpgk1-efe-hph); 6: transformant C30-3 (pPpgk1-efe-hph); 7: transformant C30-10 (pPpgk1-efe-hph); Lane 8: genomic DNA from non-transformed host strain T. reesei QM9414; Lane 9: DNA from the whole coding sequence of ethylene-forming enzyme gene. Genomic DNA digested by SalⅠand hybridized with a digoxigenin-labelled whole coding sequence of the efe gene.

Figure 1

Southern blot analysis of genomic DNA isolated from 14 transformants. (a) Lane 1: DNA molecular weight marker (λDNA/HindⅢ+EcoRⅠ); 2: transformant A10-6 (pPcbh1-efe-hph); 3: transformant A33-2 (pPcbh1-efe-hph); 4: transformant A41-1 (pPcbh1-efe-hph); 5: transformant A55-5 (pPcbh1-efe-hph); 6: transformant A55-9 (pPcbh1-efe-hph); 7: transformant B2-6 (pPgpd-efe-hph); 8: transformant B2-7 (pPgpd-efe-hph); 9: transformant B12-3 (pPgpd-efe-hph); (b) Lane 1: DNA molecular weight marker (λDNA/HindⅢ+EcoRⅠ); 2: transformant B38-2 (pPgpd-efe-hph); 3: transformant B38-4 (pPgpd-efe-hph); 4: transformant C1-3 (pPpgk1-efe-hph); 5: transformant C2-5 (pPpgk1-efe-hph); 6: transformant C30-3 (pPpgk1-efe-hph); 7: transformant C30-10 (pPpgk1-efe-hph); Lane 8: genomic DNA from non-transformed host strain T. reesei QM9414; Lane 9: DNA from the whole coding sequence of ethylene-forming enzyme gene. Genomic DNA digested by SalⅠand hybridized with a digoxigenin-labelled whole coding sequence of the efe gene.

Figure 2

RT-PCR analysis of EFE mRNA in 6 transformants. Lane 1: DNA molecular weight marker (λDNA/HindⅢ+EcoRⅠ); 2: transformant A10-6 (pPcbh1-efe-hph); 3: transformant A55-5 (pPcbh1-efe-hph); 4: transformant B12-3 (pPgpd-efe-hph); 5: transformant B38-4 (pPgpd-efe-hph); 6: transformant C1-3 (pPpgk1-efe-hph); 7: transformant C2-5 (pPpgk1-efe-hph); 8: the non-transformed parental strain T. reesei QM9414; 9: PCR production from plasmid pPcbh1-efe-hph as a positive control; 10 to 15: PCR amplified with total RNAs of aforementioned 6 transformants treated by DNaseⅠas a negative control. No signals were detected in the negative controls illustrated that the corresponding fragments from lane 2 to lane 7 were amplified with cDNAs of transformants as templates rather than low levels of genomic DNAs.

Figure 3

Effect of different carbon resources and nitrogen compounds on ethylene production in random-selecting transformants A41-4 and B38-4. The results shown are averages (± the standard deviation [±SD]) from five parallel cultures.

Figure 4

Comparison of ethylene production of 14 stable transformants and the parental strain T. reesei QM9414. A10-6, A32-3, A41-4, A55-5 and A55-9 were different transformants introduced the expression vector pPchb1-efe-hph; B2-6, B2-7, B12-3, B38-2 and B38-4 were transformed by pPgpd-efe-hph; C1-3, C2-5, C30-3 and C30-10 were transformants of pPpgk1-efe-hph; T. reesei QM9414 was used as a negative control.

Figure 5

(a) Comparison of FPA and cellobiohydrolases activities in 14 tansformants and the host strain cultured in wheat straw inducing media in the fourth day. (b) Growth curves for 3 transformants respectively came from three expression vectors and the non-tranformed T. reesie QM9414. (c) Change of extracellular protein concentration in 3 transformants and T.reesei QM9414.

Figure 5

(a) Comparison of FPA and cellobiohydrolases activities in 14 tansformants and the host strain cultured in wheat straw inducing media in the fourth day. (b) Growth curves for 3 transformants respectively came from three expression vectors and the non-tranformed T. reesie QM9414. (c) Change of extracellular protein concentration in 3 transformants and T.reesei QM9414.

Figure 5

(a) Comparison of FPA and cellobiohydrolases activities in 14 tansformants and the host strain cultured in wheat straw inducing media in the fourth day. (b) Growth curves for 3 transformants respectively came from three expression vectors and the non-tranformed T. reesie QM9414. (c) Change of extracellular protein concentration in 3 transformants and T.reesei QM9414.