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.

High-level recombinant production of squalene using selected Saccharomyces cerevisiae strains.

For recombinant production of squalene, which is a triterpenoid compound with increasing industrial applications, in microorganisms generally recognized as safe, we screened Saccharomyces cerevisiae strains to determine their suitability. A strong strain dependence was observed in squalene productivity among Saccharomyces cerevisiae strains upon overexpression of genes important for isoprenoid biosynthesis. In particular, a high level of squalene production (400 ± 45 mg/L) was obtained in shake flasks with the Y2805 strain overexpressing genes encoding a bacterial farnesyl diphosphate synthase (ispA) and a truncated form of hydroxyl-3-methylglutaryl-CoA reductase (tHMG1). Partial inhibition of squalene epoxidase by terbinafine further increased squalene production by up to 1.9-fold (756 ± 36 mg/L). Furthermore, squalene production of 2011 ± 75 or 1026 ± 37 mg/L was obtained from 5-L fed-batch fermentations in the presence or absence of terbinafine supplementation, respectively. These results suggest that the Y2805 strain has potential as a new alternative source of squalene production.

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.

Codon optimization of Saccharomyces cerevisiae mating factor alpha prepro-leader to improve recombinant protein production in Pichia pastoris.

OBJECTIVES: To evaluate different codon optimization parameters on the Saccharomyces cerevisiae-derived mating factor α prepro-leader sequence (MFLS) to improve Candida antarctica lipase B (CAL-B) secretory production in Pichia pastoris. RESULTS: Codon optimization based on the individual codon usage (ICU) and codon context (CC) design parameters enhanced secretory production of CAL-B to 7 U/ml and 12 U/ml, respectively. Only 3 U/ml was obtained with the wild type sequence while the sequence optimized using both ICU and CC objectives showed intermediate performance of 10 U/ml. These results clearly show that CC is the most relevant parameter for the codon optimization of MFLS in P. pastoris, and there is no synergistic effect achieved by considering both ICU and CC together. CONCLUSION: The CC optimized MFLS increased secretory protein production of CAL-B in P. pastoris by fourfold.

Avulsion fracture of the acromial physis in a 14-year-old boy: a case report.

Scapular fractures are uncommon and among them acromial fractures are even more uncommon. Because the vast majority of acromial fractures are either non-displaced or minimally displaced, symptomatic and nonoperative management was performed. We describe a case of avulsion fracture of the acromial physis displaced by acromioclavicular ligament treated with open reduction and internal fixation, and include a review of the literature.

Dynamic positional deformity of the hallux.

The present study reports a case with concomitant tethering of the flexor tendon and extensor tendon of the hallux after closed tibiofibular shaft fractures. We have obtained good clinical results using tenotomy of the flexor hallucis longus tendon and Z-plasty lengthening of the extensor hallucis longus tendon. Because few studies have described the clinical results and operative methods for this type of combined deformity, we report a case with dynamic positional deformity of the hallux.

Engineered heterologous FPP synthases-mediated Z,E-FPP synthesis in E. coli.

Production of Z-type farnesyl diphosphate (FPP) has not been reported in Escherichia coli. Here we present the fusion enzyme (ILRv) of E. coli E,E-FPP synthase (IspA) and Mycobacterium tuberculosis Z,E-FPP synthase (Rv1086), which can produce primarily Z,E-FPP rather than E,E-FPP, the predominant stereoisomer found in most organisms. Z,E-farnesol (FOH) was produced from E. coli harboring the bottom portion of the MVA pathway and the fusion FPP synthase (ILRv) at a titer of 115.6 mg/L in 2YT medium containing 1% (v/v) glycerol as a carbon source and 5 mM mevalonate. The Z,E-FOH production was improved by 15-fold, compared with 7.7 mg/L obtained from the co-overexpression of separate IspA and Rv1086. The Z,E-FPP was not metabolized in native metabolic pathways of E. coli. It would be of interest to produce Z,E-FPP derived sesquiterpenes from recombinant E. coli due to no loss of Z,E-FPP substrate in endogenous metabolism of the host strain.

GAL promoter-driven heterologous gene expression in Saccharomyces cerevisiae Δ strain at anaerobic alcoholic fermentation.

The removal of Gal80 protein by gene disruption turned into efficient GAL promoter-driven heterologous gene expression under anaerobic alcoholic fermentation of Saccharomyces cerevisiae. Using lipase B from Candida antarctica as a reporter, the relative strength of GAL10 promoter (P(GAL10) ) in Δgal80 mutant that does not require galactose as an inducer was compared to those of ADH1, PDC1, and PGK promoters, which have been known to work well anaerobically in actively fermenting yeast cells under high glucose concentration. P(GAL10) in the Δgal80 mutant showed 0.8-fold (ADH1), fourfold (PDC1), and 50-fold (PGK) in promoter strength.

Outcomes of the modified Brostrom procedure using suture anchors for chronic lateral ankle instability--a prospective, randomized comparison between single and double suture anchors.

The present prospective, randomized study was conducted to compare the clinical outcomes of the modified Brostrom procedure using single and double suture anchors for chronic lateral ankle instability. A total of 50 patients were followed up for more than 2 years after undergoing the modified Brostrom procedure. Of the 50 procedures, 25 each were performed using single and double suture anchors by 1 surgeon. The Karlsson scale had improved significantly to 89.8 points and 90.6 points in the single and double anchor groups, respectively. Using the Sefton grading system, 23 cases (92%) in the single anchor group and 22 (88%) in the double anchor group achieved satisfactory results. The talar tilt angle and anterior talar translation on stress radiographs using the Telos device had improved significantly to an average of 5.7° and 4.6 mm in the single anchor group and 4.5° and 4.3 mm in the double anchor group, respectively. The double anchor technique was superior with respect to the postoperative talar tilt. The single and double suture anchor techniques produced similar clinical and functional outcomes, with the exception of talar tilt as a reference of mechanical stability. The modified Brostrom procedure using both single and double suture anchors appears to be an effective treatment method for chronic lateral ankle instability.

Safety and effectiveness of intra-articular injection of a highly cross-linked hyaluronic acid, LBSA0103 (Synovian): Results from a post-marketing surveillance study in South Korea.

This study aimed to assess the safety and effectiveness of the highly cross-linked hyaluronic acid-LBSA0103-in patients with knee osteoarthritis (OA) as per the prescribing information (PI) in South Korea. A total of 3,140 subjects aged ≥19 years were enrolled in this post-marketing surveillance (PMS) study from 2013 to 2019. The subjects received one or two injections of LBSA0103. The median duration of follow-up was 308 days. Adverse events (AEs), adverse drug reactions (ADRs), and serious AEs (SAEs) were monitored. Effectiveness was evaluated based on an index of effectiveness in accordance with the guidelines established by the Ministry of Food and Drug Safety and using a 100-mm visual analog scale (VAS) for weight-bearing pain. Overall, 250 subjects (7.96%) experienced 292 AEs and of these, unexpected AEs occurred in 114 subjects (3.63% [95% CI: 3.00-4.35]). Injection site pain was the most frequent AE reported by 81 subjects (2.58% [95% confidence intervals (CI): 2.05-3.20]). One hundred subjects experienced 108 ADRs (3.18% [95% CI: 2.60, 3.86]) and 15 unexpected ADRs were experienced by 13 subjects (0.41% [95% CI: 0.22-0.71]). Seventeen subjects experienced 22 SAEs (0.54% [95% CI: 0.32-0.87]) during the entire PMS period, and all were considered "unlikely" related to the study drug. Most AEs were mild in terms of severity and resolved during the study period. LBSA0103 was also effective in relieving symptomatic pain in knee OA patients. The condition in more than 80% of the subjects was considered to be improved when assessed by the investigators. LBSA0103 resulted in a significant reduction in the mean VAS score at 12 weeks after the first and second injections (24.79 (± 20.55) mm and 17.63 (±12.31) mm, respectively; p<0.0001). In conclusion, LBSA0103, used for the treatment of knee OA in a real-world setting, was well tolerated, with an acceptable safety profile and consistent therapeutic effect.

Comparison between suture anchor and transosseous suture for the modified-Broström procedure.

BACKGROUND: This prospective, randomized study was conducted to compare clinical outcomes of the modified Broström procedure using suture anchor or transosseous suture technique for chronic ankle instability. METHODS: Forty patients were followed for more than 2 years after modified Broström procedure. Twenty procedures using a suture anchor and 20 procedures using a transosseous suture were performed by one surgeon. The clinical evaluation consisted of the Karlsson scale and the Sefton grading system. Talar tilt and anterior talar translation were measured on anterior and varus stress radiographs. RESULTS: The Karlsson scale had improved significantly to 90.8 points in the suture anchor group, and to 89.2 points in the transosseous suture group. According to Sefton grading system, 18 patients (90%) in suture anchor group and 17 patients (85%) in transosseous suture group achieved satisfactory results. The talar tilt angle and anterior talar translation improved significantly to 5.9 degrees and 4.2 mm in suture anchor group, and to 5.4 degrees and 4.1 mm in transosseous suture group, respectively. CONCLUSION: No significant differences existed in clinical and functional outcomes between the two techniques for ligament reattachment. Both modified Broström procedures using the suture anchor and transosseous suture seem to be effective treatment methods for chronic lateral ankle instability.

Metabolic engineering of Escherichia coli for α-farnesene production.

Sesquiterpenes are important materials in pharmaceuticals and industry. Metabolic engineering has been successfully used to produce these valuable compounds in microbial hosts. However, the microbial potential of sesquiterpene production is limited by the poor heterologous expression of plant sesquiterpene synthases and the deficient FPP precursor supply. In this study, we engineered E. coli to produce α-farnesene using a codon-optimized α-farnesene synthase and an exogenous MVA pathway. Codon optimization of α-farnesene synthase improved both the synthase expression and α-farnesene production. Augmentation of the metabolic flux for FPP synthesis conferred a 1.6- to 48.0-fold increase in α-farnesene production. An additional increase in α-farnesene production was achieved by the protein fusion of FPP synthase and α-farnesene synthase. The engineered E. coli strain was able to produce 380.0 mg/L of α-farnesene, which is an approximately 317-fold increase over the initial production of 1.2 mg/L.

A novel kanamycin/G418 resistance marker for direct selection of transformants in Escherichia coli and different yeast species.

We have developed a set of cloning vectors possessing a modified Tn903 kanamycin resistance gene that enables the selection of both kanamycin-resistant transformants in Escherichia coli and G418-resistant transformants in the yeasts Saccharomyces cerevisiae, Hansenula polymorpha and Pichia pastoris. Expression of this gene in yeast is controlled by the H. polymorpha glyceraldehyde-3-phosphate dehydrogenase promoter, while expression in E. coli is governed by an upstream E. coli lacZ promoter. Applicability of the vectors for gene disruption in H. polymorpha and S. cerevisiae was demonstrated by inactivation of the HpMAL1 and URA3 genes, respectively. One of the vectors possesses a H. polymorpha ARS allowing plasmid maintenance in an episomal state. The small size of the vectors (2-2.5 kb) makes them convenient for routine DNA cloning. In addition, we report a novel approach for construction of gene disruption cassettes.

Farnesol production from Escherichia coli by harnessing the exogenous mevalonate pathway.

Farnesol (FOH) production has been carried out in metabolically engineered Escherichia coli. FOH is formed through the depyrophosphorylation of farnesyl pyrophosphate (FPP), which is synthesized from isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) by FPP synthase. In order to increase FPP synthesis, E. coli was metabolically engineered to overexpress ispA and to utilize the foreign mevalonate (MVA) pathway for the efficient synthesis of IPP and DMAPP. Two-phase culture using a decane overlay of the culture broth was applied to reduce volatile loss of FOH produced during culture and to extract FOH from the culture broth. A FOH production of 135.5 mg/L was obtained from the recombinant E. coli harboring the pTispA and pSNA plasmids for ispA overexpression and MVA pathway utilization, respectively. It is interesting to observe that a large amount of FOH could be produced from E. coli without FOH synthase by the augmentation of FPP synthesis. Introduction of the exogenous MVA pathway enabled the dramatic production of FOH by E. coli while no detectable FOH production was observed in the endogenous MEP pathway-only control.

Recombinant Production of an Inulinase in a Saccharomyces cerevisiae gal80 Strain.

The inulinase gene (INU1) from Kluyveromyces marxianus NCYC2887 strain was overexpressed by using GAL10 promotor in a △gal80 strain of Saccharomyces cerevisiae. The inulinase gene lacking the original signal sequence was fused in-frame to mating factor alpha signal sequence for secretory expression. Use of the △gal80 strain allowed the galactose-free induction of inulinase expression using a glucose-only medium. Shake flask cultivation in YPD medium produced 34.6 U/ml of the recombinant inulinase, which was approximately 13-fold higher than that produced by K. marxianus NCYC2887. It was found that the use of the △gal80 strain improved the expression of inulinase in the recombinant S. cerevisiae in both the aerobic and the anaerobic condition by about 2.9- and 1.7-fold, respectively. 5 L fed-batch fermentation using YPD medium was performed under aerobic condition with glucose feeding, which resulted in the inulinase production of 31.7 U/ml at OD600 of 67. Ethanol fermentation of dried powder of Jerusalem artichoke, an inulin-rich biomass, was also performed using the recombinant S. cerevisiae expressing INU1 and K. marxianus NCYC2887. Fermentation in a 5L scale fermentor was carried out at an aeration rate of 0.2 vvm, an agitation rate of 300 rpm, and the pH was controlled at 5.0. The temperature was maintained at 30degrees C and 37degrees C, respectively, for the recombinant S. cerevisiae and K. marxianus. The maximum productivities of ethanol were 59.0 and 53.5 g/L, respectively.

Barriers to and Facilitators of Physical Activity among Korean Female Adults with Knee Osteoarthritis and Comorbidity: A Qualitative Study.

When knee osteoarthritis is combined with comorbidity, it is associated with limited physical activity. This study aimed to identify barriers to and facilitators of physical activity among Korean female adults with knee osteoarthritis and comorbidity, such as hypertension, diabetes, and dyslipidemia. A qualitative content analysis study was conducted. Ten female knee osteoarthritis participants with comorbidity were recruited at an orthopedic outpatient center in South Korea. Data were collected using in-depth interviews and were analyzed using a conventional content analysis method. Ten participants with a mean age of 70.7 years participated in this study. Four categories of barriers and three of facilitators were identified. Barriers to physical activity were physical hardships, lack of motivation, environmental restrictions, and lack of knowledge. Categories of facilitators were pain management, self-control in physical activity, and understanding the importance of physical activity. Participants did not express any social or environmental facilitators of physical exercise. Healthcare professionals should include social support and environmental facilities to achieve medical and institutional compliance. Understanding female adults with knee osteoarthritis and comorbidity would support provision of appropriately tailored interventions that account for the characteristics of the comorbidity.

Phosphate-responsive promoter of a Pichia pastoris sodium phosphate symporter.

To develop a functional phosphate-regulated promoter in Pichia pastoris, a phosphate-responsive gene, PHO89, which encodes a putative sodium (Na(+))-coupled phosphate symporter, was isolated. Sequencing analyses revealed a 1,731-bp open reading frame encoding a 576-amino-acid polypeptide with 12 putative transmembrane domains. The properties of the PHO89 promoter (P(PHO89)) were investigated using a bacterial lipase gene as a reporter in 5-liter jar fermentation experiments. P(PHO89) was tightly regulated by phosphate and was highly activated when the cells were grown in a phosphate-limited external environment. Compared to translation elongation factor 1alpha and the glyceraldehyde-3-phosphate dehydrogenase promoter, P(PHO89) exhibited strong transcriptional activity with higher specific productivity (amount of lipase produced/cell/h). Furthermore, a cost-effective and simple P(PHO89)-based fermentation process was developed for industrial application. These results demonstrate the potential for efficient use of P(PHO89) for controlled production of recombinant proteins in P. pastoris.

Skin graft using MatriDerm® for plantar defects after excision of skin cancer.

Skin cancer should be excised with sufficient margin to reduce recurrence rate. However, the surgeon always has to worry about the reconstruction method of skin defects after excision. In particular, defects in the plantar surface of the foot are difficult to reconstruct due to their position and structure, and various methods are applied by each surgeon. Surgeons think which methods are easier to apply to patients and less morbidity. To alleviate these concerns, we applied artificial dermal substitute to skin defects after skin cancer. Bowen's disease (squamous cell carcinoma in situ) and melanoma in situ on the plantar surface of the foot were subjected to wide excision with sufficient margin. After excision, a skin defect with exposed plantar fascia was applied with a matrix defect and vacuum. A granulation tissue (dermal matrix) was formed and a split-thickness skin graft was performed. Both patients had good functional results and no problems with skin donor sites. Thus, we report a skin graft method that is relatively easy to apply after skin cancer excision on the plantar surface of the foot.

In-vivo half-life and hypoglycemic bioactivity of a fusion protein of exenatide and elastin-based polypeptide from recombinant Saccharomyces cerevisiae.

Exenatide (Ex) is a 39-amino acid peptide of glucagon-like peptide-1 (GLP-1) receptor agonist that was approved by the FDA in 2005 as a Type II diabetes treatment. It shows a 53% homology with GLP-1 but has an extended half-life (ca. 2.4 h) relative to GLP-1 (ca. 2-3 min). In this study, to further extend its in vivo half-life, we constructed a fusion protein (Ex-(EBP)10-6xHis) using a biocompatible and inert elastin-based polypeptide (EBP) as a fusion partner. Valine was inserted into the guest position of the pentapeptide (VPGXG), no linker sequence was inserted in between the EBPs, and (EBP)10-6xHis tag was attached to the C-terminus of exenatide. By using a recombinant Saccharomyces cerevisiae expression system, the fusion protein was expressed and secreted to the broth and purified by Ni-NTA affinity chromatography. Compared with the native exenatide, the physical half-life of the fusion protein was ca. 3.7-fold extended while approximately 72% of the in-vitro insulin secreting activity was maintained. However, the biological half-life measured by a glucose tolerance test (GTT) and the hypoglycemic test in mice was not significantly different from that of the native form. The effects of EBPylation on bioactivity and half-life of the fusion protein are similar to those of PEGylation. The result suggests that the bioactivity and half-life should be carefully balanced to obtain optimal fusion proteins. We expect that EBPylation using an optimal repeat number of EBP can be an alternative to chemical modification for therapeutic biobetters with extended half-life.

Production of Bio-Based Isoprene by the Mevalonate Pathway Cassette in Ralstonia eutropha.

Isoprene has the potential to replace some petroleum-based chemicals and can be produced through biological systems using renewable carbon sources. Ralstonia eutropha can produce value-added compounds, including intracellular polyhydroxyalkanoate (PHA) through fatty acid and lipid metabolism. In the present study, we engineered strains of R. eutropha H16 and examined the strains for isoprene production. We optimized codons of all the genes involved in isoprene synthesis by the mevalonate pathway and manipulated the promoter regions using pLac and pJ5 elements. Our results showed that isoprene productivity was higher using the J5 promoter (1.9 ± 0.24 µg/l) than when using the lac promoter (1.5 ± 0.2 µg/l). Additionally, the use of three J5 promoters was more efficient (3.8 ± 0.18 µg/l) for isoprene production than a one-promoter system, and could be scaled up to a 5-L batch-cultivation from a T-flask culture. Although the isoprene yield obtained in our study was insufficient to meet industrial demands, our study, for the first time, shows that R. eutropha can be modified for efficient isoprene production and lays the foundation for further optimization of the fermentation process.