Developing The Technology Of Non-homolgous End Joining-Mediated Random Insertional Library In Yarrowia Lipolytica And Its Application

Random mutagenesis combined with high-throughput screening is a powerful tool for mining genetic elements and potential regulatory mechanisms linked to phenotypes.Although many mutagenesis strategies exist for diversifying nucleic acid sequences,random mutations generated by traditional physical or chemical mutagenesis often require extensive analysis to identify key mutations.CRISPR-based library screening can identify mutations relatively quickly.However,it remains challenging to design highly efficient guide RNA libraries,and inactive guide RNAs often reduce genomewide coverage and produce false negatives.Insertional mutagenesis is a convenient approach to generate mutation libraries.The insertion of specific sequences in the genome not only causes gene inactivation or affects gene expression,but the insert can also serve as a selectable marker or a tag for insertion site identification,through which it can identify insertion sites,thereby facilitating the rapid mapping of key mutations.For example,transposon-mediated insertional mutagenesis has been developed for the construction of mutagenesis libraries in many microorganisms,becoming a powerful tool for large-scale analysis of gene function.However,some microorganisms do not have suitable endogenous transposons.Therefore,it is necessary to introduce heterologous transposon systems for mutagenesis.The mutagenesis efficiency of this method is largely dependent on the transposition efficiency.Therefore,it is desirable to demonstrate powerful approach for rapid trackable mutagenesis.Yarrowia lipolytica is an unconventional oleaginous yeast that is an excellent chassis for the production of lipid-based chemicals,organic acids,flavonoids and terpenoids due to its unique physiological and metabolic properties.Unlike the model yeast Saccharomyces cerevisiae,Y.lipolytica shows a strong preference for nonhomologous end joining（NHEJ）to repair DNA double-stran d breaks（DSBs）.Endogenous DSBs are produced in many biological processes and are widely distributed throughout the genome.During NHEJ-mediated repair of DSBs,free DNA can be inserted into the junction without the need for a homologous DNA sequence as a template.Therefore,the insertion of foreign DNA offers the possibility of creating insertional mutagenesis.In this study,we mapped the distribution of NHEJ-mediated integration for the first time.Its random distribution and high coverage rate met the requirements of genome-wide mutagenesis.Mutagenesis libraries can be constructed by simply transforming DNA fragments containing the selectable marker.Then based on this approach,different types of mutagenesis libraries were constructed and applied.Among them,Using NHEJ-mediated integration to construct the random insertional mutagenesis library,the new targets that improved acetic acid tolerance and β-carotene biosynthesis were identified rapidly.On this basis,the library construction strategy was further optimized.In addition to the selectable marker,the inserted fragment also introduced a strong promoter UT8（UAS1B8-TEF1）to construct a gene activation library.And this library construction strategy was used to identify the target that activate xylose metabolism in Y.lipolytica.Subsequently,by constructing a NHEJ-mediated GFP（Green Fluorescent Protein）random expression library,the highly expressed genomic integration sites were identified,and based on this,a CRISPR-based gene expression toolkit was developed.1.Location characterization of NHEJ-mediated genome integration in Yarrowia lipolyticaFirst,we achieved efficient transformation and genome integration of foreign DNA fragments（LEU2 expression cassette）through the strong preference of Y.lipolytica for NHEJ to repair DSBs,and constructed a NHEJ-mediated mutagenesis library with a capacity of one million monoclonal.Then an enzymatic ligation-mediated intramolecular circularization and high-throughput sequencing were used to determine the integration sites,thereby mapping the distribution of NHEJ-mediated genome integration for the first time.Analysis of 9.15×105 integration sites showed that NHEJmediated integrations were randomly distributed on chromosomes with a high integration frequency.Transcriptional regulatory regions exhibited a relative integration preference,especially near the transcription start sites（TSSs）.Analysis of the flanking sequences of the integration sites revealed no sequence-specific bias.Furthermore,the mapping of nucleosome-occupied regions（NORs）and nucleosome-free regions（NFRs）in the Y.lipolytica genome was achieved for the first time using ATAC-seq technology.By further analyzing the relationship between NHEJ-mediated integration and nucleosome occupancy,the results showed that integration sites were distributed in both nucleosome-occupied and nucleosome-free regions.The distribution map of NHEJmediated genome integration exhibits random distribution and high coverage of integration sites,meeting the requirements of genome-wide mutagenesis.2.Construction of NHEJ-mediated random insertional mutagenesis library to identify targets for improving acetic acid tolerance and β-carotene biosynthesisWe constructed a NHEJ-mediated trackable random insertional mutagenesis library and achieved a rapid screening of key targets for improving acetic acid tolerance and β-carotene biosynthesis.In the study of acetic acid stress tolerance,the transcriptional activator YALI1_B10891g（homologous protein of S.cerevisiae Haal）was identified,which was involved in the regulation of weak acid stress.A stressresponsive transcriptional activator YALI1_B28150g（Msn2）and a ubiquitin-specific protease YALI1_D04469g（Ubp2）were also identified.The insertion targets were all located upstream of the gene coding region,indicating that the insertion of foreign DNA fragments improved the acetic acid tolerance by affecting the expression of the target genes.In the case of β-carotene biosynthesis,four key targets were identified:including RabGGTase（YALI1_F09177g）,a transcription factor Ndt80 that regulate meiosis（YALI1₁9390g）,a histidine-rich protein（YALI1_B07915g）and a protein with unknown function（YALI1_B10170g）.Some of these targets are located in the gene coding region,and some are located upstream of the gene coding region,such as RabGGTase,which is a Rab Escort protein,an essential gene,and the insertion occurs in the promoter region.Geranylgeranyl pyrophosphate（GGPP）is the direct precursor of β-carotene.Transcriptional analysis speculated that this mutant reduced the consumption of precursor GGPP by down-regulation the transcription of YALI1_F09177g,thereby promoting the production of β-carotene.And deletion of the transcription factor Ndt80 increased β-carotene production by 62%.On the one hand,this mutant regulates the accumulation of β-carotene by down-regulating the synthesis of ergosterol,and on the other hand,promotes the storage of β-carotene by increasing the production of lipids.In this study,the important role of this transcription factor in terpenoids synthesis was discovered for the first time.Furthermore,the identification of intergenic and intragenic targets demonstrates the ability of NHEJ-mediated mutagenesis libraries in gene disruption and gene expression regulation,especially when identifying essential genes and genes requiring moderate expression,and is a powerful complement to classical methods.3.Construction of NHEJ-mediated gene regulatory library to identify targets that activate xylose metabolism in Yarrowia lipolyticaThe preference of NHEJ-mediated integration in transcriptional regulatory regions,especially in promoter regions,enables the construction of gene regulatory libraries.To test whether this approach can construct a gene activation library,we inserted the strong hybrid promoter UT8 into different positions of the endogenous promoter of Y.lipolytica,and found that the activation of gene expression could be achieved.Therefore,a random insertional library was constructed by NHEJ-mediated integration to achieve one-step gene inactivation and regulation library construction,and the target gene YALI1_C28456g related to the activation of xylose metabolism in Y.lipolytica was successfully identified.Reverse engineering verification showed that there was an error in the open reading frame（ORF）annotation of YALI1_C28456g.After redefining the ORF,it was confirmed that the insertion of the UT8 fragment into the transcriptional regulatory region of the target gene YALI1_C28456g activated the expression of the gene,thereby realizing the growth of the mutant strain on xylose.Sequence similarity alignment,transmembrane domain prediction,subcellular localization,and RT-qPCR transcription level determination indicated that YALI1_C28456g was a xylose transporter of Y.lipolytica.In order to verify whether the mutation has transport capacity for all pentose,we also tested the effect of the mutation on the growth of arabinose,and found that the overexpression of YALI1_C28456g can also enhance the metabolism of arabinose,proving that it promotes the metabolism of pentose.4.Construction NHEJ-mediated GFP random expression library to develop CRISPR-based gene expression toolkitIn a previous study,we found that the integration of foreign genes resulted in differential expression depending on the location of genome integration.We then constructed a GFP random expression library based on NHEJ-mediated integration.Potential integration sites with high expression were identified by FluorescenceActivated Cell Sorting（FACS）technology.By characterizing the expression stability of these integration sites under different conditions,and measuring the CRISPR-Cas9mediated integration efficiency,we obtained neutral integration sites with high expression and high integration efficiency.In addition,different expression regulatory elements were also characterized,and a total of 18 promoters and 12 terminators were characterized,thus extending the expression range of the CRISPR toolkit.These genetic elements and integration sites were used to combinatorially regulate genes in the lycopene biosynthetic pathway,resulting in different levels of lycopene synthesis.In summary,this paper systematically analyzed the location distribution characteristics of NHEJ-mediated genome integration,and developed the NHEJmediated random insertional library construction technology.After optimization,this technology can achieve gene inactivation and activation or inhibition regulation.Using this library construction strategy,key genes affecting acid tolerance,β-carotene synthesis and xylose metabolism were identified,indicating the application potential of this library construction strategy in mining genetic elements related to simple phenotypes.In addition,we used this technology to construct a GFP random expression library and also identified CRISPR integration sites with high expression and high integration efficiency,and constructed a CRISPR expression regulation toolkit.The development of these technologies and toolkits has greatly expanded the molecular biology tools for understanding Y.lipolytica,and is of great significance to the mechanism research and metabolic engineering research of this yeast.