Comparative transcriptomics analysis-guided metabolic engineering of Yarrowia lipolytica for improved erythritol and fructooligosaccharides production.

Currently, fructooligosaccharides (FOS) are converted from sucrose by purified enzymes or fungal cells, but these methods are costly and time-consuming. Here, the optimal fermentation conditions for strain E326 were determined through fermentation optimization: initial glucose 200 g/L, NaCl 25 g/L, inoculum volume 20 %, dissolved oxygen 20-30 %, pH 3, and glucose feeding concentration 100 g/L, which increased erythritol titer by 1.5 times. The co-expression of HGT1 and APC11 genes alleviated the erythritol synthesis stagnation, shorten the fermentation time by 16.7 %, and increased the erythritol productivity by 17.2 %. The episomal plasmids based on yeast mitochondrial replication origins (mtORIs) were constructed to surface display fructosyltransferase, effectively utilizing waste yeast cells generated during erythritol fermentation. Under the conditions of 60℃ and pH 6, the FOS yield reached 65 %, which to our best of knowledge is so-far the highest yield of FOS obtained. These findings will contribute to the industrial production of erythritol and FOS.

The enhancement of M13 phage titration by optimizing the origin of replication.

Background and purpose: M13KO7, a modified M13 phage variant, carries the p15A replication origin and Tn903 kanamycin resistance gene. This study aimed to optimize M13KO7's replication by substituting the p15A origin with the higher-copy pMB1 origin (500-700 copy numbers). Experimental approach: A 6431-nucleotide fragment from the M13KO7 plasmid lacking the p15A replication origin and kanamycin resistance gene was amplified using a long polymerase chain reaction (PCR). The modified M13AMB1 plasmid was created by adding adenine to the 3' ends of this fragment and ligating it to the pMB1-containing fragment using T/A cloning. Afterward, to prepare the phage, pM13AMB1 was transformed into E. coli TG1 bacteria, and then, using the PEG-NaCl precipitation, the modified phage was propagated. The modified phage titer was determined utilizing the serial dilution and the qPCR methods, compared with the M13KO7 phage. Findings/Results: The results showed that in the serial dilution method, the titers of modified phage and M13KO7 phage were 4.8 × 1014 and 7 × 1012 pfu/mL, respectively. Besides, the phage titer calculated by the qPCR method for the modified phage was equal to 1.3 × 109 pfu/mL, whereas it was 4.08 × 108 pfu/mL for the M13KO7 phage. Conclusion and implications: This study provides evidence that replication origin replacement led to a significant increase in phage titers. It highlights the importance of replication optimization for molecular biology applications.

Identification of 1600 replication origins in S. cerevisiae.

There are approximately 500 known origins of replication in the yeast genome, and the process by which DNA replication initiates at these locations is well understood. In particular, these sites are made competent to initiate replication by loading of the Mcm replicative helicase prior to the start of S phase; thus, 'a site that binds Mcm in G1' might be considered to provide an operational definition of a replication origin. By fusing a subunit of Mcm to micrococcal nuclease, we previously showed that known origins are typically bound by a single Mcm double hexamer, loaded adjacent to the ARS consensus sequence (ACS). Here, we extend this analysis from known origins to the entire genome, identifying candidate Mcm binding sites whose signal intensity varies over at least three orders of magnitude. Published data quantifying single-stranded DNA (ssDNA) during S phase revealed replication initiation among the most abundant 1600 of these sites, with replication activity decreasing with Mcm abundance and disappearing at the limit of detection of ssDNA. Three other hallmarks of replication origins were apparent among the most abundant 5500 sites. Specifically, these sites: (1) appeared in intergenic nucleosome-free regions flanked on one or both sides by well-positioned nucleosomes; (2) were flanked by ACSs; and (3) exhibited a pattern of GC skew characteristic of replication initiation. We conclude that, if sites at which Mcm double hexamers are loaded can function as replication origins, then DNA replication origins are at least threefold more abundant than previously assumed, and we suggest that replication may occasionally initiate in essentially every intergenic region. These results shed light on recent reports that as many as 15% of replication events initiate outside of known origins, and this broader distribution of replication origins suggest that S phase in yeast may be less distinct from that in humans than widely assumed.

DNA replication in early mammalian embryos is patterned, predisposing lamina-associated regions to fragility.

DNA replication in differentiated cells follows a defined program, but when and how it is established during mammalian development is not known. Here we show using single-cell sequencing, that both bovine and mouse cleavage stage embryos progress through S-phase in a defined pattern. Late replicating regions are associated with the nuclear lamina from the first cell cycle after fertilization, and contain few active origins, and few but long genes. Chromosome breaks, which form spontaneously in bovine embryos at sites concordant with human embryos, preferentially locate to late replicating regions. In mice, late replicating regions show enhanced fragility due to a sparsity of dormant origins that can be activated under conditions of replication stress. This pattern predisposes regions with long neuronal genes to fragility and genetic change prior to segregation of soma and germ line. Our studies show that the formation of early and late replicating regions is among the first layers of epigenetic regulation established on the mammalian genome after fertilization.

Intrinsic bent DNA sites in the chromosomal replication origin of Xylella fastidiosa 9a5c

The features of the nucleotide sequences in both replication and promoter regions have been investigated in many organisms. Intrinsically bent DNA sites associated with transcription have been described in several prokaryotic organisms. The aim of the present study was to investigate intrinsic bent DNA sites in the segment that holds the chromosomal replication origin, oriC, of Xylella fastidiosa 9a5c. Electrophoretic behavior analyses, as well as in silico analyses of both the 2-D projection and helical parameters, were performed. The chromosomal segment analyzed contains the initial sequence of the rpmH gene, an intergenic region, the dnaA gene, the oriC sequence, and the 5' partial sequence of the dnaN gene. The analysis revealed fragments with reduced electrophoretic mobility, which indicates the presence of curved DNA segments. The analysis of the helical parameter ENDS ratio revealed three bent DNA sites (b1, b2, and b3) located in the rpmH-dnaA intergenic region, the dnaA gene, and the oriC 5' end, respectively. The chromosomal segment of X. fastidiosa analyzed here is rich in phased AT tracts and in CAnT motifs. The 2-D projection indicated a segment whose structure was determined by the cumulative effect of all bent DNA sites. Further, the in silico analysis of the three different bacterial oriC sequences indicated similar negative roll and twist >34.00° values. The DnaA box sequences, and other motifs in them, may be associated with the intrinsic DNA curvature.

Intrinsically bent DNA in replication origins and gene promoters

Intrinsically bent DNA is an alternative conformation of the DNA molecule caused by the presence of dA/dT tracts, 2 to 6 bp long, in a helical turn phase DNA or with multiple intervals of 10 to 11 bp. Other than flexibility, intrinsic bending sites induce DNA curvature in particular chromosome regions such as replication origins and promoters. Intrinsically bent DNA sites are important in initiating DNA replication, and are sometimes found near to regions associated with the nuclear matrix. Many methods have been developed to localize bent sites, for example, circular permutation, computational analysis, and atomic force microscopy. This review discusses intrinsically bent DNA sites associated with replication origins and gene promoter regions in prokaryote and eukaryote cells. We also describe methods for identifying bent DNA sites for circular permutation and computational analysis.

Genetic characterization of porcine circovirus-2 field isolates from PMWS pigs

PMWS is a new emerging disease in swine herds worldwide. Field isolates of PCV-2, a putative major causative agent of PMWS, were isolated and genetically characterized. Viral genome of two field isolates (PC201DJ and PC201SS) from pigs showing typical PMWS was sequenced. The nucleotide sequence homology with other PCV-2 isolates was ranging from 95% to 99% in complete viral genomic sequence. The highly conserved nonanucleotide motif of replication origin was identical to that of other PCV-2 isolates. To determine the genetic heterogeneity of PCV-2 isolates, the phylogenetic tree based on the complete genome of PCV-2 isolates were constructed. Two PCV-2 field isolates were closely related to Canadian isolates of PCV-2. PCV-2 isolated from field may have an origin of North America and is possibly originated from importation of breeding stocks. The result indicates that although the genome of PCV-2 is relatively stable in general, minor genetic variations exist among PCV-2 isolates from the different geographic locations. These differences of viral genome might have an important implication for genetic characteristics of PCV-2 infection. Three major immunorelevant epitopes of capsid protein showed variations in amino acid sequences. Also, the variance of amino acid sequence in antigenic epitope existed between two Korean PCV-2 isolates.