Pangenome obtained by long-read sequencing of 11 genomes reveal hidden functional structural variants in pigs.

Long-read sequencing (LRS) facilitates both the genome assembly and the discovery of structural variants (SVs). Here, we built a graph-based pig pangenome by incorporating 11 LRS genomes with an average of 94.01% BUSCO completeness score, revealing 206-Mb novel sequences. We discovered 183,352 nonredundant SVs (63% novel), representing 12.12% of the reference genome. By genotyping SVs in an additional 196 short-read sequencing samples, we identified thousands of population stratified SVs. Particularly, we detected 7,568 Tibetan specific SVs, some of which demonstrate significant population differentiation between Tibetan and low-altitude pigs, which might be associated with the high-altitude hypoxia adaptation in Tibetan pigs. Further integrating functional genomic data, the most promising candidate genes within the SVs that might contribute to the high-altitude hypoxia adaptation were discovered. Overall, our study generates a benchmark pangenome resource for illustrating the important roles of SVs in adaptive evolution, domestication, and genetic improvement of agronomic traits in pigs.

MAL31, a sugar transporter involved in pullulan biosynthesis in Aureobasidium pullulans.

To investigate the role of the sugar transporter MAL31 on pullulan biosynthesis, the coding gene mal31 was respectively disrupted and overexpressed in the parental strain A. pullulans CCTCC M 2012259 to construct mutants of A. pullulans Δmal31 and A. pullulans Mal31. Batch pullulan production significantly decreased by 69.1 % in A. pullulans Δmal31 but increased by 15.9 % in A. pullulans Mal31, as compared to the parental strain. We performed kinetics analysis, assays of key enzymes, determination of intracellular UDPG, NADH, and ATP contents, and measurement of transcriptional levels of genes associated with pullulan biosynthesis and excretion. The results confirmed that the mal31 disruption decreased the glucose consumption rate, decreased the formation rate and titer of pullulan, but increased the intracellular UDPG supply for ß-glucan accumulation. In contrast, the mal31 overexpression increased the transcriptional levels of genes associated with pullulan biosynthesis, and accelerated the rates of glucose consumption and pullulan formation, thereby increased pullulan production. Our findings revealed that MAL31 is involved in the transport of precursors for pullulan biosynthesis. This study provides an accurate operating site for genetic modification of A. pullulans for improving pullulan production and also presents a feasible technique route for the overproduction of other polysaccharides.

Improved production of ß-glucan by a T-DNA-based mutant of Aureobasidium pullulans.

To improve ß-1,3-1,6-D-glucan (ß-glucan) production by Aureobasidium pullulans, an Agrobacterium tumefaciens-mediated transformation method was developed to screen a mutant A. pullulans CGMCC 19650. Based on thermal asymmetric-interlaced PCR detection, DNA sequencing, BLAST analysis, and quantitative real-time PCR assay, the T-DNA was identified to be inserted in the coding region of mal31 gene, which encodes a sugar transporter involved in pullulan biosynthesis in the mutant. The maximal biomass and ß-glucan production under batch fermentation were significantly increased by 47.6% and 78.6%, respectively, while pullulan production was decreased by 41.7% in the mutant, as compared to the parental strain A. pullulans CCTCC M 2012259. Analysis of the physiological mechanism of these changes revealed that mal31 gene disruption increased the transcriptional levels of pgm2, ugp, fks1, and kre6 genes; increased the amounts of key enzymes associated with UDPG and ß-glucan biosynthesis; and improved intracellular UDPG contents and energy supply, all of which favored ß-glucan production. However, the T-DNA insertion decreased the transcriptional levels of ags2 genes, and reduced the biosynthetic capability to form pullulan, resulting in the decrease in pullulan production. This study not only provides an effective approach for improved ß-glucan production by A. pullulans, but also presents an accurate and useful gene for metabolic engineering of the producer for efficient polysaccharide production. KEY POINTS: • A mutant A. pullulans CGMCC 19650 was screened by using the ATMT method. • The mal31 gene encoding a sugar transporter was disrupted in the mutant. • ß-Glucan produced by the mutant was significantly improved.

Triton X-100 improves co-production of ß-1,3-D-glucan and pullulan by Aureobasidium pullulans.

The effects of several surfactants on the biosynthesis of ß-1,3-D-glucan (ß-glucan) and pullulan by Aureobasidium pullulans CCTCC M 2012259 were investigated, and Triton X-100 was found to decrease biomass formation but increase ß-glucan and pullulan production. The addition of 5 g/L Triton X-100 to the fermentation medium and bioconversion broth significantly increased ß-glucan production by 76.6% and 69.9%, respectively, when compared to the control without surfactant addition. To reveal the physiological mechanism underlying the effect of Triton X-100 on polysaccharides production, the cell morphology and viability, membrane permeability, key enzyme activities, and intracellular levels of UDPG, NADH, and ATP were determined. The results indicated that Triton X-100 increased the activities of key enzymes involved in ß-glucan and pullulan biosynthesis, improved intracellular UDPG and energy supply, and accelerated the transportation rate of precursors across the cell membrane, all of which contributed to the enhanced production of ß-glucan and pullulan. Moreover, a two-stage culture strategy with combined processes of batch fermentation and bioconversion was applied, and co-production of ß-glucan and pullulan in the presence of 5 g/L Triton X-100 additions was further improved. The present study not only provides insights into the effect of surfactant on ß-glucan and pullulan production but also presents a feasible approach for efficient production of analogue exopolysaccharides. KEY POINTS: • Triton X-100 increased ß-glucan and pullulan production under either batch fermentation or bioconversion. • Triton X-100 increased the permeability of cell membrane and accelerated the transportation rate of precursors across cell membrane. • Activities of key enzymes involved in ß-glucan and pullulan biosynthesis were increased in the presence of Triton X-100. • Intracellular UDPG levels and energy supply were improved by Triton X-100 addition.

Comparison of five methods for genomic breeding value estimation for the common dataset of the 15th QTL-MAS Workshop.

BACKGROUND: Genomic breeding value estimation is the key step in genomic selection. Among many approaches, BLUP methods and Bayesian methods are most commonly used for estimating genomic breeding values. Here, we applied two BLUP methods, TABLUP and GBLUP, and three Bayesian methods, BayesA, BayesB and BayesCπ, to the common dataset provided by the 15th QTL-MAS Workshop to evaluate and compare their predictive performances. RESULTS: For the 1000 progenies without phenotypic values, the correlations between GEBVs by different methods ranged from 0.812 (GBLUP and BayesCπ) to 0.997 (TABLUP and BayesB). The accuracies of GEBVs (measured as correlations between true breeding values (TBVs) and GEBVs) were from 0.774 (GBLUP) to 0.938 (BayesCπ) and the biases of GEBVs (measure as regressions of TBVs on GEBVs) were from 1.033 (TABLUP) to 1.648 (GBLUP). The three Bayesian methods and TABLUP had similar accuracy and bias. CONCLUSIONS: BayesA, BayesB, BayesCπ and TABLUP performed similarly and satisfactorily and remarkably outperformed GBLUP for genomic breeding value estimation in this dataset. TABLUP is a promising method for genomic breeding value estimation because of its easy computation of reliabilities of GEBVs and its easy extension to real life conditions such as multiple traits and consideration of individuals without genotypes.

Genome-wide association analyses of the 15th QTL-MAS workshop data using mixed model based single locus regression analysis.

BACKGROUND: The mixed model based single locus regression analysis (MMRA) method was used to analyse the common simulated dataset of the 15th QTL-MAS workshop to detect potential significant association between single nucleotide polymorphisms (SNPs) and the simulated trait. A Wald chi-squared statistic with df =1 was employed as test statistic and the permutation test was performed. For adjusting multiple testing, phenotypic observations were permutated 10,000 times against the genotype and pedigree data to obtain the threshold for declaring genome-wide significant SNPs. Linkage disequilibrium (LD) in term of D' between significant SNPs was quantified and LD blocks were defined to indicate quantitative trait loci (QTL) regions. RESULTS: The estimated heritability of the simulated trait is approximately 0.30. 82 genome-wide significant SNPs (P < 0.05) on chromosomes 1, 2 and 3 were detected. Through the LD blocks of the significant SNPs, we confirmed 5 and 1 QTL regions on chromosomes 1 and 3, respectively. No block was detected on chromosome 2, and no significant SNP was detected on chromosomes 4 and 5. CONCLUSION: MMRA is a suitable method for detecting additive QTL and a fast method with feasibility of performing permutation test. Using LD blocks can effectively detect QTL regions.

Retinoid production using metabolically engineered Escherichia coli with a two-phase culture system.

BACKGROUND: Retinoids are lipophilic isoprenoids composed of a cyclic group and a linear chain with a hydrophilic end group. These compounds include retinol, retinal, retinoic acid, retinyl esters, and various derivatives of these structures. Retinoids are used as cosmetic agents and effective pharmaceuticals for skin diseases. Retinal, an immediate precursor of retinoids, is derived by ß-carotene 15,15'-mono(di)oxygenase (BCM(D)O) from ß-carotene, which is synthesized from the isoprenoid building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Retinoids are chemically unstable and biologically degraded via retinoic acid. Although extensive studies have been performed on the microbial production of carotenoids, retinoid production using microbial metabolic engineering has not been reported. Here, we report retinoid production using engineered Escherichia coli that express exogenous BCM(D)O and the mevalonate (MVA) pathway for the building blocks synthesis in combination with a two-phase culture system using a dodecane overlay. RESULTS: Among the BCM(D)O tested in E. coli, the synthetic retinoid synthesis protein (SR), based on bacteriorhodopsin-related protein-like homolog (Blh) of the uncultured marine bacteria 66A03, showed the highest ß-carotene cleavage activity with no residual intracellular ß-carotene. By introducing the exogenous MVA pathway, 8.7 mg/L of retinal was produced, which is 4-fold higher production than that of augmenting the MEP pathway (dxs overexpression). There was a large gap between retinal production and ß-carotene consumption using the exogenous MVA pathway; therefore, the retinal derivatives were analyzed. The derivatives, except for retinoic acid, that formed were identified, and the levels of retinal, retinol, and retinyl acetate were measured. Amounts as high as 95 mg/L retinoids were obtained from engineered E. coli DH5α harboring the synthetic SR gene and the exogenous MVA pathway in addition to dxs overexpression, which were cultured at 29°C for 72 hours with 2YT medium containing 2.0% (w/v) glycerol as the main carbon source. However, a significant level of intracellular degradation of the retinoids was also observed in the culture. To prevent degradation of the intracellular retinoids through in situ extraction from the cells, a two-phase culture system with dodecane was used. The highest level of retinoid production (136 mg/L) was obtained after 72 hours with 5 mL of dodecane overlaid on a 5 mL culture. CONCLUSIONS: In this study, we successfully produced 136 mg/L retinoids, which were composed of 67 mg/L retinal, 54 mg/L retinol, and 15 mg/L retinyl acetate, using a two-phase culture system with dodecane, which produced 68-fold more retinoids than the initial level of production (2.2 mg/L). Our results demonstrate the potential use of E. coli as a promising microbial cell factory for retinoid production.