AGIDB: a versatile database for genotype imputation and variant decoding across species.

The high cost of large-scale, high-coverage whole-genome sequencing has limited its application in genomics and genetics research. The common approach has been to impute whole-genome sequence variants obtained from a few individuals for a larger population of interest individually genotyped using SNP chip. An alternative involves low-coverage whole-genome sequencing (lcWGS) of all individuals in the larger population, followed by imputation to sequence resolution. To overcome limitations of processing lcWGS data and meeting specific genotype imputation requirements, we developed AGIDB (https://agidb.pro), a website comprising tools and database with an unprecedented sample size and comprehensive variant decoding for animals. AGIDB integrates whole-genome sequencing and chip data from 17 360 and 174 945 individuals, respectively, across 89 species to identify over one billion variants, totaling a massive 688.57 TB of processed data. AGIDB focuses on integrating multiple genotype imputation scenarios. It also provides user-friendly searching and data analysis modules that enable comprehensive annotation of genetic variants for specific populations. To meet a wide range of research requirements, AGIDB offers downloadable reference panels for each species in addition to its extensive dataset, variant decoding and utility tools. We hope that AGIDB will become a key foundational resource in genetics and breeding, providing robust support to researchers.

Characterization of sexual maturity-associated N6-methyladenosine in boar testes.

BACKGROUND: The health and size of the testes are crucial for boar fertility. Testicular development is tightly regulated by epigenetics. N6-methyladenosine (m6A) modification is a prevalent internal modification on mRNA and plays an important role in development. The mRNA m6A methylation in boar testicular development still needs to be investigated. RESULTS: Using the MeRIP-seq technique, we identify and profile m6A modification in boar testes between piglets and adults. The results showed 7783 distinct m6A peaks in piglets and 6590 distinct m6A peaks in adults, with 2,471 peaks shared between the two groups. Enrichment of GO and KEGG analysis reveal dynamic m6A methylation in various biological processes and signalling pathways. Meanwhile, we conjointly analyzed differentially methylated and expressed genes in boar testes before and after sexual maturity, and reproductive related genes (TLE4, TSSK3, TSSK6, C11ORF94, PATZ1, PHLPP1 and PAQR7) were identified. Functional enrichment analysis showed that differential genes are associated with important biological functions, including regulation of growth and development, regulation of metabolic processes and protein catabolic processes. CONCLUSION: The results demonstrate that m6A methylation, differential expression and the related signalling pathways are crucial for boar testicular development. These results suggest a role for m6A modification in boar testicular development and provided a resource for future studies on m6A function in boar testicular development.

Genome-wide detection of genetic structure and runs of homozygosity analysis in Anhui indigenous and Western commercial pig breeds using PorcineSNP80k data.

BACKGROUND: Runs of homozygosity (ROH) are continuous homozygous regions typically located in the DNA sequence of diploid organisms. Identifications of ROH that lead to reduced performance can provide valuable insight into the genetic architecture of complex traits. Here, we systematically investigated the population genetic structure of five Anhui indigenous pig breeds (AHIPs), and compared them to those of five Western commercial pig breeds (WECPs). Furthermore, we examined the occurrence and distribution of ROHs in the five AHIPs and estimated the inbreeding coefficients based on the ROHs (FROH) and homozygosity (FHOM). Finally, we identified genomic regions with high frequencies of ROHs and annotated candidate genes contained therein. RESULTS: The WECPs and AHIPs were clearly differentiated into two separate clades consistent with their geographical origins, as revealed by the population structure and principal component analysis. We identified 13,530 ROHs across all individuals, of which 4,555 and 8,975 ROHs were unique to AHIPs and WECPs, respectively. Most ROHs identified in our study were short (< 10 Mb) or medium (10-20 Mb) in length. WECPs had significantly higher numbers of short ROHs, and AHIPs generally had longer ROHs. FROH values were significantly lower in AHIPs than in WECPs, indicating that breed improvement and conservation programmes were successful in AHIPs. On average, FROH and FHOM values were highly correlated (0.952-0.991) in AHIPs and WECPs. A total of 27 regions had a high frequency of ROHs and contained 17 key candidate genes associated with economically important traits in pigs. Among these, nine candidate genes (CCNT2, EGR2, MYL3, CDH13, PROX1, FLVCR1, SETD2, FGF18, and FGF20) found in WECPs were related to muscular and skeletal development, whereas eight candidate genes (CSN1S1, SULT1E1, TJP1, ZNF366, LIPC, MCEE, STAP1, and DUSP) found in AHIPs were associated with health, reproduction, and fatness traits. CONCLUSION: Our findings provide a useful reference for the selection and assortative mating of pig breeds, laying the groundwork for future research on the population genetic structures of AHIPs, ultimately helping protect these local varieties.

Reassessing acetyl-CoA supply and NADPH availability for mevalonate biosynthesis from glycerol in Escherichia coli.

Mevalonate is an important platform compound for the biosynthesis of isoprenoids. It can be synthesized from acetyl-CoA in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) by the introduced mvaES operon in Escherichia coli. The influences of E. coli hosts, acetyl-CoA supply, and NADPH availability were assessed and engineered to improve the production titer and yield of mevalonate from glycerol. As a result, E. coli DH5α was found to be the best host with high specific capability and titer of mevalonate from glycerol. Through the engineering of phosphoketolase-phosphotransacetylase (xPK-PTA) bypass and NADPH availability, a final titer of 7.21 g/L with a specific capability of 1.36 g/g dry cell weight was gained in flask culture. Our work could offer new information to metabolically engineer the mevalonate pathway for the efficient production of isoprenoids.

Global 3'-untranslated region landscape mediated by alternative polyadenylation during meiotic maturation of pig oocytes.

Alternative polyadenylation affects the length and composition of 3'-untranslated region (3'-UTR) and regulates mRNA stability or translational activity to affect important biological processes. However, global 3'-UTR landscape and its relationship with gamete maturation remain less studied. Here, we analysed our previously reported single-cell RNA-seq data of germinal vesicle and metaphase II stage oocytes in pigs to systematically catalogue the 3'-UTR dynamics during oocyte maturation. Two softwares (DaPars and APAtrap) were employed and identified 110 and 228 mRNAs with significantly different 3'-UTRs (adjusted p ≤ .05), respectively. Gene enrichment analyses found signalling pathways related with biological processes of female gametophyte production, methyltransferase activity and mRNA surveillance (DaPars) and cell cycle process, regulation of ERK1 and ERK2 cascade, regulation of translation, spindle organization, kinetochore, condensed chromosome and progesterone-mediated oocyte maturation (APAtrap), respectively. Moreover, 18 of 110 mRNAs (|△PDUI| ≥ 0.25 and |log2 PDUI ratio| ≥ 0.59) and 15 of 228 mRNAs (Perc. diff. ≥ 0.5) were with greater difference of 3'-UTR length or abundance, and integrative genomics viewer analysis further identified 4 (Alg10, Hadhb, Hsd17b4 and Sbds) of 18 mRNAs to be with 3'-UTR length differed ≥150 bp and 6 (Gcc1, Hnrnpa2b1, Lsm6, Prpf18, Sfr1 and Ust) of 15 mRNAs to be with 3'-UTR abundance extremely differed. Furthermore, the location, sequences and number of cis-elements were predicted, which were shown to derange cytoplasmic polyadenylation element, poly(A) site and microRNA binding sites within 3'-UTRs of Alg10, Hadhb, Hsd17b4 and Sbds mRNAs. Taken together, global 3'-UTR landscape changes dynamically with oocyte meiotic maturation, potentially involved in regulating oocyte meiotic process in pigs.

Regulatory molecule cAMP changes cell fitness of the engineered Escherichia coli for terpenoids production.

Terpenoids are a class of natural compounds with many important functions and applications. They are synthesized from a long synthetic pathway of isoprenyl unit coupling with the myriads of terpene synthases. Owing to the catalytic divergence of terpenoids synthesis, microbial production of terpenoids is compromised to the complexity of pathway engineering and suffers from the metabolic engineering burden. In this work, the adaptive Escherichia coli HP variant exhibited a general cell fitness in terpenoid synthesis. Especially, it could yield taxadiene of 193.2 mg/L in a test tube culture, which is a five-fold increase over the production in the wild type E. coli DH5α. Mutational analyses indicated that IS10 insertion in adenylate cyclase CyaA (CyaAHP) resulted in lowering intracellular cyclic AMP (cAMP), which could regulate its receptor protein CRP to rewire cell metabolism and contributed to the improved cell fitness. Our results suggested a way to manipulate cell fitness for terpenoids production and other products.

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.

Self-Phosphorization of MOF-Armored Microbes for Advanced Energy Storage.

Utilization of microbes as the carbon source and structural template to fabricate porous carbon has incentivized great interests owing to their diverse micromorphology and intricate intracellular structure, apart from the obvious benefit of "turning waste into wealth." Challenges remain to preserve the biological structure through the harsh and laborious post-synthetic treatments, and tailor the functionality as desired. Herein, Escherichia coli is directly coated with metal-organic frameworks (MOFs) through in situ assembly to fabricate N, P co-doped porous carbon capsules expressing self-phosphorized metal phosphides. While the MOF coating serves as an armoring layer for facilitating the morphology inheritance from the bio-templates and provides metal sources for generating extra porosity and electrochemically active sites, the P-rich phospholipids and N-rich proteins from the plasma membrane enable carbon matrix doping and further yield metal phosphides. These unique structural and compositional features endow the carbon capsules with great capabilities in suppressing polysulfide shuttling and catalyzing reversible oxygen conversion, ultimately leading to the superb performance of lithium-sulfur batteries and zinc-air batteries. Combining the bio-templating strategy with hierarchical MOF assembly, this work opens a new avenue for the fabrication of highly porous and functional carbon for advanced energy applications.

Extension of cell membrane boosting squalene production in the engineered Escherichia coli.

Squalene is a lipophilic and non-volatile triterpene with many industrial applications for food, pharmaceuticals, and cosmetics. Metabolic engineering focused on optimization of the production pathway suffer from little success in improving titers because of a limited space of the cell membrane accommodating the lipophilic product. Extension of cell membrane would be a promising approach to overcome the storage limitation for successful production of squalene. In this study, Escherichia coli was engineered for squalene production by overexpression of some membrane proteins. The highest production of 612 mg/L was observed in the engineered E. coli with overexpression of Tsr, a serine chemoreceptor protein, which induced invagination of inner membrane to form multilayered structure. It was also observed an increase in unsaturated fatty acid in membrane lipids composition, suggesting cellular response to maintain membrane fluidity against squalene accumulation in the engineered strain. This study potentiates the capability of E. coli for squalene production and provides an effective strategy for the enhanced production of such compounds.

Transcriptomic comparison of liver tissue between Anqing six-end-white pigs and Yorkshire pigs based on RNA sequencing.

Chinese indigenous pig and Western commercial pig breeds show different patterns of lipid metabolism, fat deposition, and fatty acid composition; for these reasons, they have become vitally important models of energy metabolism and obesity in humans. To compare the mechanisms underlying lipid metabolism between Yorkshire pigs (lean type) and Anqing six-end-white pigs (obese type), the liver transcriptomes of six castrated boars with a body weight of approximately 100 kg (three Yorkshire and three Anqing) were analyzed by RNA-seq. The total number of reads produced for each liver sample ranged from 47.05 to 62.6 million. Among 362 differentially expressed genes, 142 were up-regulated and 220 were down-regulated in Anqing six-end-white pigs. Based on these data, 79 GO terms were significantly enriched. The top 10 (the 10 with lowest corrected P-value) significantly enriched GO terms were identified, including lipid metabolic process and carboxylic acid metabolic process. Pathway analysis revealed three significantly enriched KEGG pathways including PPAR signaling pathway, steroid hormone biosynthesis, and retinol metabolism. Based on protein-protein interaction networks, multiple genes responsible for lipid metabolism were identified, such as PCK1, PPARA, and CYP7A1, and these were considered promising candidate genes that could affect porcine liver lipid metabolism and fat deposition. Our results provide abundant transcriptomic information that will be useful for animal breeding and biomedical research.

Compound Lactobacillus sp. administration ameliorates stress and body growth through gut microbiota optimization on weaning piglets.

The composition of bacteria in the gastrointestinal tract of piglets is easily affected by environmental changes, particularly during the weaning period. Compound strains of Lactobacillus reuteri and Lactobacillus salivarius were supplemented to piglets during pre- and post-weaning to determine their effects in improving the growth performance and ameliorating the diarrhea rate and stress caused by antioxidation in piglets. A larger number of L. reuteri and L. salivarius colonized the distal segment of the ileum and the total numbers of Lactobacillus spp. and Bifidobacteria were higher in the ileal mucous membrane and cecal lumen with probiotics supplementation. The numbers of antioxidants and immune molecules increased, levels of cortisol and endotoxin reduced, and growth hormone and insulin-like growth factor 1 improved in the plasma following compound bacteria (CL) supplementation. Spearman's and KEGG analysis of the bacterial operational taxonomic unit and antioxidative and immune indices and metabolic genes indicated that the body growth modulation by CL supplementation could be attributed to optimization of the intestinal bacterial composition; functional strains of L. delbrueckii, L. salivarius, L. formicilis, L. reuteri, and L. mucosae were positively correlated with body antioxidation and immunity derived by CL supplementation. Strains of L. agilis and L. pontis were diverse and negatively correlated with body antioxidation and immunity. Collectively, these results suggest that supplementation with CL could reduce stress and improve the growth performance of piglets during weaning by optimizing the intestinal bacterial composition. KEY POINTS: • The colonization of L. reuteri and L. salivarius in ileal mucous membrane optimize bacterial composition of GIT, mainly some functional strains of Lactobacillus, L. delbrueckii, L. salivarius, L. formicilis, L. reuteri, and L. mucosae. • The optimized bacterial composition of piglets in both ileal mucous membrane and cecal content improves body growth hormone level, immunity, and antioxidation, which is helpful to defend the stress. These benefits induce to increased growth performance of animal model piglets during weaning.

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.

Relationship among porcine lncRNA TCONS_00010987, miR-323, and leptin receptor based on dual luciferase reporter gene assays and expression patterns.

OBJECTIVE: Considering the physiological and clinical importance of leptin receptor (LEPR) in regulating obesity and the fact that porcine LEPR expression is not known to be controlled by lncRNAs and miRNAs, we aim to characterize this gene as a potential target of SSC-miR-323 and the lncRNA TCONS_00010987. METHODS: Bioinformatics analyses revealed that lncRNA TCONS_00010987 and LEPR have SSC-miR-323-binding sites and that LEPR might be a target of lncRNA TCONS_00010987 based on cis prediction. Wild-type and mutant TCONS_00010987-target sequence fragments and wild-type and mutant LEPR 3'-UTR fragments were generated and cloned into pmiR-RB-REPORTTM-Control vectors to construct respective recombinant plasmids. HEK293T cells were co-transfected with the SSC-miR-323 mimics or a negative control with constructs harboring the corresponding binding sites and relative luciferase activities were determined. Tissue expression patterns of lncRNA TCONS_00010987, SSC-miR-323, and LEPR in Anqing six-end-white (AQ, the obese breed) and Large White (LW, the lean breed) pigs were detected by real-time quantitative polymerase chain reaction; backfat expression of LEPR protein was detected by western blotting. RESULTS: Target gene fragments were successfully cloned, and the four recombinant vectors were constructed. Compared to the negative control, SSC-miR-323 mimics significantly inhibited luciferase activity from the wild-type TCONS_00010987-target sequence and wild-type LEPR-3'-UTR (p<0.01 for both) but not from the mutant TCONS_00010987-target sequence and mutant LEPR-3'-UTR (p>0.05 for both). Backfat expression levels of TCONS_ 00010987 and LEPR in AQ pigs were significantly higher than those in LW pigs (p<0.01), whereas levels of SSC-miR-323 in AQ pigs were significantly lower than those in LW pigs (p<0.05). LEPR protein levels in the backfat tissues of AQ pigs were markedly higher than those in LW pigs (p<0.01). CONCLUSION: LEPR is a potential target of SSC-miR-323, and TCONS_00010987 might act as a sponge for SSC-miR-323 to regulate LEPR expression.

Challenges and tackles in metabolic engineering for microbial production of carotenoids.

Naturally occurring carotenoids have been isolated and used as colorants, antioxidants, nutrients, etc. in many fields. There is an ever-growing demand for carotenoids production. To comfort this, microbial production of carotenoids is an attractive alternative to current extraction from natural sources. This review summarizes the biosynthetic pathway of carotenoids and progresses in metabolic engineering of various microorganisms for carotenoid production. The advances in synthetic pathway and systems biology lead to many versatile engineering tools available to manipulate microorganisms. In this context, challenges and possible directions are also discussed to provide an insight of microbial engineering for improved production of carotenoids in the future.

Ty1-fused protein-body formation for spatial organization of metabolic pathways in Saccharomyces cerevisiae.

Metabolite production through a multistep metabolic pathway can often be increased by efficient substrate channeling created by spatial sequestration of the metabolic reactions. Here, Tya, a structural component in the Ty1 retrotransposon element that forms virus-like particles (VLPs) in Saccharomyces cerevisiae, was used to spatially organize enzymes involved in a metabolic pathway into a multi-enzyme protein body in yeast. As a proof of principle, Tya fusion to three key enzymes involved in biosynthesis of the isoprenoids farnesene and farnesol was tested to assess its potential to improve productivity. The Tya-fusion protein resulted in three and fourfold increases in farnesene and farnesol production, respectively, as compared with that observed in a non-fused control. Specifically, two-phase partitioning fed-batch fermentations of S. cerevisiae ATCC200589 overexpressing Tya-fused enzymes (tHmg1, IspA, and α-farnesene synthase) yielded 930 ± 40 mg/L of farnesene after 7 days. Additionally, we observed that the Tya-fusion proteins tended to partition into particulate fractions upon 100,000g ultracentrifugation, suggesting the formation of large aggregates of protein bodies, with their particulate structure also observed by transmission electron microscopy. The dramatic increase in the biosynthetic productivity of metabolites via use of a Tya-fusion protein suggested that this approach might be useful for the creation of multi-enzyme complexes to improve metabolic engineering in yeast.

Improved S-adenosylmethionine and glutathione biosynthesis by heterologous expression of an ATP6 gene in Candida utilis.

ATP is indispensable to the biosynthesis of both S-adenosylmethionine (SAM) and glutathione (GSH) in yeast cells. To improve ATP supply for overproduction of SAM and GSH in Candida utilis CCTCC M 209298, an exogenous ATP6 gene from Arabidopsis thaliana was expressed in the parental strain to construct the mutant C. utilis ATP6 by genomic integration. The maximal production of SAM and GSH in the mutant increased by 46.6 and 28.7%, respectively, when compared with those obtained in the parental strain. The mechanism underlying improved SAM and GSH biosynthesis by exogenous ATP6 gene expression revealed that the mutant had higher activities of key enzymes involved in SAM and GSH biosynthesis as well as energy metabolism. Increased NADH availability and F0 F1 -ATPase activity subsequently resulted in improved ATP regeneration and intracellular ATP supply for SAM and GSH overproduction. The present study not only developed an effective method for improving SAM and GSH biosynthesis by energy metabolism regulation, but also offered a novel approach for efficient production of similar energy-consuming products in eukaryotic cells.

The patterns of genomic variances and covariances across genome for milk production traits between Chinese and Nordic Holstein populations.

BACKGROUND: With the development of SNP chips, SNP information provides an efficient approach to further disentangle different patterns of genomic variances and covariances across the genome for traits of interest. Due to the interaction between genotype and environment as well as possible differences in genetic background, it is reasonable to treat the performances of a biological trait in different populations as different but genetic correlated traits. In the present study, we performed an investigation on the patterns of region-specific genomic variances, covariances and correlations between Chinese and Nordic Holstein populations for three milk production traits. RESULTS: Variances and covariances between Chinese and Nordic Holstein populations were estimated for genomic regions at three different levels of genome region (all SNP as one region, each chromosome as one region and every 100 SNP as one region) using a novel multi-trait random regression model which uses latent variables to model heterogeneous variance and covariance. In the scenario of the whole genome as one region, the genomic variances, covariances and correlations obtained from the new multi-trait Bayesian method were comparable to those obtained from a multi-trait GBLUP for all the three milk production traits. In the scenario of each chromosome as one region, BTA 14 and BTA 5 accounted for very large genomic variance, covariance and correlation for milk yield and fat yield, whereas no specific chromosome showed very large genomic variance, covariance and correlation for protein yield. In the scenario of every 100 SNP as one region, most regions explained <0.50% of genomic variance and covariance for milk yield and fat yield, and explained <0.30% for protein yield, while some regions could present large variance and covariance. Although overall correlations between two populations for the three traits were positive and high, a few regions still showed weakly positive or highly negative genomic correlations for milk yield and fat yield. CONCLUSIONS: The new multi-trait Bayesian method using latent variables to model heterogeneous variance and covariance could work well for estimating the genomic variances and covariances for all genome regions simultaneously. Those estimated genomic parameters could be useful to improve the genomic prediction accuracy for Chinese and Nordic Holstein populations using a joint reference data in the future.

Discovery of porcine miRNA-196a/b may influence porcine adipogenesis in longissimus dorsi muscle by miRNA sequencing.

Intramuscular fat (IMF) is one of the fat traits that has economic importance in the pork industry. Longissimus dorsi muscle contains IMF and is suitable for studying adipogenesis. To discover further potential regulatory miRNAs that may influence adipogenesis, we analyzed miRNA in the longissimus dorsi muscle of Yorkshire (YY, lean-type) and Chinese Wannanhua (WH, fatty) pigs using miRNA sequencing (miRNA-seq). From this dataset, we identified 598 unique miRNAs comprising 325 pre-miRNAs and 273 novel pre-miRNAs through comparison with known miRNAs in miRBase version 21. We found 42 miRNAs including nine up- and 33 down-regulated between the YY and WH pigs. Moreover, we found two miRNAs, miR-196a/b (miR-196a, miR-196b-5p), that had the highest level of expression in WH pigs, and miR-196a/b may influence porcine adipogenesis in longissimus dorsi muscle through an adipocytokine signaling pathway.

Combinatorial engineering of hybrid mevalonate pathways in Escherichia coli for protoilludene production.

BACKGROUND: Protoilludene is a valuable sesquiterpene and serves as a precursor for several medicinal compounds and antimicrobial chemicals. It can be synthesized by heterologous expression of protoilludene synthase in Escherichia coli with overexpression of mevalonate (MVA) or methylerythritol-phosphate (MEP) pathway, and farnesyl diphosphate (FPP) synthase. Here, we present E. coli as a cell factory for protoilludene production. RESULTS: Protoilludene was successfully produced in E. coli by overexpression of a hybrid exogenous MVA pathway, endogenous FPP synthase (IspA), and protoilludene synthase (OMP7) of Omphalotus olearius. For improving protoilludene production, the MVA pathway was engineered to increase synthesis of building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) by sequential order permutation of the lower MVA portion (MvL), the alteration of promoters and copy numbers for the upper MVA portion (MvU), and the coordination of both portions, resulting in an efficient entire MVA pathway. To reduce the accumulation of mevalonate observed in the culture broth due to lower efficiency of the MvL than the MvU, the MvL was further engineered by homolog substitution with the corresponding genes from Staphylococcus aureus. Finally, the highest protoilludene production of 1199 mg/L was obtained from recombinant E. coli harboring the optimized hybrid MVA pathway in a test tube culture. CONCLUSIONS: This is the first report of microbial synthesis of protoilludene by using an engineered E. coli strain. The protoilludene production was increased by approx. Thousandfold from an initial titer of 1.14 mg/L. The strategies of both the sequential order permutation and homolog substitution could provide a new perspective of engineering MVA pathway, and be applied to optimization of other metabolic pathways.

Isoprene production by Escherichia coli through the exogenous mevalonate pathway with reduced formation of fermentation byproducts.

BACKGROUND: Isoprene, a volatile C5 hydrocarbon, is an important platform chemical used in the manufacturing of synthetic rubber for tires and various other applications, such as elastomers and adhesives. RESULTS: In this study, Escherichia coli MG1655 harboring Populus trichocarpa isoprene synthase (PtispS) and the exogenous mevalonate (MVA) pathway produced 80 mg/L isoprene. Codon optimization and optimal expression of the ispS gene via adjustment of the RBS strength and inducer concentration increased isoprene production to 199 and 337 mg/L, respectively. To augment expression of MVA pathway genes, the MVA pathway was cloned on a high-copy plasmid (pBR322 origin) with a strong promoter (Ptrc), which resulted in an additional increase in isoprene production up to 956 mg/L. To reduce the formation of byproducts derived from acetyl-CoA (an initial substrate of the MVA pathway), nine relevant genes were deleted to generate the E. coli AceCo strain (E. coli MG1655 ΔackA-pta, poxB, ldhA, dld, adhE, pps, and atoDA). The AceCo strain harboring the ispS gene and MVA pathway showed enhanced isoprene production of 1832 mg/L in flask culture with reduced accumulation of byproducts. CONCLUSIONS: We achieved a 23-fold increase in isoprene production by codon optimization of PtispS, augmentation of the MVA pathway, and deletion of genes involved in byproduct formation.