Study On Cloning And Expression Of A Thermotolerant And Alkaline Lipase Gene

Talaromyces thermophilus lipase (TTL) showed strong tolerance toward high temperature and alkaline conditions, which endowed itself great potential in paper making, deinking, detergents addition, biodiesel production and chiral resolution. Whereas, the low capacity for protein expression by T. thermophilus made the large scale production of TTL difficult. In this work, the ttl gene was successfully expressed in recombinant Pichia pastoris and Trichoderma reesei via genetic engineering, which would greatly benefit the production and application of thermo-tolerant and alkaline lipase. Major results were as followed:ttl gene was optimized and synthesized according to preference of P. pastoris, and it would be further ligated after the AOX1 promoter and a signal sequence of pPIC9K to construct the expression plasmid pPIC9K-TTL. This plasmid was transformed into the genome of P. pastoris via electroporation, and 220 transformants were attained using G418 antibiotic screening. Stable integration of ttl gene into recombinant P. pastoris was verified through PCR cloning. In lab scale fermentation, it was found 1.5% methanol,3.5×108 cell/mL P. pastoris and pH 6.0 were optimal for fermentation of recombinant P. pastoris, within which the lipase activity of recombinant P. pastoris increased to 156 IU/mL.SDS-PAGE detected a clear band at 39 kDa in fermentation broth, which corresponded to the size of TTL and further proved that the foreign ttl gene was successfully extracellularly expressed by P. pastoris.Heterologous expression of ttl gene in T. reesei was carried out. T. reesei is a popular filamentous fungi for heterologous gene expression, while its prerequisite is establishment of an efficient transformation system. In protoplast mediated transformation (PMT), the highest transformation efficiency resulted from 1 h enzymolysis of 8 h pre-germinated spores by 10 mg/mL snailase to release protoplasts, and then 5 x 108 cell/mL protoplasts were applied for transformation using PEG6000. In Agrobacterium tumefaciens mediated transformation (ATMT), the optimal conditions were as followed:mixing the 3 h pre-germinated T. reesei spores with Agrobacterium EHA105 at OD6000.8, sprayed this mixture on nitrocellulose filter of induction medium for 2 d cultivation at pH 5.3 and 24 ℃. Compared with PMT, ATMT required less time and led to higher transformation frequency, furthermore, the ATMT transformants showed higher stability and protein expression capacity, proving that ATMT was an efficient tool for transformation of T. reesei.The lipase gene was further optimized and synthesized according to codon preferences of T. reesei, and inserted into the expression cassette controlled by cbhl promoter and terminator to construct the binary expression plasmid pCB-H-PstT with hygromycin resistance. This plasmid was transformed into T. reesei cells via PMT or ATMT, and recombinant transformants were obtained via a newly designed screening methodology. Lipase activity of recombinant T. reesei transformants would reach 241 IU/mL in fermentation for 72 h in shaking flasks. PCR and SDS-PAGE analysis proved that ttl gene was successfully extracellularly expressed by T. reesei.For overcoming the uncertainty of random insertion during transformation and further increasing the expression level of heterologous alkaline lipase by T. reesei, homologous integration was implemented. Applying the genomic DNA of fresh T. reesei mycelium as templates, the left flanking sequence (1.4 kb) and right flanking sequence (1.5 kb) of cbhl was cloned using specific primers. These flanking sequences were further ligated with the lipase expression cassette PstT to construct the plasmid pCB-H-LER for homologous integration. This plasmid was transferred into T. reesei genomes to generate recombinant transformants. It was found that 3% lactose,0.9% yeast extract and pH 5.5 were the most preferred conditions for lipase production, within which lipase activity of transformant reached 375 IU/mL and no cellobiohydrolase activity was detected. SDS-PAGE analysis showed that there was a 39 kDa band in fermentation supernatant corresponding to TTL and no band corresponding to cellobiohydrolase was detected. Result proved that foreign lipase gene was stably integrated into the cbhl locus of T. reesei, endowing it the ability to stably extracellularly express TTL.Property research of recombinant lipase found that properties of recombinant lipases from P. pastoris and T. reesei were nearly the same. The optimum pH and temperature of lipase produced by P. pastoris were 9.5 and 60 ℃ respectively, and it showed obvious lipase activity in the pH range from 8.0 to 10.5 and temperature range from 40 ℃ to 70 ℃. TTL also exhibited strong tolerance toward alkaline conditions and high temperatures that it would keep more than 70% of the maximum activity after treatment in 60 ℃ and pH 11 for 1 h. Recombinant lipase showed so strong resistance to denaturation caused by fatty acids’ accumulation that it would stably hydrolyze the olive oil emulsion. Ca2+ would slightly increase TTL activity and Na+ and K+ had nearly no effect on TTL, while, TTL was suppressed by Mn2+, Fe2+, Cu2+ and Zn2+. Additionally, TTL showed strong tolerance toward ethanol, butanol, methanol and isopropanol, but it was obviously inhibited by acetone. There was inhibition effect from surfactants on TTL, especially Tween 20.The T. thermophilus lipase gene was successfully cloned and expressed by P. pastoris and filamentous fungi T. reesei, which paved the way for industrial application of the thermotolerant and alkaline lipase. Results from this study were meaningful and promissing froms scientific and industrial aspect.