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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. [ABSTRACT FROM AUTHOR]

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 |log2PDUI 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Apoptosis of granulosa cells affects follicular atresia and reproduction and is regulated by miRNAs and the expression of certain genes. For the present study, we investigated the regulatory relationship between microRNA‐222 (miR‐222) and THBS1 in porcine follicular granulosa cells (pGCs) and its effects on apoptosis to provide empirical data for developing methods to improve pig fecundity. Results revealed that miR‐222 promotes the proliferation of pGCs. MiRNA mimics and luciferase reporter assays revealed that miR‐222 functions as an anti‐apoptotic factor in pGCs. MiR‐222 mimics in pGCs result in the upregulation of the anti‐apoptotic BCL‐2 gene, down‐regulation of the proapoptotic caspase‐3 gene, and inhibition of apoptosis. MiR‐222 inhibitors reduced BCL‐2 and had no significant effect on caspase‐3. MiR‐222 mimics promoted estrogen levels. Inhibition of THBS1 inhibited pGC apoptosis. Transfection of THBS1‐siRNA reduced the proapoptotic BAX gene. MiR‐222 can directly target the 3′‐untranslated region of the THBS1 gene. MiR‐222 mimics suppressed THBS1 mRNA and proteins, but these were upregulated by the miR‐222 inhibitor. Transfection of THBS1‐siRNA resulted in the inhibition of the miR‐222 inhibitor, which suggests that miR‐222 inhibits pGC apoptosis by targeting THBS1. These findings suggest that miR‐222 and THBS1 play important roles in follicular atresia, ovarian development, and female reproduction. [ABSTRACT FROM AUTHOR]

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 F0F1‐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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

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 mi RNAs that may influence adipogenesis, we analyzed mi RNA in the longissimus dorsi muscle of Yorkshire ( YY, lean-type) and Chinese Wannanhua ( WH, fatty) pigs using mi RNA sequencing (mi RNA-seq). From this dataset, we identified 598 unique mi RNAs comprising 325 pre-mi RNAs and 273 novel pre-mi RNAs through comparison with known mi RNAs in mi RBase version 21. We found 42 mi RNAs including nine up- and 33 down-regulated between the YY and WH pigs. Moreover, we found two mi RNAs, 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. [ABSTRACT FROM AUTHOR]

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., (© 2016 Stichting International Foundation for Animal Genetics.)