Yarrowia lipolytica is an important oleaginous industrial microorganism used to produce biofuels and other value-added compounds. Although several genetic engineering tools have been developed for Y. lipolytica, there is no efficient method for genomic integration of large DNA fragments. In addition, methods for constructing multigene expression libraries for biosynthetic pathway optimization are still lacking in Y. lipolytica. In this study, we demonstrate that multiple and large DNA fragments can be randomly and efficiently integrated into the genome of Y.lipolytica in a homology-independent manner. This homology-independent integration generates variation in the chromosomal locations of the inserted fragments and in gene copy numbers, resulting in the expression differences in the integrated genes or pathways. Because of these variations, gene expression libraries can be easily created through one-step integration. As a proof of concept, a LIP2 (producing lipase) expression library and a library of multiple genes in the β-carotene biosynthetic pathway were constructed, and high-production strains were obtained through library screening. Our work demonstrates the potential of homology-independent genome integration for library construction, especially for multivariate modular libraries for metabolic pathways in Y. lipolytica, and will facilitate pathway optimization in metabolic engineering applications.
Highlights d Lateral roots are potential Pseudomonas syringae pv. tomato DC3000 entry sites d The ARF7-LBD module regulates Pto DC3000-triggered lateral root formation d SA-suppressed lateral root formation is partially dependent on PR1 and PR2 d ARF7 regulates the expression of PR1 and PR2 in lateral root development
Yarrowia lipolytica has been extensively used for producing essential chemicals and enzymes. Like most other eukaryotes, non-homologous end joining (NHEJ) is the major repair pathway for DNA double strand breaks in Y. lipolytica. Although numerous studies have attempted to achieve targeted genome integration through homologous recombination (HR), this process requires the construction of homologous arms, which is time-consuming. This study aimed to develop a homology-independent and CRISPR/Cas9-mediated targeted genome integration tool in Y. lipolytica. Through optimizing the cleavage efficiency of Cas9, targeted integration of a hyg fragment was achieved with12.9% efficiency, which was further improved by manipulation of the fidelity of NHEJ repair, cell cycle, and the integration sites. Thus, targeted integration rate reached to 55% through G1 phase synchronization. This tool was successfully applied for the rapid verification of intronic promoters and iterative integration of four genes pathway for canthaxanthin biosynthesis. This homology-independent integration tool does not require homologous templates and selection markers, and achieves one-step targeted genome integration of the 8417 bp DNA fragment, potentially replacing current HR-dependent genome editing methods for Y. lipolytica. IMPORTANCE This study describes the development and optimization of a homology-independent targeted genome integration tool mediated by CRISPR/Cas9 in Yarrowia lipolytica. This tool does not require the construction of homologous templates, and can be used to rapidly verify genetic elements and iteratively integrate of multiple genes pathway in Y. lipolytica. This tool may serve as a potential supplement for current HR-dependent genome editing methods for eukaryotes.
Hepatitis A virus (HAV) is usually transmitted by an oral-fecal route and is prevalent not only in developing countries but also in developed countries. In the present study, the phylogenetic characterization of the VP1/2A junction region (321 nucleotides) of China HAV isolates was examined. Anti-HAV IgM-positive serum samples were collected from 8 provinces, including 20 cities or counties in China from 2003 to 2008; 337 isolates from 406 HAV patients' serum samples were amplified by RT-PCR, sequenced at the VP1/2A junction region and aligned with the published sequences from GenBank to establish phylogenetic analysis. All China HAV isolates in this study belonged to genotype I, with 98.8% (333/337) of samples clustering in sub-genotype IA and 1.2% (4/337) in sub-genotype IB. In addition, sub-genotype IA isolates clustered into four groups (92.7-100% nucleotide identity), and the samples collected from all China HAV isolates in this investigation showed 87.5-100% nucleotide identity, but the amino acids in this region were more conserved (95.2-100% identity). Few unique amino acid changes could be deduced (VP1-253: Glu → Gly; 2A-34: Pro → Ala; 2A-33: Leu → Phe). Genetically identical or similar HAV strains existed in some investigated areas in China during different years, suggesting that an indigenous strain has been circulating in those regions. This report provides new data on the genetic relatedness and molecular epidemiology of HAV isolates from China as well as the distribution of sub-genotype IA and IB in this part of the world.
5-Aminolevulinic acid (5-ALA) is a key metabolic intermediate of the heme biosynthesis pathway, which has broad application prospects in agriculture and medicine. However, segregational instability of plasmid-based expression systems and low yield have hampered large-scale manufacture of 5-ALA. In this study, two important genes of the 5-ALA C5 biosynthesis pathway, hemA and hemL, were integrated into Escherichia coli MG1655 for chemically induced chromosomal evolution (CIChE). The highest hemA and hemL copy-number, 98 per genome, was obtained in CIChE strain MG136. The 5-ALA titer of this strain reached 2724 mg/L in optimized condition. Then, after undergoing adaptative evolution and the deletion of recA, strain MG136a ΔrecA::FRT could stably produce 4550 mg/L 5-ALA from glucose, 450 times the amount produced by hemA-hemL single copy strain MG1655-hemAL. This study constructed a plasmid-free E. coli strain for 5-ALA production, which will provide the basis for further manipulation of metabolic regulation and optimization of fermentation.
Hepatitis A virus (HAV) is the most common cause of infectious hepatitis throughout the world, spread largely by the fecal-oral route. To characterize the genetic diversity of the virus circulating in China where HAV in endemic, we selected the outbreak cases with identical sequences in VP1-2A junction region and compiled a panel of 42 isolates. The VP3-VP1-2A regions of the HAV capsid-coding genes were further sequenced and analyzed. The quasispecies distribution was evaluated by cloning the VP3 and VP1-2A genes in three clinical samples. Phylogenetic analysis demonstrated that the same genotyping results could be obtained whether using the complete VP3, VP1, or partial VP1-2A genes for analysis in this study, although some differences did exist. Most isolates clustered in sub-genotype IA, and fewer in sub-genotype IB. No amino acid mutations were found at the published neutralizing epitope sites, however, several unique amino acid substitutions in the VP3 or VP1 region were identified, with two amino acid variants closely located to the immunodominant site. Quasispecies analysis showed the mutation frequencies were in the range of 7.22x10-4 -2.33x10-3 substitutions per nucleotide for VP3, VP1, or VP1-2A. When compared with the consensus sequences, mutated nucleotide sites represented the minority of all the analyzed sequences sites. HAV replicated as a complex distribution of closely genetically related variants referred to as quasispecies, and were under negative selection. The results indicate that diverse HAV strains and quasispecies inside the viral populations are presented in China, with unique amino acid substitutions detected close to the immunodominant site, and that the possibility of antigenic escaping mutants cannot be ruled out and needs to be further analyzed.
The present study used a P300-based Concealed Information Test (CIT) to detect individual and collaborative crimes and to explore whether or not the P300 index is effective in identifying collaborative crime members. Participants were divided into two groups to either steal a ring alone (individual group) or collaboratively with another companion participant (collaborative group) before taking the Complex Trial Protocol test that is regarded as an accurate version of the P300-based CIT. The ERP results revealed that both groups showed significantly larger P300s to probe (the ring) than to all irrelevant stimuli (other jewelery), but the P300 amplitude difference of probe stimulus versus irrelevant stimuli in the collaborative group was significantly less than that in the individual group. For the individual diagnosis, using P300 index, the detection rate was significantly inferior for collaborative crime than individual crime, probably related to weakness of collaborative encoding. The ROC curve comparisons showed the individual guilty was effectively discriminated from the simulated-innocent (AUC = .84) and from the collaborative guilty (AUC = .83), but the collaborative guilty was not discriminable from the simulated-innocent (AUC = .66). These findings suggest that collaborative encoding of crime-related information impacts the efficiency of the P300 index, and that the P300-based CIT is not applicable when used to identify collaborative crime perpetrators.
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