Metabolic engineering for the synthesis of polyesters: A 100-year journey from polyhydroxyalkanoates to non-natural microbial polyesters.

As concerns increase regarding sustainable industries and environmental pollutions caused by the accumulation of non-degradable plastic wastes, bio-based polymers, particularly biodegradable plastics, have attracted considerable attention as potential candidates for solving these problems by substituting petroleum-based plastics. Among these candidates, polyhydroxyalkanoates (PHAs), natural polyesters that are synthesized and accumulated in a range of microorganisms, are considered as promising biopolymers since they have biocompatibility, biodegradability, and material properties similar to those of commodity plastics. Accordingly, substantial efforts have been made to gain a better understanding of mechanisms related to the biosynthesis and properties of PHAs and to develop natural and recombinant microorganisms that can efficiently produce PHAs comprising desired monomers with high titer and productivity for industrial applications. Recent advances in biotechnology, including those related to evolutionary engineering, synthetic biology, and systems biology, can provide efficient and effective tools and strategies that reduce time, labor, and costs to develop microbial platform strains that produce desired chemicals and materials. Adopting these technologies in a systematic manner has enabled microbial fermentative production of non-natural polyesters such as poly(lactate) [PLA], poly(lactate-co-glycolate) [PLGA], and even polyesters consisting of aromatic monomers from renewable biomass-derived carbohydrates, which can be widely used in current chemical industries. In this review, we present an overview of strain development for the production of various important natural PHAs, which will give the reader an insight into the recent advances and provide indicators for the future direction of engineering microorganisms as plastic cell factories. On the basis of our current understanding of PHA biosynthesis systems, we discuss recent advances in the approaches adopted for strain development in the production of non-natural polyesters, notably 2-hydroxycarboxylic acid-containing polymers, with particular reference to systems metabolic engineering strategies.

The Effects of Topical Application of Polycal (a 2:98 (g/g) Mixture of Polycan and Calcium Gluconate) on Experimental Periodontitis and Alveolar Bone Loss in Rats.

The aim of this study was to observe whether Polycal has inhibitory activity on ligation-induced experimental periodontitis and related alveolar bone loss in rats following topical application to the gingival regions. One day after the ligation placements, Polycal (50, 25, and 12.5 mg/mL solutions at 200 µL/rat) was topically applied to the ligated gingival regions daily for 10 days. Changes in bodyweight, alveolar bone loss index, and total number of buccal gingival aerobic bacterial cells were monitored, and the anti-inflammatory effects were investigated via myeloperoxidase activity and levels of the pro-inflammatory cytokines IL-1ß and TNF-α. The activities of inducible nitric oxide synthase (iNOS) and lipid peroxidation (MDA) were also evaluated. Bacterial proliferation, periodontitis, and alveolar bone loss induced by ligature placements were significantly inhibited after 10 days of continuous topical application of Polycal. These results indicate that topical application of Polycal has a significant inhibitory effect on periodontitis and related alveolar bone loss in rats mediated by antibacterial, anti-inflammatory, and anti-oxidative activities.

Sustainable production and degradation of plastics using microbes.

Plastics are indispensable in everyday life and industry, but the environmental impact of plastic waste on ecosystems and human health is a huge concern. Microbial biotechnology offers sustainable routes to plastic production and waste management. Bacteria and fungi can produce plastics, as well as their constituent monomers, from renewable biomass, such as crops, agricultural residues, wood and organic waste. Bacteria and fungi can also degrade plastics. We review state-of-the-art microbial technologies for sustainable production and degradation of bio-based plastics and highlight the potential contributions of microorganisms to a circular economy for plastics.

Inflammatory cytokines are suppressed by light-emitting diode irradiation of P. gingivalis LPS-treated human gingival fibroblasts: inflammatory cytokine changes by LED irradiation.

Human gingival fibroblasts (hGFs) play an important role in the inflammatory reaction to lipopolysaccharide (LPS) from P. gingivalis, which infects periodontal connective tissue. In addition, although light-emitting diode (LED) irradiation has been reported to have biostimulatory effects, including anti-inflammatory activity, the pathological mechanisms of these effects are unclear. This study examined the effects of 635-nm irradiation of P. gingivalis LPS-treated human gingival fibroblasts on inflammatory cytokine profiles and the mitogen-activated protein kinase (MAPK) pathway, which is involved in cytokine production. Gingival fibroblasts treated or not treated with P. gingivalis LPS were irradiated with 635-nm LED light, and cytokine profiles in the supernatant were assessed using a human inflammation antibody array. Expression of cyclooxyginase-2 (COX-2) protein and phosphorylation of extracellular signal-regulated kinase (ERK 1/2), p38, and c-Jun-N-terminal kinase (JNK) were assessed by Western-blot analysis to determine the effects on the MAPK pathway, and prostaglandin E(2) (PGE(2)) in the supernatant was measured using an enzyme-linked immunoassay. COX-2 protein expression and PGE(2) production were significantly increased in the LPS-treated group and decreased by LED irradiation. LPS treatment of gingival fibroblasts led to the increased release of the pro-inflammatory-related cytokines interleukin-6 (IL-6) and IL-8, whereas LED irradiation inhibited their release. Analysis of MAPK signal transduction revealed a considerable decrease in p38 phosphorylation in response to 635-nm radiation either in the presence or absence of LPS. In addition, 635-nm LED irradiation significantly promoted JNK phosphorylation in the presence of LPS. LED irradiation can inhibit activation of pro-inflammatory cytokines, mediate the MAPK signaling pathway, and may be clinically useful as an anti-inflammatory tool.

Gastric lymphatic basin dissection for sentinel node biopsy using hybrid natural orifice transluminal endoscopic surgery (NOTES).

The aim of the present study was to describe a method of gastric lymphatic basin dissection for sentinel node biopsy using natural orifice transluminal endoscopic surgery with laparoscopic assistance (hybrid NOTES) in a porcine model. Lymph node dissection was performed in three healthy female domestic farm pigs (each around 40 kg) between October, 2007, and December, 2007. The pigs were administered a general anesthetic and laparoscopy-guided transvaginal colpotomy was performed. A two-channel endoscope was then inserted through the incision into the peritoneal cavity via the transvaginal route. An endoscope was inserted simultaneously into the mouth and indocyanine green solution was injected into the submucosal layer of the gastric wall at four sites. Dyed omentum and lymphatics were dissected using a laparoscopic dissector and the grasping forceps of a transvaginal endoscope. Lymphatics and omentum (mean 13.3 cm, range 8-20 cm) were removed transvaginally. The mean number of detected and resected sentinel nodes was 2.6 (range 1-4, diameter 2~12 mm). Sentinel lymphatic basin dissection was performed successfully and without intraoperative complications in all three cases. Hybrid NOTES is technically feasible, and this procedure may represent an alternative to laparoscopic sentinel lymph node dissection of the stomach.

Microbial Polyhydroxyalkanoates and Nonnatural Polyesters.

Microorganisms produce diverse polymers for various purposes such as storing genetic information, energy, and reducing power, and serving as structural materials and scaffolds. Among these polymers, polyhydroxyalkanoates (PHAs) are microbial polyesters synthesized and accumulated intracellularly as a storage material of carbon, energy, and reducing power under unfavorable growth conditions in the presence of excess carbon source. PHAs have attracted considerable attention for their wide range of applications in industrial and medical fields. Since the first discovery of PHA accumulating bacteria about 100 years ago, remarkable advances have been made in the understanding of PHA biosynthesis and metabolic engineering of microorganisms toward developing efficient PHA producers. Recently, nonnatural polyesters have also been synthesized by metabolically engineered microorganisms, which opened a new avenue toward sustainable production of more diverse plastics. Herein, the current state of PHAs and nonnatural polyesters is reviewed, covering mechanisms of microbial polyester biosynthesis, metabolic pathways, and enzymes involved in biosynthesis of short-chain-length PHAs, medium-chain-length PHAs, and nonnatural polyesters, especially 2-hydroxyacid-containing polyesters, metabolic engineering strategies to produce novel polymers and enhance production capabilities and fermentation, and downstream processing strategies for cost-effective production of these microbial polyesters. In addition, the applications of PHAs and prospects are discussed.

Biocatalytic synthesis of polylactate and its copolymers by engineered microorganisms.

Poly(lactate), also called poly(lactic acid) or poly(lactide) [PLA], has been one of the most attractive bio-based polymers since it possesses desirable material properties for its use in general performance plastics in addition to biodegradability and biocompatibility. PLA has been produced by biological and chemical hybrid process comprising microbial fermentation for lactate (LA) production followed by purification and chemical polymerization process of LA. Recently, the direct one-step fermentative processes for production of PLA and several LA-containing polyesters have been developed by employing metabolically engineered microorganisms. Since natural microorganisms cannot produce the LA-containing polymers, several engineering strategies have been employed together based on the polyhydroxyalkanoate (PHA) biosynthesis system. In this chapter, we summarize strategies and procedures on developing the engineered microorganisms producing PLA and its copolymers, cultivating the cells, and extracting the polymers from the cells. Focuses were given on construction of enzymatic polymerization process of LA: design of metabolic pathway for PLA by mimicking PHA biosynthetic pathway, examination of possible enzymes, and engineering of the enzymes for better performances. This synthetic pathway has been established in a microorganism producing LA that enabled one-step fermentative production of LA-containing polyesters from carbohydrates derived from renewable biomass. Polymer production has been further enhanced by implementing strain engineering to concentrate the metabolic fluxes toward PLA formation. In addition, various monomers such as glycolate, 2-hydroxybutyrate, and phenyllactate have been copolymerized with LA by the microbial system. These fermentative production systems developed by using the engineered microorganisms can be versatile and sustainable platforms for the production of LA-containing polyesters and other non-natural polymers.

Systems Metabolic Engineering Strategies for Non-Natural Microbial Polyester Production.

Plastics, used everyday, are mostly synthetic polymers derived from fossil resources, and their accumulation is becoming a serious concern worldwide. Polyhydroxyalkanoates (PHAs) are naturally produced polyesters synthesized and intracellularly accumulated by many different microorganisms. PHAs are good alternatives to petroleum-based plastics because they possess a wide range of material properties depending on monomer types and molecular weights. In addition, PHAs are biodegradable and can be produced from renewable biomass. Thus, producing PHAs through the development of high-performance engineered microorganisms and efficient bioprocesses gained much interest. In addition, non-natural polyesters comprising 2-hydroxycarboxylic acids as monomers have been produced by fermentation of metabolically engineered bacteria. For example, poly(lactic acid) and poly(lactic acid-co-glycolic acid), which have been chemically synthesized using the corresponding monomers either fermentatively or chemically produced, can be produced by metabolically engineered bacteria by one-step fermentation. Recently, PHAs containing aromatic monomers could be produced by fermentation of metabolically engineered bacteria. Here, metabolic engineering strategies applied in developing microbial strains capable of producing non-natural polyesters in a stepwise manner are reviewed. It is hoped that the detailed strategies described will be helpful for designing metabolic engineering strategies for developing diverse microbial strains capable of producing various polymers that can replace petroleum-derived polymers.

Protective effects of calcium gluconate on osteoarthritis induced by anterior cruciate ligament transection and partial medial meniscectomy in Sprague-Dawley rats.

BACKGROUND: This study aimed to determine whether calcium gluconate exerts protective effects on osteoarthritis (OA) induced by anterior cruciate ligament (ACL) transection and partial medial meniscectomy. METHODS: Calcium gluconate was administered by mouth daily for 84 days to male ACL transected and partial medial meniscectomized Sprague-Dawley rats 1 week after operation. RESULTS: Eighty-four days of treatment with 50 mg/kg calcium gluconate led to a lower degree of articular stiffness and cartilage damage compared to the OA control, possibly through inhibition of overexpressed cyclooxygenase (COX)-2 and related chondrocyte apoptosis. Similar favorable effects on stiffness and cartilage were detected in calcium gluconate-administered rats. Additionally, calcium gluconate increased 5-bromo-2'-deoxyuridine (BrdU) uptake based on observation of BrdU-immunoreactive cells on both the femur and tibia articular surface cartilages 84 days after intra-joint treatment with calcium gluconate. CONCLUSIONS: Taken together, our results demonstrate that calcium gluconate has a protective effect against OA through inhibition of COX-2 and related chondrocyte apoptosis.