Agrobacterium Tumefaciens-mediated Transformation-based Genetic Recombination System For Rhodosporidium Toruloides

Rhodosporidium toruloides has highly developed pathways for isoprene and fatty acid synthesis.Therefore,it has considerable potential industrial applications in the intracellular synthesis of lipid and carotenoids.Establishing an effective genetic operating system in R.toruloides is necessary to improve its productivity and to investigate the molecular mechanisms of carotenoid synthesis by this strain.We previously obtained multiomics information about R.toruloides and established Agrobacterium tumefaciens-mediated transformation?ATMT?to lay the foundation for the metabolic engineering of R.toruloides.We then investigated the mechanism of genetic recombination in R.toruloides,and we found that the probability of non-homologous end joining?NHEJ?was considerably higher than that of homologous recombination?HR?.The rational design of R.toruloides was restricted by random integration of ATMT and feasible genetic manipulation technology.Therefore,in the present study,we illustrated the recombination mechanism in R.toruloides and established a recombinase system for the rational design and modification of its metabolic properties.This study consists of the following parts:1)The target gene was knocked out through Agrobacterium-mediated HR.The knockout vector pZPK-CRT-H with CRTI?Phytoene dehydrogenase gene?homologous arm was designed and constructed.Haploid NP11 was used as the starting strain,and the target chromosome was integrated through ATMT.The CRTI gene knockout transformants were screened on the basis of phenotype.The CRTI was targeted by a homologous knockout cassette,and 2%white-colored transformants were obtained,called NP11-ACRT.Vectors pZPK-CRT-1 and pZPK-CRT-2 were constructed with strong promoters PGPD and PADH2,respectively.Both promoters control CRTI expression.The final red transformants were then obtained.Fermentation and composition analysis showed that the intracellular carotenoid content?309 ?g/g?of the transformants increased by 30%.Therefore,we successfully developed a homologous genomic integration approach for targeting the CRTI locus in R.toruloides.We also confirmed the function of RHTO₀4602 in carotenoid biosynthesis.The established method will be helpful in the metabolic engineering of R.toruloides,a non-model species.2)The Cre/loxP recombinase system in R.toruloides was established.First,a double-resistant,double-expression cassette vector pZPK-H-BLE-2 was constructed,and its function was verified through ATMT.The recombinant site vector pZPK-Loxp-B with the antibiotic expression cassette and the recombinant expression vector pZPK-H-GPD-CRE/CREP were constructed by replacing BLE on pZPK-H-BLE-2 with the recombinase gene.Referring to R.toruloides bias,the codon-optimized Cre was synthesized and transformed through ATMT.Transformants expressing Cre could not grow on plates containing double antibiotics.This characteristic corresponded with the expected phenotype associated with the deletion of the resistance gene between loxP recombination sites.Thus,a gene recombination method based on Cre/loxP technology was established in R.toruloides.3)For the first time,the FLP/FRT system was applied to genetically manipulate R.toruloides.The recombinase plasmid pZPK-H-GPD-FLP was designated as the codon-optimized FLP under a native strong constitutive promoter PGPD.The recombinant site vector pZPK-FRT-B and recombinase plasmid pZPK-GPD-FLP were integrated into the genome of R.toruloides,and the phenotype associated with the insertion of the resistance gene between the FRT recombination site was retained.Three nuclear localization signals?NLS?were selected to fuse with the flippase.To analyze the NLS information of R.toruloides,we fused the expression vector recombinase FLP and transcription factor RHTO 01582 NLS sequence NLS3.We also constructed the vector pZPK-GPD-FLP-NLS3 and then transformed it into R.toruloides engineered strain NP11-FRT-B,which contained the recombinant site expression cassettes.Transformants expressing the phenotype of FRT were successfully obtained,and the recombination method based on FLP/FRT was established.To control FLP expression,we constructed the self-knockout vector pZPK-FRT-NAT-ADH2-FLP-NLS3 and the engineered strain NP11-H-ADH2-FLP-NLS3,which can express FLP when grown on glucose-deficient medium.The resistance gene could also be deleted by recombination.Thus,we established and perfected a gene recombination system based on FLP/FRT technology in R.toruloides.This system lays the foundation for complicated gene operation.4)We utilized the FLP/FRT recombinase system for general applications.MDH,HPS,PHI and PTA,four genes crucial to methanol metabolism,were successively transformed into R.toruloides by using the FLP/FRT system.In addition,carotenoid synthetic genes?CarB and CarRP?derived from Blakeslea trispora were integrated into the knockout CRTI strain NP11-?CRT,and the engineered strain regained the ability to synthesize carotenoids.Results showed that FLP/FRT can be used to engineer multiple genes in R.toruloides with limited available selection markers.To investigate the feasibility of applying FLP/FRT to other red yeast strains,we transformated the FLP/FRT system to the strains CGMCC 2.1515 and CGMCC 2.1609.Strains integrated foreign DNA in chromosomes by ATMT did not exhibit the expected site-specific gene knockout function.FLP/FRT technique was used in R.toruloides to recycle antibiotic marker genes and multigene expression,further indicating that the FLP/FRT technique is an important tool for the design and editing of the R.toruloides genome.