Construction And Preliminary Verification Of A 18S Ribosomal DNA-mediated Integrative Expression Vector In Kluyveromyces Lactis

Kluyveromyces lactis has been approved as GRAS by FDA,it is a favorable host for heterologous proteins production,offers considerable advantages,such as rapid growth to high density on a wide variety of carbon source,high secretion capacity,proper protein glycosylation and so on,which has been extensively used in food and pharmaceutical fields.However,some restrictions and drawbacks are often encounterded,low production titers and poor activity of heterologous proteins expressed in K.lactis,the limited choice of expression vectors for heterologous gene,and immaturity in basic theoretical study.Above all,it is certainly necessary to construct efficient and suitable molecular tools to provide the basis for genetics research and molecular transformation in K.lactis.In this study,we investigated the function of different promoters and signal peptides in K.lactis,and selected excellent functional elements to construct stable integrative expression vector with 18 S rDNA sequence for homologous recombination.The main contents and results as follows:The ability of different promoters that regulate expression of heterologous gene were evaluated with Enhanced Green Fluorescent Protein gene(egfp)as a reporter gene in K.lactis,including promoters from K.lactis(pKlADH4,pKlMAL22),S.cerevisiae(pScGAL7,pScPGK,pScTEF)and S.passalidarum(pSpMAL1,pSpMAL6,pSpGAL1).Based on the reformed vector pKLAC1*,the recombinant expression vectors pKLAC1*-promoter-egfp were constructed,then electrotransformed into K.lactis GG799 to obtain single-copy integrated recombinants by PCR with specific primers.The results of fluorescence tests showed that EGFP was successfully expressed with all eight promoters in various degree,the fluorescence intensity of pScGAL7 was 2.92±0.07,much more stronger than pKlLAC4(2.13±0.35)which is the most widely used currently,and under the inducting condition of galactose,the expression of EGFP improved 57%,reached to 4.57±0.28,indicating the potential ability of efficiently transcription initiation for heterologous genes.The ability of different signal peptides that guide secretory expression of heterologous protein were evaluated with codon optimized adenylate deaminase gene(AMPD)derived from Streptomyces murinus as a reporter gene in K.lactis.Five different signal peptides were compared,including the signal peptides derived from S.cerevisiae(SUC2,PHO5,KT),K.marxianus(INUSS)and K.lactis(?MF).The recombinant vectors pKLAC1-sp-AMPD were constructed,then electrotransformed into K.lactis GG799 to obtain single-copy integrated recombinants by PCR with specific primers.The AMP deaminase activity in the fermentation supernatant of recombinants were compared and the results showed that all five signal peptides sucessfully guided secretory expression,what's more,?MF is more efficient than others,its AMP deaminase activity reached to 243.89±9.56 U·m L-1.Based on the function analysis of promoters and signal peptides,pScGAL7 and ?MF were chosen as functional elements.The recombinant expression vector pTRGA-amdS was based on the plasmid pMD 19-T simple,containing K.lactis-derived 18 S rDNA sequence for homologous recombination,the Aspergillus nidulans-derived acetamidase gene(amdS)as selection marker,and AMPD as reporter gene.The recombinant expression vector was linearized by Sac II,removing E.coli ori and Amp resistant gene,then electransformed into K.lactis GG799 to obtain transformants.Real-time quantitative PCR analysis indicated that the copy number of the integrated AMPD gene ranged from 1 to 3 and positively correlated with AMP deaminase activity,indicated heterologous gene can be successfully expressed by this vector in K.lactis.When the inducer galactose was used as carbon source,the AMP deaminase activity in the culture supernant of the recombinant with three AMPD gene copies reached to 590±13.33 U·m L-1,improved 32.6%.What's more,the integrants were mitoically stable for 58 generations under the non-selective pressure condition(98.58%).The new expression vector with a integrative locus of rDNA lays a theoretical foundation for further practical application.