| Yarrowia lipolytica as a non-conventional yeast,is a generally recognized as safe microorganism.Due to its unique physiological characteristics,it has been widely used to construct cell factory.In metabolic engineering,the selection marker gene is the basic element of genetic vector and engineering cell factories require genetic markers to select the cells with desired genetic modifications.Besides,the precise modification of key genes in metabolic pathways is also crucial.However,the commonly used selection markers in Y.lipolytica are limited and the successive genetic manipulations are often restricted by the number of available selection markers.Although Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)gene editing system has been widely applied in numerous organisms with high efficiency and accuracy,the low editing efficiency of Cas9 and poor homologous recombination rate of Y.lipolytica hinders its precise genetic manipulation.Therefore,development of the genetic markers and construction of the efficient CRISPR editing system are important for the engineering of Y.lipolytica strains in constructing efficient cell factories.In this study,by testing different selection markers and the working concentration of antibiotics,we identified four genetic markers that can be used in Y.lipolytica.Firstly,we developed a dominant marker dsdA encoding D-serine deaminase for genetic manipulation in Y.lipolytica.It confers to Y.lipolytica the ability to use D-serine as nitrogen source.In addition,the selection condition of several unfrequently used dominant markers was further analyzed.We found that the expression of resistance gene enables the selection of zeocin-resistant transformants(bleoR),G418-resistant transformants(kanMX)and mycophenolic acid-resistant transformants(guaB)in Y.lipolytica,but the expression of Cu2+ resistance gene CRF1 did not get positive transformants.Therefore,we successfully developed four dominant selection markers.In order to construct an efficient CRISPR genome editing system in Y.lipolytica,we optimized the elements of the CRISPR/Cas9.The cutting efficiency of Cas9 was tested through determining the gene deletion efficiency of URA3.We found that when sgRNA was expressed under the promoter SCR1p-tRNA,and Cas9 was integrated into the YALI1_E15321g,the gene deletion efficiency was 92.5%,37.5%higher than that of TEFin promoter expressing sgRNA,22.5%higher than the CEN plasmid expressing Cas9.Besides,using endogenous tRNAGly processing mechanism,we constructed a tRNAGly-based multiplex sgRNA expression system,and the efficiency of simultaneous gene deletion of TRP1 and URA3 reached to 57.5%.Then,to achieve efficient precise genome integration by CRISPR/Cas9 in Y.lipolytica,DNA recombination machinery in Y.lipolytica was engineered to enhance homologous recombination rate.However.the expression of homologous recombination related genes RAD52 and SAE2 did not enhance the integration efficiency.Knocking out the non-homologous end-joining key gene KU70,the site-specific integration efficiency reached to 92.5%.Besides that,we found the integration efficiency of wild-type Cas9 in the CEN plasmid expression was 45%lower than that of chromosomal integration when knockout KU70.To enhance the integration efficiency of the CEN plasmid expressing Cas9,we introduced D147Y and P4 11T in Cas9,and the integration efficiency reached to 75%in the strain without KU70 deletion,which was 32.5%higher than wild-type Cas9,while KU70 deletion did not further improve the integration efficiency.The genetic markers developed here will help to expand the genetic tools and facilitate genetic manipulation of Y.lipolytica.In addition,the CRISPR-base genome editing system was constructed by optimizing the gene editing elements and recombination machinery of Y.lipolytica,the efficient gene knockout and site-specific integration were achieved.These findings will facilitate the engineering of Y.lipolytica and construct efficient cell factories for the production of various chemicals. |
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