Study Of Heterologous Expression Of Streptomyces Phospholipase D

Phospholipase D(PLD,EC 3.1.4.4)is a well-known enzyme for the modification of phospholipids by introducing functional polar head groups.It has a great potential in pharmaceutical and food industry for the enzymatic synthesis of multifunctional bioactive phospholipids,such as phosphatidylserine,phosphatidyl-batilol,phosphatidyl-glucose,cardiolipin analogs,phosphatidyl-tyrosol,phosphatidyl-terpenes,and phosphatidyl-serinol.Although PLD from Streptoverticillium cinnamoneum has been successfully over-expressed in recombinant Streptomyces lividans with a high yield of 5.5×104 U·L-1(118 mg·L-1),the limited supply of PLD is still on an obstacle to the widespread use of enzymatic processing due to high price.Meanwhile,high-production of Streptomyces PLD in Escherichia coli or Pichia pastoris is still difficult,due to its high cytotoxicity which causes severe cell lysis upon expression of active PLD,which likely degrades phospholipids contained in the membrane of the host cells.In this study,in a secretory manner similar to Streptomyces,extracelluar production of Streptomyces PLDs in P.pastoris,Brevibacillus and E.coli were investigated for selecting an appropriate heterogenous expression system.In P.pastoris,although the strategies of temperature down shifting and differences in pH between cell growth and PLD optimum activity were effective for enhancing PLD expression,the level of PLD producton was quite low,up to 0.08 U·mL-1.In Brevibacillus choshinensis,secretory PLD production was successfully attained using weak constitutive promoter P5,up to 0.88 U·mL-1.In E.coli,extracelluar PLD activity was slightly improved by co-expressing colicin E1 lysis protein(Kil),but not by fusing PLD to the Yebf carrier protein,compared to periplasmic production by fusing the PelB signal peptide to the N-terminus of PLD.Additionally,E.coli cell surface display of PLD was attained by fusing PLD to the Ag43 auto-transporter protein,and the PLD activity of whole cells reached 1.3 U·mL-1.The specific PLD yields of four fusion expression system in E.coli were similar since using the same PT7 promoter.In addition,it was slightly higher in the periplasmic production.Taken account into the PLD yield and specific yield,the periplasmic production of PLD in E.coli was further investigated.To address the issue of PLD cytotoxicity to E.coli in the periplasmic production of PLD which caused plasmid instability,short-time PLD synthesis,cell lysis and PLD leakage,the integrated optimization of PLD production in upstream processing was performed.Effective strategies includes the use of plasmids containing the pSC101 par region in recA-E.coli,the use of moderately strong and tightly regulated promoter PBAD,optimization of codon usage and amino acid supplementation,maintaining the best cellular state by supplementing nutrition,and saturation induction at low-temperature(18 ?).According to the strategies,the plasmids for expressing PLD maintained completely stability in the strains,and a large amount of periplasmic PLD(1.2×105 U·L-1,81.5 mg·L-1)was obtained in the batch culture,which accounted for 99.5%of the total activity.Meanwhile,the synthesis time of PLD was prolonged to 9 h with glycerol consumption,in contrast to that almost all the recombinant cells were rapidly killed after the induction of 2-3 h in the reported literature.Moreover,since more than 90%expressed PLD was maintained in the periplasm of E.coli,a simple and efficient extract method was developed.The total PLD in the periplasm was released and the specific activity was at an acceptable level in the presence of 10 mM EDTA,0.05%(w/v)sodium deoxycholate DOC,and 200 mM Tris-HCl(pH 7.5).Furthermore,optimization of extract parameters,such as extracting volume and time,showed that the ratio of extractant:wet cell weight(WCW)around 18-20 mL·g-1 for 30-40 min was efficient enough.Subsequently,large-scale purification of PLD was achieved through a four-step downstream processing,including extraction with the EDTA and DOC method,ammonium sulfate precipitation,anion exchange and Ni2+-affinity chromatography.As a result,the PLD was purified to 327 fold and had a specific activity of 1471.5 U·mg-1,and it had the same molecular weight as expected on SDS-PAGE and Western Blotting.Finally,from a cross-protection perspective,the effects of various cell stress responses induced by reductant,alcohol,ribosomal antibiotics,salts and polyols chemicals on PLD expression and cell growth were investigated.Among them,tetracycline mimicking cold shock and osmotic agents(salts and polyols)enhanced PLD expression.Especially high NaCl stress(0.4-0.5 M),profoundly increased PLD production(5-fold increase),and simultaneously alleviated the growth inhibition imposed by PLD expression.This phenomenon also occurred in addition of KC1 and LiCl,but not in addition of iso-osmolal amounts of sorbitol or mannitol.These results suggest that the enhancement is dependent on the specific efficiency of alkali metal ions but not osmotic stress.Ultimately,highly enhanced PLD production via 0.4 M NaCl stress was achieved in a 3 L fermentor,up to 1.1×106 U·L-1(748 mg·L-1),and the extracellular activity was 1.6×104 U·L-1,accounting for 1.5%of the total activity.The yield of PLD activity is 90 times and 20 times higher than that from previously reported studies in E.coli and in Streptomyces lividans,respectively.Meanwhile,cell growth and glycerol consumption was coupled to PLD production in the induction phase whereby PLD synthesis time was prolonged to 36 h.Subsequently,the senescent phase lasted for 34 h,but the extracellular activity was only 5.0×104 U·L-1,accounting for 4.8%of the total activity.Hence,severe cell lysis and enzyme leakage failed to occur.In general,this study has successfully circumvented the problem of cytotoxicity of PLD to E.coli,and overcome the barriers of plasmid instability,short-time PLD synthesis,cell lysis and PLD leakage,resulting in high production of PLD.Since large-scale PLD purification has been successfully achieved,the study provides the basis for the widespread use of PLD-enzymatic processing in pharmaceutical and food industry.