Functional Analysis Of Two Lipases And Research On Regulation Of Phosphatidic Acid In Oil Metabolism

Biodiesel is one of the substitutes for the world’s finite non-renewable energy resources, with the characteristics of renewable energy and non-pollution for environment. Lipase can transesterify TAG or esterify fatty acid to biodiesel, which is a best current way to make biodiesel without second pollution brought by acid or alkali catalyzer. However, the high cost of biodiesel production still exists mainly caused by the finite lipase efficiency, moderate thermostability and the high cost of oil materials which occupied the 60%-75% of the production cost of biodiesel. Therefore, research on improving the efficiency of potential lipase, discovering the new lipase resources and the regulation of transcriptional factors for lipid biosynthesis in Arabidopsis thaliana will be helpful to disclose the relationship of lipase structure and function, and the metabolism of lipid biosynthetic regulation in plant seeds. In this thesis, three-site mutagenesis was made for Candida Antarctica Lipase B （CALB）, a most widely-used current lipase for biodiesel production, to study its properties. One novel lipase gene was cloned from Serratia liquefaciens S33 DB-1. And six transcriptional factors belonging to three families were used to screen the regulator for lipid biosynthesis in Arabidopsis seeds.1) The three-site CALB mutagenesis of Asn292Tyr, Val210Ile and Ala281Glu （designated muCALB） was synthesized in vitro based on the preferred codons of Pichia pastoris, so did wild-type CALB as control. The muCALB or CALB gene fused with the secretory signaling peptideα-factor was successfully integrated into the heterologous host, Pichia pastoris GS115, respectively. The recombinant was induced by methanol to secrete active lipase. The growth curve of recombinant strain showed that the lipase level reached highest after induction for 48 h though the cell identity was still lower to be about 7. Based on computer-aided molecular modeling with SYBYL 7.0, the biomimetic affinity ligand of 4-aminobenzamidine and M-aminophenylboronic acid specifically binding to CALB was designed to purify the recombinant lipase. The CALB and muCALB can reach 99% purity after purification with affinity ligand of 4-aminobenzamidine and M-aminophenylboronic acid. The hydrolytic activity of muCALB on substrate ofρ-nitrophenyl esters was changed and concomitantly decreased from 17.3 U mL-1 to 7.1 U mL-1 with the increase of its acyl chain length from C10 to C18. The transesterification activity of crude muCALB enzyme on soybean oil and methanol was increased 2- to 3-fold, compared with wild-type CALB. Especially, its transesterification activity reached 3.02% at 55℃and 3.08-fold improvement, compared with 0.98% activity of wild-type CALB. The thermostability by hydrolysis assay showed the residue activity was 20% and 10-fold improvement, compared with the only 2% residue activity of wild-type CALB after thermal treatment for 210 min at 85℃.2) Genomic-walking method was used to clone a novel lipase gene （designated SLLipA） from Serratia liquefaciens S33 DB-1. The BLAST finder showed SLLipA contained an open reading frame （ORF） of 1845 bp encoding 615 amino acids with a conserved GXSXG motif. Alignment of the coding protein SLLipA showed it has 74% high identity with Serratia marcescens （Acc. No. DQ841349）. SLLipA gene fused with the secretory signaling peptideα-factor was successfully integrated into the heterologous host, Pichia pastoris GS115. The recombinant strain formed big halo on the plate of Rhodamine-Triglyceride-Agarose was selected to study its function. The growth curve of its recombinant strain showed that the lipase activity was secreted when induction for 12 h and reached high level of 16 U mL-1 after induction for 48 h. The hydrolytic activity of SLLipA on substrate ofρ-nitrophenyl esters was specific and preferred to long acyl chain ester of para-nitrophenyl stearate （ρ-NPS）, then to the middle acyl chain esters of para-nitrophenyl laurate （ρ-NPL） and para-nitrophenyl myristate （ρ-NPM）. SLLipA did not show the transesterification activity when catalyzing the substrate of soybean oil and methanol.3) The five transcriptional factors of GL2, WER, FUS3, MYB （At4G01060） and MYB （At2G30420） from Arabisopsis thaliana and one GmMYB73 from Glycine max drove by the seed specific promoter of beta-conglycinin were transformed into Arabidopsis using the plant binary vector of 35S-FAST. Seeds of the T1 transformants were used to analyze the oil content by GC. The results showed the oil content of transformants by GL2, WER and GmMYB73, respectively, changed a lot including higher of some lines and lower of others, suggesting that these three might be related to affect the lipid biosynthesis in Arabidopsis.4) The proteins of WER, GL2, FUS3, MYB （At4G01060） and MYB （At2G30420） fused 6XHis were expressed in Reselta （DE3） and used to investigate the interaction between protein and lipid by fatty western blot, respectively. Binding data showed that 6XHis-WER and 6XHis-GL2 specifically bound with egg yolk PA and other type PA including diPA（18:1）, diPA（18:2）, diPA（16:0-18:1） and diPA（16:0-18:2）, and didn’t bind with other lipids including PC, PE, PG, PI, PS, LPC, LPA, DAG（8:0）, DAG（18:0-18:1） and DAG（2:0-18:1）. And 6XHis-FUS3, 6XHis-MYB （At4G01060） and 6XHis-MYB （At2G30420） didn’t show any binding to any of all these lipids, suggesting the high-affinity interaction between 6XHis-WER / 6XHis-GL2 and PA had high-affinity specificity.5) Phosphatidic acid （PA） has been recognized as a lipid second messager. Surface plasmon resonance （SPR） was employed to quantitate the interaction between 6XHis-WER and liposome of PA/phosphatidycholine （PC） or PC alone. The data disclosed a high-affinity interaction with a 1.76E-11（1/s） of average best-fit data by nonlinear regression using GraphPrism software.Next, identification of the segment of WER necessary for PA binding was determined using a deletion mutagenesis strategy. A series of 6XHis-tag WER deletion mutants of WER （1-65）, WER （66-116）, WER （117-203）, WER （1-116） and WER （66-203） were initially generated and used in liposomal binding assays. Binding assays revealed that WER residues between 1 and 65 were sufficient for the interaction with the lipid PA, consistent with the SPR data of a high specificity binding of WER （1-65）. To further narrow the segment of WER necessary for PA-specific binding domain, C-terminal deletion of 14 residues from WER （1-65） was generated and assayed for PA/PC and PC alone by liposomal binding. Further revealed that segment between 1 and 51 was not bound to PA, then suggested to determine that WER residues between 51 and 65 were required to confer PA binding to WER.To identify critical residues involved in PA binding within the localized PA binding region of WER, point mutations of WER_K51A, WER_R52A, WER_R58A, WER_R60A, WER_K51A+R52A and WER_R58A+R60A were generated by site-directed mutagenesis and used in liposomal binding assay. Binding assays revealed that none of them was bound to PA, which was also confirmed by SPR. When these data are taken together, it is confirmed that WER motif （51KRCGKSCRLRWMNYL65） is sufficient for PA binding and residues of K51, R52, R58 and R60 are critical for PA binding.6) To observe the subcellular localization of WER and mutants of WERK51A+R52A and WERR58A+R60A, tobacco （Nicotiana Benthamiana） was used to infiltrate the agrobacterial GV3101 containing the construct of Pro35S::eYFP-wtWER, pro35S::eYFP-mutWERK51A+R52A and pro35S::eYFP-mutWERR58A+R60A, respectively, together with C58 containing P19 construct, into its leaves. Subcellular localization revealed that WER and mutants were localized in the nucleus, but WER mainly distributed into the nucleolus.