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PHA, a family of natural biopolymers aiming to replace non-degradable plastics for short-term usages, has been developed to include various structures such as short-chain-length (scl) and medium-chain-length (mcl) monomers as well as their copolymers. However, PHA market has been grown slowly since 1980s due to limited variety with good mechanical properties and the high production cost. Here, we review most updated strategies or approaches including metabolic engineering, synthetic biology and morphology engineering on expanding PHA diversity, reducing production cost and enhancing PHA production. The extremophilic Halomonas spp. are taken as examples to show the feasibility and challenges to develop next generation industrial biotechnology (NGIB) for producing PHA more competitively., (Copyright © 2019 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a promising biopolyester with good mechanical properties and biodegradability. Large-scale production of PHBV is still hindered by the high production cost. CRISPR/Cas9 method was used to engineer the TCA cycle in Halomonas bluephagenesis on its chromosome for production of PHBV from glucose as a sole carbon source. Two TCA cycle related genes sdhE and icl encoding succinate dehydrogenase assembly factor 2 and isocitrate lysase were deleted, respectively, in H. bluephagenesis TD08AB containing PHBV synthesis genes on the chromosome, to channel more flux to increase the 3-hydroxyvalerate (3HV) ratio of PHBV. Due to a poor growth behavior of the mutant strains, H. bluephagenesis TY194 equipped with a medium strength P porin -194 promoter was selected for further studies. The sdhE and/or icl mutant strains of H. bluephagenesis TY194 were constructed to show enhanced cell growth, PHBV synthesis and 3HV molar ratio. Gluconate was used to activate ED pathway and thus TCA cycle to increase 3HV content. H. bluephagenesis TY194 (ΔsdhEΔicl) was found to synthesize 17mol% 3HV in PHBV. Supported by the synergetic function of phosphoenolpyruvate carboxylase and Vitreoscilla hemoglobin encoded by genes ppc and vgb inserted into the chromosome of H. bluephagenesis TY194 (ΔsdhE) serving to enhance TCA cycle activity, a series of strains were generated that could produce PHBV containing 3-18mol% 3HV using glucose as a sole carbon source. Shake flask studies showed that H. bluephagenesis TY194 (ΔsdhE, G7::P porin -ppc) produced 6.3 g/L cell dry weight (CDW), 65% PHBV in CDW and 25mol% 3HV in PHBV when grown in glucose and gluconate. 25mol% 3HV was the highest reported via chromosomal expression system. PHBV copolymers with different 3HV molar ratios were extracted and characterized. Next-generation industrial biotechnology (NGIB) based on recombinant H. bluephagenesis grown under unsterile and continuous conditions, allows production of P(3HB-0∼25mol% 3HV) in a convenient way with reduced production complexity and cost., (Copyright © 2019 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Bacterial polyhydroxyalkanoates (PHA) are a family of intracellular polyester granules with sizes ranging from 100 to 500 nm. Due to their small sizes, it has been very difficult to separate the PHA granules from the bacterial broths. This study aims to engineer the PHA size control mechanism to obtain large PHA granular sizes beneficial for the separation. It has been reported that phasin (PhaP) is an amphiphilic protein located on the surface of PHA granules functioning to regulate sizes and numbers of PHA granules in bacterial cells, deletions on PhaPs result in reduced PHA granule number and enhanced granule sizes. Three genes phaP1, phaP2 and phaP3 encoding three PhaP proteins were deleted in various combinations in halophilic bacterium Halomonas bluephagenesis TD01. The phaP1-knockout strain generated much larger PHA granules with almost the same size as their producing cells without significantly affecting the PHA accumulation yet with a reduced PHA molecular weights. In contrast, the phaP2- and phaP3-knockout strains produced slightly larger sizes of PHA granules with increased PHA molecular weights. While PHA accumulation by phaP3-knockout strains showed a significant reduction. All of the PhaP deletion efforts could not form PHA granules larger than a normal size of H. bluephagenesis TD01. It appears that the PHA granular sizes could be limited by bacterial cell sizes. Therefore, genes minC and minD encoding proteins that block formation of cell fission rings (Z-rings) were over-expressed in various phaP deleted H. bluephagenesis TD01, resulting in large cell sizes of H. bluephagenesis TD01 containing PHA granules with sizes of up to 10 μm that has never been observed previously. It can be concluded that PHA granule sizes are limited by the cell sizes. By engineering a large cell morphology large PHA granules can be produced by PhaP deleted mutants., (Copyright © 2019 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Microbial polyhydroxyalkanoates (PHA) are a family of biopolyesters with properties similar to petroleum plastics such as polyethylene (PE) or polypropylene (PP). Polyhydroxybutyrate (PHB) is the most common PHA known so far. Clustered regularly interspaced short palindromic repeats interference (CRISPRi), a technology recently developed to control gene expression levels in eukaryotic and prokaryotic genomes, was employed to regulate PHB synthase activity influencing PHB synthesis. Recombinant Escherichia coli harboring an operon of three PHB synthesis genes phaCAB cloned from Ralstonia eutropha, was transformed with various single guided RNA (sgRNA with its guide sequence of 20-23 bases) able to bind to various locations of the PHB synthase PhaC, respectively. Depending on the binding location and the number of sgRNA on phaC, CRISPRi was able to control the phaC transcription and thus PhaC activity. It was found that PHB content, molecular weight, and polydispersity were approximately in direct and reverse proportion to the PhaC activity, respectively. The higher the PhaC activity, the more the intracellular PHB accumulation, yet the less the PHB molecular weights and the wider the polydispersity. This study allowed the PHB contents to be controlled in the ranges of 1.47-75.21% cell dry weights, molecular weights from 2 to 6 millions Dalton and polydispersity of 1.2 to 1.43 in 48 h shake flask studies. This result will be very important for future development of ultrahigh molecular weight PHA useful to meet high strength application requirements.

Objective To observe the effects of Huatan Tongluo Recipe (HTTLR) on the proliferation of IL-β p induced rheumatoid arthritis synovial fibroblast ( RASFB) and secretion of necrosis factor α (TNF-α) and acidic fibroblast growth factor (aFGF) in vitro. Methods RASFB cell line was cultured in vitro and stimulated by IL-1β. The proliferation of RASFB was detected using WST-1 after adding IL-1β with final concentrations of 1 , 5, 10, 20 μg/L for 24 and 48 h respectively. Then 20 μg/L IL-1β recruited as induction dose was set up as IL-1β group. High, middle, low dose HTTLR groups were set up by adding HT- TLR decoction with final concentration of 5%, 2%, 1% (V/V) , respectively for 24 and 48 h. A blank con- trol group was also set up. The proliferation rates were compared. Contents of TNF-α and aFGF were detected in each group using ELISA. mRNA expressions of TNF-α and aFGF were detected using RT-PCR. Results The proliferation rates of RASFB at 24 h and 48 h were lower at 1 μg/L IL-1 β than at 5, 10, 20 μg/L IL-1β (P <0. 01). The proliferation rate of RASFB was higher at 10 and 20 μg/L IL-1β than at 5 μg/L IL-1β (P <0. 01). Besides, the proliferation rate of RASFB was higher at 20 μg/L IL-1β than at 10 μg/L IL-1 β (P <0. 01). The proliferation rate of RASFB was higher at 48 h than at 24 h (P <0. 01). Com- pared with the high dose HTTLR group, the proliferation rate of RASFB was lowered in middle and low dose HTTLR groups (P <0. 01). Besides, IL-1β induced proliferation rate of RASFB was obviously reduced in the middle dose HTTLR group (P <0. 01). Compared with the blank control group, mRNA ex- pressions of TNF-α and aFGF and their contents were elevated in the IL-1β group at 24 and 48 h (P < 0. 05). Compared with the IL-1 β group, mRNA expressions of TNF-α- and aFGF and their contents, except TNF-α- mRNA expression in the low dose HTTLR group at 24 h, were all obviously lowered in 3 dose HTTLR groups at 24 h and 48 h (P <0. 05). Compared with the high dose HTTLR group, mRNA expressions of TNF-(α and aFGF increased in middle and low dose HTTLR groups at 24 h and 48 h; TNF-α content in the low dose HTTLR group at 24 h; contents of TNF-α and aFGF in middle and low dose HTTLR groups at 24 h and 48 h all increased (P <0. 05). Conclusion The mechanism of HTTLR treatment for RA might be related to inhibiting RASFB proliferation, and decreasing mRNA expressions of TNF-α and aFGF as well as their protein secretion.

Halomonas spp. have been studied as a low cost production host for producing bulk materials such as polyhydroxyalkanoates (PHA) bioplastics, since they are able to grow at high pH and high NaCl concentration under unsterile and continuous conditions without microbial contamination. In this paper, Halomonas strain TD is used as a host to produce a protein named PHA phasin or PhaP which has a potential to be developed into a bio-surfactant. Four Halomonas TD expression strains are constructed based on a strong T7-family expression system. Of these, the strain with phaC deletion and chromosomal expression system resulted in the highest production of PhaP in soluble form, reaching 19% of total cellular soluble proteins and with a yield of 1.86 g/L in an open fed-batch fermentation process. A simple "heat lysis and salt precipitation" method is applied to allow rapid PhaP purification from a mixture of cellular proteins with a PhaP recovery rate of 63%. It clearly demonstrated that Halomonas TD could be used for high yield expression of a bio-surfactant protein PhaP for industrial application in an economical way., (Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Industrial biotechnology aims to produce chemicals, materials and biofuels to ease the challenges of shortage on petroleum. However, due to the disadvantages of bioprocesses including energy consuming sterilization, high fresh water consumption, discontinuous fermentation to avoid microbial contamination, highly expensive stainless steel fermentation facilities and competing substrates for human consumption, industrial biotechnology is less competitive compared with chemical processes. Recently, halophiles have shown promises to overcome these shortcomings. Due to their unique halophilic properties, some halophiles are able to grow in high pH and high NaCl containing medium under higher temperature, allowing fermentation processes to run contamination free under unsterile conditions and continuous way. At the same time, genetic manipulation methods have been developed for halophiles. So far, halophiles have been used to produce bioplastics polyhydroxyalkanoates (PHA), ectoines, enzymes, and bio-surfactants. Increasing effects have been made to develop halophiles into a low cost platform for bioprocessing with advantages of low energy, less fresh water consumption, low fixed capital investment, and continuous production., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Clustered regularly interspaced short palindromic repeats interference (CRISPRi) is used to edit eukaryotic genomes. Here, we show that CRISPRi can also be used for fine-tuning prokaryotic gene expression while simultaneously regulating multiple essential gene expression with less labor and time consumption. As a case study, CRISPRi was used to control polyhydroxyalkanoate (PHA) biosynthesis pathway flux and to adjust PHA composition. A pathway was constructed in Escherichia coli for the production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] from glucose. The native gene sad encoding E. coli succinate semi-aldehyde dehydrogenase was expressed under the control of CRISPRi using five specially designed single guide RNAs (sgRNAs) for regulating carbon flux to 4-hydroxybutyrate (4HB) biosynthesis. The system allowed formation of P(3HB-co-4HB) consisting of 1-9mol% 4HB. Additionally, succinate, generated by succinyl-coA synthetase and succinate dehydrogenase (respectively encoded by genes sucC, sucD and sdhA, sdhB) was channeled preferentially to the 4HB precursor by using selected sgRNAs such as sucC2, sucD2, sdhB2 and sdhA1 via CRISPRi. The resulting 4HB content in P(3HB-co-4HB) was found to range from 1.4 to 18.4mol% depending on the expression levels of down-regulated genes. The results show that CRISPRi is a feasible method to simultaneously manipulate multiple genes in E. coli., (Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Poly(3-hydroxypropionate) (P3HP) is the strongest family member of microbial polyhydroxyalkanoates (PHA) synthesized by bacteria grown on 1,3-propandiol or glycerol. In this study synthesis pathways of P3HP and its copolymer P3HB3HP of 3-hydroxybutyrate (3HB) and 3-hydroxypropionate (3HP) were assembled respectively to allow their synthesis from glucose, a more abundant carbon source. Recombinant Escherichia coli was constructed harboring the P3HP synthetic pathway consisting of heterologous genes encoding glycerol-3-phosphate dehydrogenase (gpd1), glycerol-3-P phosphatase (gpp2) from Saccharomyces cerevisiae that catalyzes formation of glycerol from glucose, and genes coding glycerol dehydratase (dhaB123) with its reactivating factors (gdrAB) from Klebsiella pneumoniae that transfer glycerol to 3-hydroxypropionaldehyde, as well as gene encoding propionaldehyde dehydrogenase (pdup) from Salmonella typhimurium which converts 3-hydroxypropionaldehyde to 3-hydroxypropionyl-CoA, together with the gene of PHA synthase (phaC) from Ralstonia eutropha which polymerizes 3-hydroxypropionyl-CoA into P3HP. When phaA and phaB from Ralstonia eutropha respectively encoding β-ketothiolase and acetoacetate reductase, were introduced into the above P3HP producing recombinant E. coli, copolymers poly(3-hydroxybutyrate-co-3-hydroxypropionate) (P3HB3HP) were synthesized from glucose as a sole carbon source. The above E. coli recombinants grown on glucose LB medium successfully produced 5g/L cell dry weight containing 18% P3HP and 42% P(3HB-co-84mol% 3HP), respectively, in 48h shake flask studies., (Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Background: With the rapid development of synthetic biology, the demand for assembling multiple DNA (genes) fragments into a large circular DNA structure in one step has dramatically increased. However, for constructions of most circular DNA, there are two contradictions in the ligation/assembly and transformation steps. The ligation/assembly consists of two different reactions: 1) the ligation/assembly between any two pieces of a linear form DNA; 2) the cyclization (or self-ligation) of a single linear form DNA. The first contradiction is that the bimolecular ligation/assembly requires a higher DNA concentration while the cyclization favors a lower one; the second contradiction is that a successful transformation of a ligation/assembly product requires a relatively high DNA concentration again. This study is the first attempt to use linear plasmid and Cyclization After Transformation (CAT) strategy to neutralize those contradictions systematically., Results: The linear assembly combined with CAT method was demonstrated to increase the overall construction efficiency by 3-4 times for both the traditional ligation and for the new in vitro recombination-based assembly methods including recombinant DNA, Golden Gate, SLIC (Sequence and Ligation Independent Cloning) and Gibson Isothermal Assembly. Finally, the linear assembly combined with CAT method was successfully applied to assemble a pathway of 7 gene fragments responsible for synthesizing precorrin 3A which is an important intermediate in VB12 production., Conclusion: The linear assembly combined with CAT strategy method can be regarded as a general strategy to enhance the efficiency of most existing circular DNA construction technologies and could be used in construction of a metabolic pathway consisting of multiple genes.

Microbial fermentation is the key to industrial biotechnology. Most fermentation processes are sensitive to microbial contamination and require an energy intensive sterilization process. The majority of microbial fermentations can only be conducted over a short period of time in a batch or fed-batch culture, further increasing energy consumption and process complexity, and these factors contribute to the high costs of bio-products. In an effort to make bio-products more economically competitive, increased attention has been paid to developing open (unsterile) and continuous processes. If well conducted, continuous fermentation processes will lead to the reduced cost of industrial bio-products. To achieve cost-efficient open and continuous fermentations, the feeding of raw materials and the removal of products must be conducted in a continuous manner without the risk of contamination, even under 'open' conditions. Factors such as the stability of the biological system as a whole during long cultivations, as well as the yield and productivity of the process, are also important. Microorganisms that grow under extreme conditions such as high or low pH, high osmotic pressure, and high or low temperature, as well as under conditions of mixed culturing, cell immobilization, and solid state cultivation, are of interest for developing open and continuous fermentation processes., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Since halophile Halomonas spp. can grow contamination free in seawater under unsterile and continuous conditions, it holds great promise for industrial biotechnology to produce low-cost chemicals in an economic way. Yet, metabolic engineering methods are urgently needed for Halomonas spp. It is commonly known that chromosomal expression is more stable yet weaker than plasmid one is. To overcome this challenge, a novel chromosomal expression method was developed for halophile Halomonas TD01 and its derivatives based on a strongly expressed porin gene as a site for external gene integration. The gene of interest was inserted downstream the porin gene, forming an artificial operon porin-inserted gene. This chromosome expression system was proven functional by some examples: First, chromosomal expression of heterologous polyhydroxybutyrate (PHB) synthase gene phaC Re from Ralstonia eutropha completely restored the PHB accumulation level in endogenous phaC knockout mutant of Halomonas TD01. The integrated phaC Re was expressed at the highest level when inserted at the locus of porin compared with insertions in other chromosome locations. Second, an inducible expression system was constructed in phaC-deleted Halomonas TD01 by integrating the lac repressor gene (lacI) into the porin site in the host chromosome. The native porin promoter was inserted with the key 21 bp DNA of lac operator (lacO) sequence to become an inducible promoter encoded in a plasmid. This inducible system allowed on-off switch of gene expression in Halomonas TD strains. Thus, the stable and strong chromosomal expression method in Halomonas TD spp. was established.

The halophile Halomonas TD01 and its derivatives have been successfully developed as a low-cost platform for the unsterile and continuous production of chemicals. Therefore, to increase the genetic engineering stability of this platform, the DNA restriction/methylation system of Halomonas TD01 was partially inhibited. In addition, a stable and conjugative plasmid pSEVA341 with a high-copy number was constructed to contain a LacI(q)-Ptrc system for the inducible expression of multiple pathway genes. The Halomonas TD01 platform, was further engineered with its 2-methylcitrate synthase and three PHA depolymerases deleted within the chromosome, resulting in the production of the Halomonas TD08 strain. The overexpression of the threonine synthesis pathway and threonine dehydrogenase made the recombinant Halomonas TD08 able to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) or PHBV consisting of 4-6 mol% 3-hydroxyvalerate or 3 HV, from various carbohydrates as the sole carbon source. The overexpression of the cell division inhibitor MinCD during the cell growth stationary phase in Halomonas TD08 elongated its shape to become at least 1.4-fold longer than its original size, resulting in enhanced PHB accumulation from 69 wt% to 82 wt% in the elongated cells, further promoting gravity-induced cell precipitations that simplify the downstream processing of the biomass. The resulted Halomonas strains contributed to further reducing the PHA production cost., (Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Many bacteria have been found to produce various polyhydroxyalkanoates (PHA) biopolyesters. In many cases, it is not easy to control the structures of PHA including homopolymers, random copolymers and block copolymers as well as ratios of monomers in the copolymers. It has become possible to engineer bacteria for controllable synthesis of PHA with the desirable structures by creating new PHA synthesis pathways. Remarkably, the weakening of β-oxidation cycle in Pseudomonas putida and Pseudomonas entomophila led to controllable synthesis of all kinds of PHA structures including monomer ratios in random and/or block copolymers when fatty acids are used as PHA precursors. Introduction of functional groups into PHA polymer chains in predefined proportions has become a reality provided fatty acids containing the functional groups are taken up by the bacteria for PHA synthesis. This allows the formation of functional PHA for further grafting. The PHA diversity is further widened by the endless possibility of controllable homopolymerization, random copolymerization, block copolymerization and grafting on functional PHA site chains., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Genetic engineering of Halomonas spp. was seldom reported due to the difficulty of genetic manipulation and lack of molecular biology tools. Halomonas TD01 can grow in a continuous and unsterile process without other microbial contaminations. It can be therefore exploited for economic production of chemicals. Here, Halomonas TD01 was metabolically engineered using the gene knockout procedure based on markerless gene replacement stimulated by double-strand breaks in the chromosome. When gene encoding 2-methylcitrate synthase in Halomonas TD01 was deleted, the conversion efficiency of propionic acid to 3-hydroxyvalerate (3HV) monomer fraction in random PHBV copolymers of 3-hydroxybutyrate (3HB) and 3HV was increased from around 10% to almost 100%, as a result, cells were grown to accumulate 70% PHBV in dry weight (CDW) consisting of 12mol% 3HV from 0.5g/L propionic acid in glucose mineral medium. Furthermore, successful deletions on three PHA depolymerases eliminate the possible influence of PHA depolymerases on PHA degradation in the complicated industrial fermentation process even though significant enhanced PHA content was not observed. In two 500L pilot-scale fermentor studies lasting 70h, the above engineered Halomonas TD01 grew to 112g/L CDW containing 70wt% P3HB, and to 80g/L CDW with 70wt% P(3HB-co-8mol% 3HV) in the presence of propionic acid. The cells grown in shake flasks even accumulated close to 92% PHB in CDW with a significant increase of glucose to PHB conversion efficiency from around 30% to 42% after 48h cultivation when pyridine nucleotide transhydrogenase was overexpressed. Halomonas TD01 was also engineered for producing a PHA regulatory protein PhaR which is a robust biosurfactant., (Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Objective: To assess the effectiveness and safety on post-stroke dysphagia in chronic stage treated with magnetic-ball sticking therapy at the auricular points., Methods: Ninety cases of post-apoplexy dysphagia in chronic stage were randomized into an auricular points group and an acupuncture group. In the auricular points group, the magnetic-ball sticking therapy was applied to subcortex (pizhixia, AT4), brainstem (naogan, AT(3,4i)), mouth (kou, CO1), cheek (mianjia, LO(5,6i)), tongue (she, LO2) and throat (yanhou, TG3) on one ear each time, and were changed on the other ear once every 3 days. In the acupucnture group, acupuncture was applied to Feng-chi (GB 20), Yifeng (TE 17), Shanglianquan (Extra), Jinjin (EX-HN 12), Yuye (EX-HN 13), Shuigou (GV 26) and Tongli (TH 5), etc. The needles were retained for 30 min in each treatment. The treatment was gi-yen once a day in the two groups and the treatment of 6 days made one session. There was 1 day at an interval among the sessions. Totally, 3 sessions of treatment were required. The video fluoroscopic swallowing study (VFSS) was performed for 4 kinds of food with different properties and shapes in each patient. The main indices were Rosenbek penetration-aspiration score, oral-retaining score and throat-retaining score. The efficacy, and the incidences of aspiration pneumonia and malnutrition were compared between the two groups. The nutrition indices were compared before and after treatment between the two groups, such as the skinfold thickness of triceps brachii muscle, serum albumin and peralbumin., Results: In 21 days of treatment, in the auricular points group, the 1 mL liquid loversol Rosenbek penetration-aspiration score (1.51 +/- 0.69), oral-retaining score (1.17 +/- 0.38) and throat-retaining score (1.30 +/- 0.66) were all lower than those (2.51 +/- 0.67, 1.63 +/- 0.72, 1.67 +/- 0.7) in the acupuncture group separately. The 10 mL liquid loversol Rosenbek penetration-aspiration score (2.27 +/- 0.65), oral-retaining score (1.60 +/- 0.50) and throat-retaining score (1.49 +/- 0.51) were all lower than those (4.19 +/- 0.73, 2.30 +/- 0.51, 2.41 +/- 0.50) in the acupuncture group separately. The 10 mL paste loversol Rosenbek penetration-aspiration score (1.68 +/- 0.81), oral-retaining score (1.11 +/- 0.31) and throat-retaining score (1.10 +/- 0.31) were all lower than those (3.91 +/- 0.68, 1.63 +/- 0.76, 1.60 +/- 0.76) in the acupuncture group separately. The 1/4 cake-form loversol Rosenbek penetration-aspiration score (2.60 +/- 0.65), oral-retaining score (1.40 +/- 0.50) and throat-retaining score (1.74 +/- 0.49) were all lower than those (4.14 +/- 1.10, 2.40 +/- 0.73, 2.30 +/- 0.83) in the acupuncture group separately. The incidence of aspiration pneumonia was 14.9% (7/47) in the auricular points group, which was lower than 55.0% (22/40) in the acupuncture group (P < 0.01). The incidence of malnutrition was 8. 5% (4/47) in the auricular points group, which was lower than 50.0% (20/40) in the acupuncture group (P < 0.01). In 21 days of treatment, the results of the skinfold thickness of triceps brachii muscle and serum albumin in the auricular points group were better than those in the acupuncture group (both P < 0.05)., Conclusion: The magnetic-ball sticking therapy at auricular points achieves the definite efficacy on post-stoke dysphagia in chronic stage and decreases the incidences of aspiration pneumonia and malnutrition. The efficacy of this therapy is better than acupuncture.

The DNA repair gene X-ray cross-complementary group 4 (XRCC4), an important caretaker of the overall genome stability, is thought to play a major role in human tumorigenesis. We investigated the association between an important polymorphic variant of this gene at codon 247 (rs373409) and diffusely infiltrating astrocytoma (DIA) risk and prognosis. This hospital-based case-control study investigated this association in the Guangxi population. In total, 242 cases with DIA and 358 age-, sex-, and race-matched healthy controls were genotyped using TaqMan-PCR technique. We found a significant difference in the frequency of XRCC4 genotypes between cases and controls. Compared with the homozygote of XRCC4 codon 247 Ala alleles (XRCC4-AA), the genotypes of XRCC4 codon 247 Ser alleles (namely XRCC4-AS or -SS) increased DIA risk (odds ratios [OR], 1.82 and 2.89, respectively). Furthermore, XRCC4 polymorphism was correlated with tumor dedifferentiation of DIA (r = 0.261, p < 0.01). Additionally, this polymorphism modified the overall survival of DIA patients (the median survival times were 26, 14, and 8 months for patients with XRCC4-AA, -AS, and -SS, respectively). Like tumor grade, XRCC4 codon 247 polymorphism was an independent prognostic factor influencing the survival of DIA. These results suggest that XRCC4 codon 247 polymorphism may be associated with DIA risk and prognosis among the Guangxi population.

Alzheimer's disease (AD) is induced by many reasons, including decreased cellular utilization of glucose and brain cell mitochondrial damages. Degradation product of microbially synthesized polyhydroxybutyrate (PHB), namely, 3-hydroxybutyrate (3HB), can be an alternative to glucose during sustained hypoglycemia. In this study, the derivative of 3HB, 3-hydroxybutyrate methyl ester (HBME), was used by cells as an alternative to glucose. HBME inhibited cell apoptosis under glucose deprivation, rescued activities of mitochondrial respiratory chain complexes that were impaired in AD patients and decreased the generation of ROS. Meanwhile, HBME stabilized the mitochondrial membrane potential. In vivo studies showed that HBME crossed the blood brain barrier easier compared with charged 3HB, resulting in a better bioavailability. AD mice treated with HBME performed significantly better (p < 0.05) in the Morris water maze compared with other groups, demonstrating that HBME has a positive in vivo pharmaceutical effect to improve the spatial learning and working memory of mice. A reduced amyloid-β deposition in mouse brains after intragastric administration of HBME was also observed. Combined with the in vitro and in vivo results, HBME was proposed to be a drug candidate against AD, its working mechanism appeared to be mediated by various effects of protecting mitochondrial damages., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Polyhydroxyalkanoates (PHA), a family of diverse bio-polyesters, are produced by many bacteria as an energy and carbon storage material. PHA synthesis regulatory protein PhaR was reported to attach on the surface of intracellular PHA granules for convenience of synthesis regulation. PhaR was found to have an amphiphilic property. However, no study was conducted to exploit this property for applications as bio-surfactant and bactericide agent. Purified PhaR showed a higher emulsification ability than that of the widely used chemical surfactants including SDS, Tween 20, sodium oleate, and liquefied detergent (LD). PhaR also showed a higher emulsification ability than bio-surfactants rhamnose and PHA granules associated protein termed phasin or PhaP. Non-purified PhaR, namely, the native inclusion bodies and cell lysates, also demonstrated to be an excellent surfactant. PhaR was found highly stable even at 95 °C. In addition, PhaR was revealed to be a promising bactericidal agent against Gram positive and negative bacteria. PhaR can be conveniently produced by recombinant Escherichia coli. It has shown to be a bio-surfactant with excellent emulsification ability and strong bactericidal capacity at elevated temperature as high as 95 °C. Therefore, PhaR could be used in areas including food, beverage, pharmaceutical and cosmetics industries., (Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.)

The mechanism study on behaviors of cells influenced by biomaterial surface properties can provide profound guidances for functional tissue engineering scaffolds design. In this study, regulation of integrin-mediated cell-substrate interactions using rat osteoblasts incubated on PHA films was investigated. Compared with tissue culture plate (TCP), poly-3-hydroxybutyrate (PHB), copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV) and copolymer of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx), osteoblasts inoculated on a terpolymer of 3-hydroxybutyrate, 3-hydroxyvalerate and 3-hydroxyhexanoate (PHBVHHx) were found to have higher apoptosis rates. Several integrin subunits in osteoblasts grown on PHBVHHx showed altered expressions. Simultaneously, extracellular matrics (ECM) were also remodeled on the material surface. Osteoblasts showed a higher expression of integrin subunit β3 and αv on PHBVHHx films compared with that on TCP. On the other hand, less vitronectin, osteopontin and fibronectin, the main ligands for integrin β3 were expressed and deposited in ECM. The unligated integrin β3 could recruit caspase-8 to the membrane and activate its downstream signaling which was proven by the caspase-8 activation assay. It was therefore concluded that the induced apoptosis of osteoblasts on PHBVHHx was regulated by recruitment of caspase-8 to the unligated integrin β3., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

3-Hydroxyalkanoic acids (3HA) are precious precursors for synthesis of value added chemicals. According to their carbon chain lengths, 3HA can be divided into two groups: short-chain-length (SCL) 3HA consisting of 3-5 carbon atoms and medium-chain-length (MCL) 3HA containing 6-14 carbon atoms. To produce MCL 3HA, a metabolic engineered pathway expressing tesB gene, a thioesterase encoding gene that has been reported to catalyze acyl-CoA to free fatty acids, was constructed in Pseudomonas entomophila L48. When tesB of Escherichia coli encoding thioesterase II was introduced into polyhydroxyalkanoate (PHA) synthase and β-oxidation pathway deleted mutant of P. entomophila LAC31 derived from wild type P. entomophila L48, 6.65g/l 3-hydroxytetradecanoic acid (3HTD) and 4.6g/l 3-hydroxydodecanoic acid (3HDD) were obtained, respectively, when tetradecanoic acid or dodecanoic acid as related carbon sources was added in shake flask cultures. Moreover, 1.8g/l of 3-hydroxydecanoic (3HD) acid was also produced by P. entomophila LAC31 harboring PTE1 gene cloned from Saccharomyces cerevisiae using corresponding fatty acid decanoic acid. Interestingly, shake flask studies indicated that PTE1 harboring strain showed advantages over tesB expressing one for 3HDD and 3HD production, while tesB favored 3HTD production by P. entomophila LAC31. For the first time our study revealed that fine chemicals 3HTD, 3HDD or 3HD could be efficiently produced by metabolic engineered β-oxidation in Pseudomonas spp grown on related fatty acids., (Copyright © 2013 Elsevier Inc. All rights reserved.)

A couple of DNA ligation-independent cloning (LIC) methods have been reported to meet various requirements in metabolic engineering and synthetic biology. The principle of LIC is the assembly of multiple overlapping DNA fragments by single-stranded (ss) DNA overlaps annealing. Here we present a method to generate single-stranded DNA overlaps based on Nicking Endonucleases (NEases) for LIC, the method was termed NE-LIC. Factors related to cloning efficiency were optimized in this study. This NE-LIC allows generating 3'-end or 5'-end ss DNA overlaps of various lengths for fragments assembly. We demonstrated that the 10 bp/15 bp overlaps had the highest DNA fragments assembling efficiency, while 5 bp/10 bp overlaps showed the highest efficiency when T4 DNA ligase was added. Its advantage over Sequence and Ligation Independent Cloning (SLIC) and Uracil-Specific Excision Reagent (USER) was obvious. The mechanism can be applied to many other LIC strategies. Finally, the NEases based LIC (NE-LIC) was successfully applied to assemble a pathway of six gene fragments responsible for synthesizing microbial poly-3-hydroxybutyrate (PHB).

Background: Microbial polyhydroxyalkanoates (PHA) are biopolyesters consisting of diverse monomers. PHA synthase PhaC2Ps cloned from Pseudomonas stutzeri 1317 is able to polymerize short-chain-length (scl) 3-hydroxybutyrate (3HB) monomers and medium-chain-length (mcl) 3-hydroxyalkanoates (3HA) with carbon chain lengths ranging from C6 to C12. However, the scl and mcl PHA production in Escherichia coli expressing PhaC2Ps is limited with very low PHA yield., Results: To improve the production of PHA with a wide range of monomer compositions in E. coli, a series of optimization strategies were applied on the PHA synthase PhaC2Ps. Codon optimization of the gene and mRNA stabilization with a hairpin structure were conducted and the function of the optimized PHA synthase was tested in E. coli. The transcript was more stable after the hairpin structure was introduced, and western blot analysis showed that both codon optimization and hairpin introduction increased the protein expression level. Compared with the wild type PhaC2Ps, the optimized PhaC2Ps increased poly-3-hydroxybutyrate (PHB) production by approximately 16-fold to 30% of the cell dry weight. When grown on dodecanoate, the recombinant E. coli harboring the optimized gene phaC2PsO with a hairpin structure in the 5' untranslated region was able to synthesize 4-fold more PHA consisting of 3HB and medium-chain-length 3HA compared to the recombinant harboring the wild type phaC2Ps., Conclusions: The levels of both PHB and scl-mcl PHA in E. coli were significantly increased by series of optimization strategies applied on PHA synthase PhaC2Ps. These results indicate that strategies including codon optimization and mRNA stabilization are useful for heterologous PHA synthase expression and therefore enhance PHA production.

Recombinant Escherichia coli was constructed for co-production of hydrogen and polyhydroxybutyrate (PHB) due to its rapid growth and convenience of genetic manipulation. In particular, anaerobic metabolic pathways dedicated to co-production of hydrogen and PHB were established due to the advantages of directing fluxes away from toxic compounds such as formate and acetate to useful products. Here, recombinant E. coli expressing hydrogenase 3 and/or acetyl-CoA synthetase showed improved PHB and hydrogen production when grown with or without acetate as a carbon source. When hydrogenase 3 was over-expressed, hydrogen yield was increased from 14 to 153 mmol H(2)/mol glucose in a mineral salt (MS) medium with glucose as carbon source, accompanied by an increased PHB yield from 0.55 to 5.34 mg PHB/g glucose in MS medium with glucose and acetate as carbon source., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Despite recent advances in genomic sequencing and DNA chemical synthesis, construction of large gene clusters containing DNA fragments is still a difficult and expensive task. To tackle this problem, we developed a gene cluster extraction method based on in vitro single-strand overlapping annealing (SSOA). It starts with digesting the target gene cluster in an existing genome, followed by recovering digested chromosome fragments. Subsequently, the single-strand DNA overhangs formed from the digestion process would be specifically annealed and covalently joined together with a circular and a linear vector, respectively. The SSOA method was successfully applied to clone a 18 kb DNA fragment encoding NADH:ubiquinone oxidoreductase. Genomic DNA fragments of different sizes including 11.86, 18.33, 28.67, 34.56, and 55.99 kb were used to test the cloning efficiency. Combined with genetic information from KEGG and the KEIO strain collection, this method will be useful to clone any specific region of an E. coli genome at sizes less than ~28 kb. The method provides a cost-effective way for genome assembly, alternative to chemically synthesized gene clusters.

Copolyesters of 3-hydroxypropionate (3HP) and 4-hydroxybutyrate (4HB), abbreviated as P(3HP-co-4HB), was synthesized by Escherichia coli harboring a synthetic pathway consisting of five heterologous genes including orfZ encoding 4-hydroxybutyrate-coenzyme A transferase from Clostridium kluyveri, pcs' encoding the ACS domain of tri-functional propionyl-CoA ligase (PCS) from Chloroflexus aurantiacus, dhaT and aldD encoding dehydratase and aldehyde dehydrogenase from Pseudomonas putida KT2442, and phaC1 encoding PHA synthase from Ralstonia eutropha. When grown on mixtures of 1,3-propanediol (PDO) and 1,4-butanediol (BDO), compositions of 4HB in microbial P(3HP-co-4HB) were controllable ranging from 12 mol% to 82 mol% depending on PDO/BDO ratios. Nuclear magnetic resonance (NMR) spectra clearly indicated the polymers were random copolymers of 3HP and 4HB. Their mechanical and thermal properties showed obvious changes depending on the monomer ratios. Morphologically, P(3HP-co-4HB) films only became fully transparent when monomer 4HB content was around 67 mol%. For the first time, P(3HP-co-4HB) with adjustable monomer ratios were produced and characterized., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Human islet amyloid polypeptide (hIAPP) shows an antimicrobial activity towards two types of clinically relevant bacteria. The potency of hIAPP varies with its aggregation states. Circular dichroism was employed to determine the interaction between hIAPP and bacteria lipid membrane mimic. The antimicrobial activity of each aggregate species is associated with their ability to induce membrane disruption. Our findings provide new evidence revealing the antimicrobial activity of amyloid peptide, which suggest a possible connection between amyloid peptides and antimicrobial peptides.

Heat pump systems using treated sewage water as the heat source were used in the Beijing Olympic Village for domestic heating and cooling. However, considerable biofouling occurred in the plate heat exchangers used in the heat pump system, greatly limiting the system efficiency. This study investigates the biofouling characteristics using a plate heat exchanger in parallel with a flow cell system to focus on the effect of calcium ions on the biofilm development. The interactions between the microorganisms and Ca(2+) enhances both the extent and the rate of biofilm development with increasing Ca(2+) concentration, leading to increased heat transfer and flow resistances. Three stages of biofouling development were identified in the presence of Ca(2+) from different biofouling mass growth rates with an initial stage, a rapid growth stage and an extended growth stage. Each growth stage had different biofouling morphologies influenced by the Ca(2+) concentration. The effects of Ca(2+) on the biofouling heat transfer and flow resistances had a synergistic effect related to both the biofouling mass and the morphology. The effect of Ca(2+) on the biofouling development was most prominent during the rapid growth stage., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Poly(4-hydroxybutyrate) [poly(4HB)] is a strong thermoplastic biomaterial with remarkable mechanical properties, biocompatibility and biodegradability. However, it is generally synthesized when 4-hydroxybutyrate (4HB) structurally related substrates such as γ-butyrolactone, 4-hydroxybutyrate or 1,4-butanediol (1,4-BD) are provided as precursor which are much more expensive than glucose. At present, high production cost is a big obstacle for large scale production of poly(4HB)., Results: Recombinant Escherichia coli strain was constructed to achieve hyperproduction of poly(4-hydroxybutyrate) [poly(4HB)] using glucose as a sole carbon source. An engineering pathway was established in E. coli containing genes encoding succinate degradation of Clostridium kluyveri and PHB synthase of Ralstonia eutropha. Native succinate semialdehyde dehydrogenase genes sad and gabD in E. coli were both inactivated to enhance the carbon flux to poly(4HB) biosynthesis. Four PHA binding proteins (PhaP or phasins) including PhaP1, PhaP2, PhaP3 and PhaP4 from R. eutropha were heterologously expressed in the recombinant E. coli, respectively, leading to different levels of improvement in poly(4HB) production. Among them PhaP1 exhibited the highest capability for enhanced polymer synthesis. The recombinant E. coli produced 5.5 g L(-1) cell dry weight containing 35.4% poly(4HB) using glucose as a sole carbon source in a 48 h shake flask growth. In a 6-L fermentor study, 11.5 g L(-1) cell dry weight containing 68.2% poly(4HB) was obtained after 52 h of cultivation. This was the highest poly(4HB) yield using glucose as a sole carbon source reported so far. Poly(4HB) was structurally confirmed by gas chromatographic (GC) as well as (1)H and (13)C NMR studies., Conclusions: Significant level of poly(4HB) biosynthesis from glucose can be achieved in sad and gabD genes deficient strain of E. coli JM109 harboring an engineering pathway encoding succinate degradation genes and PHB synthase gene, together with expression of four PHA binding proteins PhaP or phasins, respectively. Over 68% poly(4HB) was produced in a fed-batch fermentation process, demonstrating the feasibility for enhanced poly(4HB) production using the recombinant strain for future cost effective commercial development.

Microbial polyhydroxyalkanoates (PHAs) are a family of polyesters with biodegradability, biocompatibility and adjustable mechanical properties that are under intensive development for bioimplant applications. In this research, a fusion protein of PHA repressor protein (PhaR) and Lys-Gln-Ala-Gly-Asp-Val (KQAGDV) oligopeptide (PhaR-KQAGDV) was utilized to enhance the PHA cytocompatability via a mechanism of PhaR hydrophobically binding to PHA coupled with KQAGDV oligopeptide, a specific ligand to the integrins on the cell surface, for promotion of cell adhesion. The PhaR-KQAGDV fusion protein successfully produced and purified from recombinant E. coli was used to coat the surfaces of several PHA including poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), respectively. The PhaR was observed to bind efficiently on all PHA surfaces measured by the fluorescence intensity of PhaR-EGFP as compared to the uncoated (PhaR negative) PHA films. The PHA surface hydrophilicity measured by water contact angles was significantly improved after PhaR-KQAGDV coating. Observations under confocal microscope and scanning electron microscopy, together with CCK-8 assays clearly demonstrated that adhesion and proliferation of human vascular smooth muscle cells (HvSMCs) inoculated on PHA films were much better on PhaR-KQAGDV coated surfaces than the non-coated control ones. The convenient physical coating approach for enhanced PHA cytocompatibility provides an advantage for PHA based tissue engineering., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Folate derivatives are crucial growth factors for Ketogulonigenium vulgare which is used in mixed culture with Bacillus megaterium for the industrial production of 2-keto-L-gulonic acid (2-KGA), the precursor of L-ascorbic acid (L-AA) or vitamin C (Vc). To improve the growth and 2-KGA production, five genes involved in folate biosynthesis identified in a folate gene cluster from Lactococcus lactis MG1363, including folB, folKE, folP, folQ and folC, were over-expressed in K. vulgare. Intracellular folate concentration in the recombinant strain harboring folate biosynthesis genes cluster under the control of P(sdh) (sorbose dehydrogenase gene sdh promoter from K. vulgare) was 8 times higher than that of the wildtype K. vulgare DSM 4025 (P<0.001). In shake flask studies, the cell density and 2-KGA production of the recombinant K. vulgare Rif (pMCS2PsdhfolBC) were increased by 18% (P<0.001) and 14% (P<0.001), respectively, under a relatively stable pH 7 condition. In fermentor studies, enhancements around 25% cell density (P<0.001) and approximately 35% 2-KGA productivity (P<0.001) were observed in comparison with the controls without over-expressing the folate biosynthesis genes. This was the first successful study of metabolic engineering on K. vulgare for enhanced 2-KGA production., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Microbial polyhydroxyalkanoates (PHA) are a family of structurally diverse polyesters produced by many bacteria. Deleting key steps from the beta-oxidation cycle in Pseudomonas putida makes it possible to achieve precise substrate based design of PHA homopolymers, copolymers, and block polymers, allowing the study of structure-property relationship in a clear way. The PHA homopolymer synthesis also allows the microbial or chemical production of pure monomers of PHA in a convenient way without separating the mixed monomers. After used as bioplastics, PHA can be methyl esterified to become biofuels, which further extends the PHA application value. The microbial production of PHA with diverse structures is entering a new developing phase., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Halophilic bacteria have shown their significance in industrial production of polyhydroxyalkanoates (PHA) and are gaining more attention for genetic engineering modification. Yet, little information on the genomics and PHA related genes from halophilic bacteria have been disclosed so far., Results: The draft genome of moderately halophilic bacterium, Halomonas sp. TD01, a strain of great potential for industrial production of short-chain-length polyhydroxyalkanoates (PHA), was analyzed through computational methods to reveal the osmoregulation mechanism and the evolutionary relationship of the enzymes relevant to PHA and ectoine syntheses. Genes involved in the metabolism of PHA and osmolytes were annotated and studied in silico. Although PHA synthase, depolymerase, regulator/repressor and phasin were all involved in PHA metabolic pathways, they demonstrated different horizontal gene transfer (HGT) events between the genomes of different strains. In contrast, co-occurrence of ectoine genes in the same genome was more frequently observed, and ectoine genes were more likely under coincidental horizontal gene transfer than PHA related genes. In addition, the adjacent organization of the homologues of PHA synthase phaC1 and PHA granule binding protein phaP was conserved in the strain TD01, which was also observed in some halophiles and non-halophiles exclusively from γ-proteobacteria. In contrast to haloarchaea, the proteome of Halomonas sp. TD01 did not show obvious inclination towards acidity relative to non-halophilic Escherichia coli MG1655, which signified that Halomonas sp. TD01 preferred the accumulation of organic osmolytes to ions in order to balance the intracellular osmotic pressure with the environment., Conclusions: The accessibility of genome information would facilitate research on the genetic engineering of halophilic bacteria including Halomonas sp. TD01.

Conversion of 3-hydroxypropionate (3HP) from 1,3-propanediol (PDO) was improved by expressing dehydratase gene (dhaT) and aldehyde dehydrogenase gene (aldD) of Pseudomonas putida KT2442 under the promoter of phaCAB operon from Ralstonia eutropha H16. Expression of these genes in Aeromonas hydrophila 4AK4 produced up to 21 g/L 3HP in a fermentation process. To synthesize homopolymer poly(3-hydroxypropionate) (P3HP), and copolymer poly(3-hydroxypropionate-co-3-hydroxybutyrate) (P3HP4HB), dhaT and aldD were expressed in E. coli together with the phaC1 gene encoding polyhydroxyalkanoate (PHA) synthase gene of Ralstonia eutropha, and pcs' gene encoding the ACS domain of the tri-functional propionyl-CoA ligase (PCS) of Chloroflexus aurantiacus. Up to 92 wt% P3HP and 42 wt% P3HP4HB were produced by the recombinant Escherichia coli grown on PDO and a mixture of PDO+1,4-butanediol (BD), respectively., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A medium-chain-length (MCL) polyhydroxyalkanoates (PHAs) producer Pseudomonas entomophila L48 was investigated for microbial production of 3-hydroxydodecanote homopolymer. Pseudomonas entomophila L48 was found to produce MCL PHA consisting of 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO), 3-hydroxydecanoate (3HD), and 3-hydroxydodecanoate (3HDD) from related carbon sources fatty acids. In this study, some of the genes encoding key enzymes in β-oxidation cycle of P. entomophila such as 3-hydroxyacyl-CoA dehydrogenase, 3-ketoacyl-CoA thiolase, and acetyl-CoA acetyltransferase were deleted to study the relationship between β-oxidation and PHA synthesis in P. entomophila. Among the mutants constructed, P. entomophila LAC26 accumulated over 90 wt % PHA consisting of 99 mol % 3HDD. A fed-batch fermentation process carried out in a 6 L automatic fermentor produced 7.3 g L(-1) PHA consisting of over 97 mol % 3HDD fraction. Properties of MCL PHA were significantly improved along with increasing 3HDD contents. P(2.1 mol % 3HD-co-97.9 mol % 3HDD) produced by P. entomophila LAC25 had the widest temperature range between T(g) and T(m), which were -49.3 and 82.4 °C, respectively, in all MCL PHA reported so far. The new type of PHA also represented high crystallinity caused by side-chain crystallization compared with short side chain PHA. For the first time, P(3HDD) homopolymers were obtained.

Polyhydroxyalkanoates (PHA) synthesis genes phbC and orfZ cloned from Ralstonia eutropha H16 were transformed into beta-oxidation weakened Pseudomonas putida KTOY08ΔGC, a mutant of P. putida KT2442. The recombinant P. putida strain termed KTHH06 was able to produce a short-chain-length PHA block copolymer consisting of poly(3-hydroxybutyrate) (P3HB) as one block and poly(4-hydroxybutyrate) (P4HB) as another block. One-dimensional and two-dimensional nuclear magnetic resonance (NMR) clearly indicated the polymer was a diblock copolymer consisting of 20 mol % P3HB as one block and 80 mol % P4HB as another one. Differential scanning calorimetric (DSC) showed that P3HB block melting temperatures (T(m)) in the block copolymer P3HB-b-P4HB was shift to low temperature compared with homopolymer P3HB and a blend of P3HB and P4HB. The block copolymer with a number average molecular weight of 50000 Da and a polydispersity of 3.1 demonstrated a better yield and tensile strength compared with that of its related random copolymer and blend of homopolymers of P3HB and P4HB.

Arene cis-diols are interesting chemicals because of their chiral structures and great potentials in industrial synthesis of useful chiral chemical products. Pseudomonas putida KT2442 was genetically modified to transform benzoic acid (benzoate) to 1,2-dihydroxy-cyclohexa-3,5-diene-1-carboxylic acid (DHCDC) or named benzoate cis-diol. BenD gene encoding cis-diol dehydrogenase was deleted to generate a mutant named P. putida KTSY01. Genes benABC encoding benzoate dioxygenase were cloned into plasmid pSYM01 and overexpressed in P. putida KTSY01. The recombinant bacteria P. putida KTSY01 (pSYM01) showed strong ability to transform benzoate to DHCDC. DHCDC of 2.3 g/L was obtained with a yield of 73% after 24 h of cultivation in shake flasks incubated under optimized growth conditions. Transformation of benzoate carried out in a 6-L fermentor using a benzoate fed-batch process produced over 17 g/L DHCDC after 48 h of fermentation. The average DHCDC production rate was 0.356 g L(-1) h(-1). DHCDC purified from the fermentation broth showed a purity of more than 95%, and its chemical structure was confirmed by nuclear magnetic resonance.

Toluene dioxygenase (TDO) catalyzes asymmetric cis-dihydroxylations of aromatic compounds. Pseudomonas putida KT2442 (pSPM01) harboring TDO genes could effectively biotransform a wide-range of aromatic substrates into their cis-diols products. In shake-flask culture, approximately 2.7gl(-1) benzene cis-diols, 8.8gl(-1) toluene cis-diols and 6.0gl(-1) chlorobenzene cis-diols were obtained from the biotransformation process. Furthermore, vgb gene encoding Vitreoscilla hemoglobin protein (VHb) which enhances oxygen microbial utilization rate under low dissolved oxygen concentration was integrated into P. putida KT2442 genome. The oxidation ability of the mutant strain P. putida KTOY02 (pSPM01) harboring TDO gene was increased in the presence of VHb protein. As a result, approximately 3.8, 15.1 or 6.8gl(-1) different cis-diols production was achieved in P. putida KTOY02 (pSPM01) grown in shake-flasks when benzene, toluene or chlorobenzene was used as the substrate. The above results indicate that P. putida KT2442 could be used as a cell factory to biotransform aromatic compounds.

Treatment of cells with estrogens and several pure ERalpha antagonists rapidly induces down-regulation of the alpha-type estrogen receptor (ERalpha) in the nucleus by mechanisms that are sensitive to the proteasome inhibitors, MG132 and clasto-lactacystin-beta-lactone. Hence, it is believed that these ER ligands induce down-regulation of ERalpha by proteasome-dependent mechanisms, which serve to control both the amount of transcriptional activity and the level of ligand-bound ERalpha in cells. In this study, we observed that treatment of cultured MCF-7 and T47D human breast cancer cells with the low affinity ER ligand, 4,4'-dihydroxy-trans-stilbene (4,4'-DHS), inhibited the transcriptional activity of ERalpha and induced slow and gradual decrease in the amount of ERalpha protein (henceforth referred to as down-regulation of ERalpha). The 4,4'-DHS-induced down-regulation of ERalpha in MCF-7 cells involved a mechanism that was insensitive to the two most specific proteasome inhibitors, clasto-lactacystin-beta-lactone and epoxomycin, but sensitive to MG132 at concentrations exceeding that required for maximal inhibition of the proteasome in MCF-7 cells. Therefore, 4,4'-DHS appears to induce down-regulation of ERalpha by a proteasome-independent mechanism. Here, we present data to show that both 4-OH and 4'-OH are critical for the ability of 4,4'-DHS to induce down-regulation of ERalpha and suggest that 4,4'-DHS provides a useful scaffold for development of novel ERalpha antagonists.

Polyhydroxyalkanoates (PHA) were biodegradable thermoplastics. Due to their broad applications, direct biosynthesis of PHA from inexpensive substrates, such as carbohydrates, is actively pursued. It has been recently revealed that (R)-3-hydroxyacyl-ACP: CoA transacylase (PhaG) played an important role in this pathway. In this study, a polymerase chain reaction (PCR) protocol was developed for the rapid and specific identification of phaG gene from various bacteria. Using the PCR strategy, the complete open reading frames of two phaG genes from Pseudomonas stutzeri 1317 and Pseudomonas nitroreducens 0802 were cloned from the genomic DNA and functionally expressed in Pseudomonas putida PHAGN-21. Furthermore, this strategy was successful applied in non-Pseudomonas strains, such as Burkholderia. These results suggest that PhaG-mediated pathway of medium-chain-length polyhydroxyalkanoates was widespread among bacteria.

3-Hydroxydecanoic acid (3HD) was produced in Escherichia coli by mobilizing (R)-3-hydroxydecanoyl-acyl carrier protein-coenzyme A transacylase (PhaG, encoded by the phaG gene). By employing an isogenic tesB (encoding thioesterase II)-negative knockout E. coli strain, CH01, it was found that the expressions of tesB and phaG can up-regulate each other. In addition, 3HD was synthesized from glucose or fructose by recombinant E. coli harboring phaG and tesB. This study supports the hypothesis that the physiological role of thioesterase II in E. coli is to prevent the abnormal accumulation of intracellular acyl-coenzyme A.

Objective: To study the effect of Chinese herbal medicine 1023 Recipe in blocking cancer transformation of experimental oral precancerous lesion and its mechanism., Methods: We treated the experimental oral precancerous lesion in hamster's cheek pouch using 1023 Recipe (consisting of Radix Astragali, Gynostemma Pentaphyllum, Rhizoma Chuanxiong and selenium-rich green tea) for 6 weeks, and observed its effect in blocking cancer transformation, detected 2 kinds of agglutinin receptors (receptors of wheat germ agglutinin and Ricinus communis agglutinin) in the mucosa of the hamster's cheek pouch., Results: The rate of cancer transformation in 1023 Recipe treated group was lower than that in the control group without treatment (P<0.05). Agglutinin receptors in the two groups were different significantly., Conclusion: 1023 Recipe is effective in treating hyperplasia, and can prevent its cancer transformation. The mechanism may be that 1023 Recipe can induce precancerous lesions to differentiate into normal tissues.

The whole polyhydroxyalkanoate (PHA) synthesis gene locus of Pseudomonas stutzeri strain 1317 containing PHA synthase genes phaC1Ps, phaC2Ps and PHA depolymerase gene phaZPs was cloned using a PCR cloning strategy. The sequence analysis results of the phaC1Ps, phaC2Ps and phaZPs showed high homology to the corresponding pha loci of the known Pseudomonas strains, respectively. PhaC1Ps and PhaC2Ps were functionally expressed in recombinant Escherichia coli strains and their substrate specificity was compared. The results demonstrated that PhaC1Ps and PhaC2Ps from P. stutzeri 1317 had different substrate specificities when expressed in E. coli. In details, PhaC2Ps could incorporate both short-chain-length 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates (mcl 3HA) into PHA, while PhaC1Ps only favored mcl 3HA for polymerization., (Copyright 2004 Federation of European Microbiological Societies)

A lab-scale composting experiment was carried out using vegetable and flower stalks waste to study the effectiveness of ligno-cellulolytic microorganisms (LCMs) obtained from the previous isolation on composting process, especially on enhancement of biodegradation rate of these organic materials. The addition of LCMs to compost showed promised to be a valuable asset by rendering timely benefits in efficiency, maturity, and quality of the composting. This was evidenced by a significant increase of temperature, O2 consumption and CO2 emission, and population density of LCMs in compost mass compared with that of biotic (addition of culture of horse feces) and abiotic (1% molasses amendment) treatments, as well as control trial. The phytotoxicity assay showed that the substrate became mature after 60 days' composting. The LCMs inoculation enhanced the biodegradation of the composting materials as evidenced by an increasing screening ratio (1.2 cm sieve pore) of 34.5% in the treated trail, compared with that of control, which elucidated that big advantage of adding selected inoculants over other treatment, and screening ratio is a reasonable index to compare the quality of different compost. However, the inoculation seemed to have no significant effect on the moisture content, pH, and the final organic carbon of the composting materials.

The present investigation describes the production of extracellular matrix of rabbit articular cartilage chondrocytes grown on scaffolds of polyhydroxybutyrate (PHB) blended with poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx) for up to 7 days. The mRNA level of type II collagen of chondrocytes seeded on all scaffolds consisting of PHBHHx were obviously higher than that of PHB-only scaffold throughout the culture period, suggesting the positive effect of PHBHHx on extracellular matrix production. Second-harmonic generation (SHG) imaging technique, combined with confocal fluorescence microscopy (CFM) revealed that PHBHHx in PHB scaffold provided better surface properties for anchoring type II collagen filaments and their penetration into internal layers of the scaffolds. Glycosaminoglycan (GAG), a major composition of extracellular matrix, showed a sharp increase in construct of 1:2 PHB/PHBHHx scaffold after 7 day cultivation, while only a small increase was observed in all other tested scaffolds. At the same time, total collagen contents in all scaffolds containing PHBHHx increased with time, with the maximum collagen production of 742.1+/-99.2mg/g dry weight observed in construct of 1:2 PHB/PHBHHx scaffold inoculated for 7 days, this was almost 4-fold higher than that in scaffold of PHB only. It appears that the presence of right proportion of PHBHHx in the composite system of PHB/PHBHHx highly favored the production of extracellular matrix of articular cartilage chondrocytes.

Pathways for extracellular production of chiral D-(-)-3-hydroxybutyric acid (3HB) and D-(-)-3-hydroxyalkanoic acid (mcl-3HA) were constructed by co-expression of genes of beta-ketothiolase (phbA), acetoacetyl-CoA reductase (phbB) and 3-hydroxyacyl-ACP CoA transacylase (phaG), respectively, in Escherichia coli strain DH5alpha. The effect of acrylic acid and glucose on production of both 3HB and mcl-3HA was investigated. It was found that the addition of acrylic acid significantly increased production of 3HB and mcl-3HA consisting of 3-hydroxyoctanoic acid and 3-hydroxydecanoic acid in a ratio of 1:3 from 199 mg x l(-1) to 661 mg x l(-1) and from 27 mg x l(-1) to 135 mg x l(-1), respectively, in shake flask studies when glucose was present in the medium at the very beginning of fermentation. The timing of glucose addition had no effect on 3HB production. In contrast, mcl-3HA production was affected by glucose addition, an mcl-3HA concentration of 193 mg x l(-1) was obtained when glucose was added to the culture at 12 h. A more than seven-fold increase was obtained when compared with that in medium containing glucose at the beginning of fermentation. However, a decrease in production of 3HB and mcl-3HA was found when glucose was added at 12 h to the culture containing acrylic acid. The repressive effect of acrylic acid on acetic acid production was also evaluated and discussed.

Cis-1,2-dihydroxycyclohexa-3,5-diene (DHCD) can be used as a valuable chiral intermediates for applications in pharmaceuticals, aerospace, electrical and fine chemical industries. By on-line detection of toluene dioxygenase (TDO) activity in whole recombinant Escherichia coli JM109 (pKST11) cells that harbored TDO gene under a tac promoter, effects of IPTG and various benzene addition strategies on bioransformation of benzene to DHCD were investigated. When IPTG was used at the beginning of fermentation, the growth of cells was inhibited and TDO activity only maintained for 4 hours while same experiments with addition of IPTG at 6h or 8h generated TDO activity for 18 hours. Suitable induction time for IPTG was in the cell logarithmic growth phase and 0.5 mmol/L IPTG was sufficient for inducing maximum TDO activities. Benzene strongly inhibited the activity of TDO which catalyses the conversion of benzene to DHCD. It was found that both cell growth and TDO activity was remarkably inhibited by feeding of benzene vapor, only 7.5 g/L DHCD was obtained. While the benzene inhibition effect was ameliorated by two-liquid phase culture fermentation in which liquid paraffin was used as second phase in the broth. Using different initial ratios of paraffin to benzene in fed-batch culture, DHCD contents were increased to 22.6 g/L, which was 3-fold more compared with that in benzene vapor culture. A further improvement of DHCD production was achieved when the mixture of liquid paraffin and benzene was added continuously by peristaltic pump, the DHCD contents were increased to a final concentration of 36.8 g/L. It was proven that the key to improving DHCD production by recombinants is to prolong TDO activity in cells, which can be achieved by using suitable addition benzene strategies.