One-step Integration Of Multiple Genes Into The Pichia Pastoris By CRISPR

Pichia pastoris(Komagataella phaffii)is widely used in industrial production and laboratory research because of its notable advantages in protein expression.Recently,a variety of pharmaceutical compounds and intermediates have been successfully synthesized in P.pastoris,such as 6-methylsalicylic acid,terreic acid,lovastatin,etc.Thus P.pastoris is a good chassis cell for production of useful compounds via assembly of complicated biosynthetic pathway with multiple enzymes.For gene recombinantion in P.pastoris,the foreign gene is usuallly integrated into genome because the free plasmid cannot stably exist.Commonly,efficiency of homologous recombination is low in P.pastoris and selection markers are quite limited.It caused many problems when constructing biosynthetic pathway such as complicated design and long construction period,which limits the study and application of this host.Vogl group has established an efficient CRISPR/Cas9 based gene editing platform in P.pastoris CBS7435 through a large number of experimental explorations,achieving gene knockout and mutation and developing methods for marker recycling.Besides,some other methods have been developed,such as marker recovery and plasmid construction with multiple expression cassettes.Nevertheless,it is still cannot solve the problems of long period for strain construction and low efficiency for assembly of complicated biosynthetic pathway.In this work,we employed nonhomologous end joining(NHEJ)defective strain Aku70 as a host to develop an efficient multiple genes one-step integration method with marker-free,providing a new method for gene expression in P.pastoris.Homologous recombination efficiency is usually weak in P.pastoris,because genes of double-strand break of Cas9 are more likely repaired by NHEJ.Thus reducing the NHEJ should facilitate the efficiency of homologous recombination.KU70 is a key gene working for NHEJ in cells.Therefore,we knocked out KU70 in P.pastoris GS115 using CRISPR/Cas9 technology,resulting in a NHEJ defective strain Aku70.This strain is histidine defective after losing plasmid carrying Cas9 coding gene,gRNA and histidine marker after cultured in YPD medium.The results showed that homologous recombination efficiency is obviously promoted in ?ku70,which was then used as a parent strain for the following constructions.Firstly,integration site play an important role for gene knock-in and a good integration site should not affect the growth and metabolism of the host strain.In this study,the integration sites were selected from the upstream of identified promoters or downstream of terminators in the genome.This will avoid negative affects of gene integration on the endogenous expression cassette in cells.Ten gRNA targets were designed in the range of 100 bp upstream of promoters or downstream of terminator,and eGFP was used as a reporter protein to test the homologous recombination efficiency of each gRNA target.A series of efficient integration sites were screened finally.Therein,the efficiency of the pAOX1up-g2,pTEF1up-g1 and pFLD1up-g1 target even reached 94.7%,95.8%,91.7%,respectively.Secondly,to avoid the effects of chromosomal rearrangement,the selected sites should distributed in different chromosomes.Here,the pAOX1up-g2,pTEF1up-g1 and pFLD1up-g1 target was distributed in different chromosomes.The high efficiency targets were combined,and eGFP and mCherry were used as reporter proteins to test the efficiency of two genes one-step integration,and it even reached up to 65%.Similarly,eGFP,mCherry and BFP were used as reporter proteins to test the efficiency of three genes one-step integration and it reached about 20%finally.These results demonstrated that the CRISPR/Cas9 method worked well for one-step integration of multiple genes into the P.pastoris.Then,we involved biosynthetic pathway of a fungal polyketide terreic acid to test the practicability of the multiple genes one-step integration method.Two intermediates of 6-methylsalicylic acid(6-MSA)and 3-methylcatechol were used as the reported products.The recombinant strains were cultured in a shake flask and the products were extracted for HPLC analysis.The results showed that the recombinant strains obtained by one-step integration method successfully synthesized the target products,which proved that this method has application potential and practical value.In addition,plasmid carrying Cas9 coding gene,gRNA and selection marker was successfully losed in the producing strains after cultured in YPD medium,which achieved the recovery of marker and verified the feasibility of integration without markers.Generally,this study constructed a series of universal vector tools for one-step integration for multiple genes integration,which achieves efficient maker-free genes knockin in P.pastoris,which greatly shortens the construction period and simplifies construction design.It provides new methods for the application of P.pastoris expression system.