The development of biodegradable polymers is crucial for addressing environmental issues and waste management challenges, and a medium-chain-length polyhydroxyalkanoate(MCL-PHA) exhibits significant application potential in diverse industrial and environmental contexts owing to its versatility and biodegradability. Here, Pseudomonas putida was metabolically engineered to produce MCL-PHA from crude glycerol. To increase the precursor pool, we first deleted the phaC1ZC2 operon and introduced a plasmid-based overexpression of phaC2 and phaG, and the MCL-PHA content derived from glycerol increased to 18.27 wt% at 60 h. Subsequently, by optimizing the acoA expression through promoter selection and UTR design, the MCL-PHA content further increased to 19.93 wt% at 72 h. Additionally, a notable increase in MCL-PHA production was achieved using PhaC2 designed to have no substrate-trapping effect (PhaC2A477A478). This improvement was guided by filling structural data gaps using AlphaFold2 and docking simulations that revealed the substrate-trapping phenomenon. High-level production of MCL-PHA was achieved through fed-batch fermentation using the final engineered P. putida from refined glycerol, which yielded 34.9 g/L of MCL-PHA with 44.64 wt% at 180 h. Furthermore, using crude glycerol as the sole carbon source enabled the production of 49.5 g/L of MCL-PHA with 45.41 wt% at 180 h in fed-batch culture.

Background The necessity to develop high lipid-producing microalgae is emphasized for the commercialization of microalgal biomass, which is environmentally friendly and sustainable. Nannochloropsis are one of the best industrial microalgae and have been widely studied for their lipids, including high-value polyunsaturated fatty acids (PUFAs). Many reports on the genetic and biological engineering of Nannochloropsis to improve their growth and lipid contents have been published. Results We performed insertional mutagenesis in Nannochloropsis salina , and screened mutants with high lipid contents using fluorescence-activated cell sorting (FACS). We isolated a mutant, Mut68, which showed improved growth and a concomitant increase in lipid contents. Mut68 exhibited 53% faster growth rate and 34% higher fatty acid methyl ester (FAME) contents after incubation for 8 days, resulting in a 75% increase in FAME productivity compared to that in the wild type (WT). By sequencing the whole genome, we identified the disrupted gene in Mut68 that encoded trehalose-6-phosphate (T6P) synthase (TPS). TPS is composed of two domains: TPS domain and T6P phosphatase (TPP) domain, which catalyze the initial formation of T6P and dephosphorylation to trehalose, respectively. Mut68 was disrupted at the TPP domain in the C-terminal half, which was confirmed by metabolic analyses revealing a great reduction in the trehalose content in Mut68. Consistent with the unaffected N-terminal TPS domain, Mut68 showed moderate increase in T6P that is known for regulation of sugar metabolism, growth, and lipid biosynthesis. Interestingly, the metabolic analyses also revealed a significant increase in stress-related amino acids, including proline and glutamine, which may further contribute to the Mut68 phenotypes. Conclusion We have successfully isolated an insertional mutant showing improved growth and lipid production. Moreover, we identified the disrupted gene encoding TPS. Consistent with the disrupted TPP domain, metabolic analyses revealed a moderate increase in T6P and greatly reduced trehalose. Herein, we provide an excellent proof of concept that the selection of insertional mutations via FACS can be employed for the isolation of mutants with improved growth and lipid production. In addition, trehalose and genes encoding TPS will provide novel targets for chemical and genetic engineering, in other microalgae and organisms as well as Nannochloropsis .

Bacterial cellulose (BC) has outstanding physical and chemical properties, including high crystallinity, moisture retention, and tensile strength. Currently, the major producer of BC is Komagataeibacter xylinus. However, due to limited tools of expression, this host is difficult to engineer metabolically to improve BC productivity. In this study, a regulated expression system for K. xylinus with synthetic ribosome binding site (RBS) was developed and used to engineer a BC biosynthesis pathway. A synthetic RBS library was constructed using green fluorescent protein (GFP) as a reporter, and three synthetic RBSs (R4, R15, and R6) with different strengths were successfully isolated by fluorescence-activated cell sorting (FACS). Using synthetic RBS, we optimized the expression of three homologous genes responsible for BC production, pgm, galU, and ndp, and thereby greatly increased it under both static and shaking culture conditions. The final titer of BC under static and shaking conditions was 5.28 and 3.67 g/l, respectively. Our findings demonstrate that reinforced metabolic flux towards BC through quantitative gene expression represents a practical strategy for the improvement of BC productivity.

Bacterial cellulose consisting of pure cellulose nanofibers, is a promising biomaterial with versatile applications. The primary producer is Komagataeibacter xylinus, however, its use is limited because the bacteria undergo irreversible conversion into a non-cellulose-producing mutant (Cel(-)) during shaking cultivation. Here, by investigating the bcs operon in Cel(-) mutants, we found that an IS element-inserted mutation in bcsA, is responsible for the conversion into Cel(-). To prevent IS element insertion into bcsA, putative IS element recognition sequences were modified. As a result, the engineered strain retained its own bcsA gene over seven sub-cultivations, and showed 1.7-fold higher productivity than wild-type strains without any differences of physical and chemical properties.

Background Methane, a main component of natural gas and biogas, has gained much attention as an abundant and low-cost carbon source. Methanotrophs, which can use methane as a sole carbon and energy source, are promising hosts to produce value-added chemicals from methane, but their metabolic engineering is still challenging. In previous attempts to produce lactic acid (LA) from methane, LA production levels were limited in part due to LA toxicity. We solved this problem by generating an LA-tolerant strain, which also contributes to understanding novel LA tolerance mechanisms. Results In this study, we engineered a methanotroph strain Methylomonas sp. DH-1 to produce d-lactic acid (d-LA) from methane. LA toxicity is one of the limiting factors for high-level production of LA. Therefore, we first performed adaptive laboratory evolution of Methylomonas sp. DH-1, generating an LA-tolerant strain JHM80. Genome sequencing of JHM80 revealed the causal gene watR, encoding a LysR-type transcription factor, whose overexpression due to a 2-bp (TT) deletion in the promoter region is partly responsible for the LA tolerance of JHM80. Overexpression of the watR gene in wild-type strain also led to an increase in LA tolerance. When d form-specific lactate dehydrogenase gene from Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 was introduced into the genome while deleting the glgA gene encoding glycogen synthase, JHM80 produced about 7.5-fold higher level of d-LA from methane than wild type, suggesting that LA tolerance is a critical limiting factor for LA production in this host. d-LA production was further enhanced by optimization of the medium, resulting in a titer of 1.19 g/L and a yield of 0.245 g/g CH4. Conclusions JHM80, an LA-tolerant strain of Methylomonas sp. DH-1, generated by adaptive laboratory evolution was effective in LA production from methane. Characterization of the mutated genes in JHM80 revealed that overexpression of the watR gene, encoding a LysR-type transcription factor, is responsible for LA tolerance. By introducing a heterologous lactate dehydrogenase gene into the genome of JHM80 strain while deleting the glgA gene, high d-LA production titer and yield were achieved from methane.

Methylomonas sp. DH-1, newly isolated from the activated sludge of a brewery plant, has been used as a promising biocatalytic platform for the conversion of methane to value-added chemicals. Methylomonas sp. DH-1 can efficiently convert methane and propane into methanol and acetone with a specific productivity of 4.31 and 0.14 mmol/g cell/h, the highest values ever reported, respectively. Here, we present the complete genome sequence of Methylomonas sp. DH-1 which consists of a 4.86 Mb chromosome and a 278 kb plasmid. The existence of a set of genes related to one-carbon metabolism and various secondary metabolite biosynthetic pathways including carotenoid pathways were identified. Interestingly, Methylomonas sp. DH-1 possesses not only the genes of the ribulose monophosphate cycle for type I methanotrophs but also the genes of the serine cycle for type II. Methylomonas sp. DH-1 accumulated 80 mM succinate from methane under aerobic conditions, because DH-1 has 2-oxoglutarate dehydrogenase activity and the ability to operate the full TCA cycle. Availability of the complete genome sequence of Methylomonas sp. DH-1 enables further investigations on the metabolic engineering of this strain for the production of value-added chemicals from methane.

PurposeShort metaphyseal fitting femoral stems convey stress to proximal femur and have no distal fixation. They have advantages in that there is no thigh pain and no bone loss due to stress shielding, but there is a concern for weakened fixation. So the authors evaluated whether short metaphyseal fitting femoral stems, which have only metaphyseal and no diaphyseal fixation, can acquire sufficient stability.Materials & methods39 cases of 36 patients who undervent uncemented total hip arthroplasty with DePuy ProximaTM (Johnson & Johnson orthopaedics, New Milton, UK) short metaphyseal fitting femoral stems from August 2009 to September 2011 were retrospectively evaluated. There were 19 male and 20 female cases. The mean follow-up period was 35.8(21.8∼49.2) months. Harris hip scores, WOMAC scores, UCLA scores, and presence of femoral pain were evaluated to assess clinical outcome. Femoral radiolucency in coronal and sagittal views of the hip, femoral stem loosening, and displacement was measured to evaluate ...

PurposeCementless cup with structural allograft is one of option for acetabular revision in the cases which has severe bone loss. This study was performed to verify that the structural allograft with cementless cup could be one of good options for revision of acetabular cup with severe bone defect and to verify that the allograft resorption affect the stability of cementless acetabular cup.Materials and MethodsWe reviewed 25 cases of 25 patients who underwent acetabular cup rvision using cementless porous coated hemispherical cup with structural allograft from May 1992 to July 2011 July 2011. There were nine males and sixteen females with an average age of 50.0 years. The average follow-up period was 76.7(28∼212) months. The clinical evaluation was performed using Harris Hip Score(HHS) and UCLA activity score. Radiologically, the degree of resorption of grafted bone, incorporation of allograft bone with normal bone, osteolysis and cup loosening were evaluated.ResultsClinically, the average Harris hip scor...

BACKGROUND: Methane is the major component of natural and shale gas. Methane can be converted into methanol via a bioprocess using methanotrophs, and methanol is a valuable chemical feedstock for the production of value-added chemicals. This work demonstrates highly effective bioconversion of methane to methanol using a newly isolated novel methanotroph, Methylomonas sp. DH-1.RESULTS: A novel methanotroph strain was isolated from activated sludge from a brewery plant and characterized using phylogenetic analysis, electron microscopy and chemotaxonomic analysis. This aerobic, Gram-negative, non-motile rod-shaped type I methanotroph was designated as Methylomonas sp. DH-1. The growth condition of Methylomonas sp. DH-1 and batch methane-to-methanol bioconversion conditions such as methane concentration, pH, biocatalyst loading, concentration of formate and MDH inhibitor were analyzed and optimized. Methanol was produced from methane with a 1.340 g L-1 titer, a 0.332 g L-1 h(-1) volumetric conversion rate and a 0.0752 g g(-1) cell h(-1) specific methanol conversion rate.CONCLUSION: It was demonstrated that isolation and application of a new methanotroph strain is a practical way of improving bioconversion efficiency in the conversion of methane to methanol. Moreover, one promising feature of Methylomonas sp. DH-1 for methanol production was its extremely high tolerance to methanol up to 7%(v/v), which is advantageous for high-titer methanol production. (C) 2016 Society of Chemical Industry

Propane is the major component of liquefied petroleum gas (LPG). Nowadays, the use of LPG is decreasing, and thus utilization of propane as a chemical feedstock is in need of development. An efficient biological conversion of propane to acetone using a methanotrophic whole cell as the biocatalyst was proposed and investigated. A bio-oxidation pathway of propane to acetone in Methylomonas sp. DH-1 was analyzed by gene expression profiling via RNA sequencing. Propane was oxidized to 2-propanol by particulate methane monooxygenase and subsequently to acetone by methanol dehydrogenases. Methylomonas sp. DH-1 was deficient in acetone-converting enzymes and thus accumulated acetone in the absence of any enzyme inhibition. The maximum accumulation, average productivity and specific productivity of acetone were 16.62 mM, 0.678 mM/h and 0.141 mmol/g cell/h, respectively, under the optimized conditions. Our study demonstrates a novel method for the bioconversion of propane to acetone using methanotrophs under mild reaction condition.

Methane-assimilating bacteria, methanotrophs, can play an important role in producing various value-added chemicals and biofuels from methane, which is considered a next-generation carbon feedstock. The capability to engineer the metabolic pathway of methanotrophs is a key success factor for enhancing methane-to-product conversion efficiency. Recently, OMICS studies on several model methanotrophs have been conducted and provided strategies to engineer methanotrophs. Here, we present a review on the current progresses and future prospects of metabolic engineering of methanotrophs and its application to chemical and biofuel production from methane. (c) 2016 Society of Chemical Industry and John Wiley & Sons, Ltd

Backgound:ligament advanced reinforcement system is a graft choice for ligament reconstruction,we used ligament advanced reinforcement system Y-shape double bundles to reconstruction the posterior cruciate ligament,it's necessary to know the clinical effect of this technique. Method:A retrospective analysis was conducted among 11 patients with posterior cruciate ligament injuries, who were enrolled from Department of Orthopaedics in Guangzhou General Hospital of Guangzhou Military Area Command of Chinese PLA from January to July in 2007. Arthroscopic reconstructions of posterior cruciate ligament were performed by using the ligament advanced reinforcement system (LARS) Y-shape bundles artificial ligament. Y-shape double cords of LARS artificial ligament were fixed at 90°and 30° flexion respectively. All 11 patients received the follow-ups. Result:There were no complications occurred, such as synovitis, ligament rupture or limited motion. X-ray results proved the extrused screws were positioned well. Posterior drawer test, Lachman’s test and pivot shift test were negative after surgery. According to the international knee documentation committee activity grades, grade D were 8 cases and grade C were 3 cases preoperatively; while grade A were 10 cases and grade B was 1 case postoperatively, with the significant differences (χ2=9.142, P < 0.05). The clinical assessment judging by lysholm knee functional scoring system showed that, average scores before operation was (65.2±5.6) and increased to an average of (90.7±3.6) postoperatively (t=10.572, P < 0.05). Conclusion:LARS artificial ligament is good grafts for posterior cruciate ligament reconstruction, can do early functional exercise and have good clinical effect. Huang Huayang, E⁃mail: johnhzy@sina.com tel:+86 13802968969 地址:中国 广东省广州市流花路 111 号广州军区广州总医院 1 号楼 5 楼骨病关

The purpose of this study was to analyse the factors affecting the conversion from posterior cruciate ligament (PCL)-retaining (CR) to PCL-substituting (PS) prostheses during total knee arthroplasty (TKA).

Computer assisted surgery (CAS) is used to improve accuracy of neutral mechanical axis and ligament balancing in primary TKA. However, its efficiency in revision TKA is not clear. The application of CAS in revision TKA provide a significant improvement of prosthesis alignment, balanced flexion and extension gaps, data about joint line position throughout the surgery. However, it requires further investigations about long-term clinical results and survival rate.

BACKGROUND:The purpose of the present study was to compare the clinical and radiographic results and survival rates between computer-assisted and conventional closing wedge high tibial osteotomies (HTOs).METHODS:Data from a consecutive cohort comprised of 75 computer-assisted HTOs and 75 conventional HTOs were retrospectively reviewed. The Knee Society knee and function scores, Hospital for Special Surgery (HSS) score and femorotibial angle (FTA) were compared between the two groups. Survival rates were also compared with procedure failure.RESULTS:The knee and function scores at one year postoperatively were slightly better in the computer-assisted group than those in conventional group (90.1 vs. 86.1) (82.0 vs. 76.0). The HSS scores at one year postoperatively were slightly better for the computer-assisted HTOs than those of conventional HTOs (89.5 vs. 81.8). The inlier of the postoperative FTA was wider in the computer-assisted group than that in the conventional HTO group (88.0% vs. 58.7%), and mean postoperative FTA was greater in the computer-assisted group that in the conventional HTO group (valgus 9.0° vs. valgus 7.6°, p<0.001). The five- and 10-year survival rates were 97.1% and 89.6%, respectively. No difference was detected in nine-year survival rates (p=0.369) between the two groups, although the clinical and radiographic results were better in the computer-assisted group that those in the conventional HTO group.CONCLUSIONS:Mid-term survival rates did not differ between computer-assisted and conventional HTOs. A comparative analysis of longer-term survival rate is required to demonstrate the long-term benefit of computer-assisted HTO.LEVEL OF EVIDENCE:III.

Recently, methane has attracted much attention as an alternative carbon feedstock since it is the major component of abundant shale and natural gas. In this work, we produced methanol from methane using whole cells of Methylosinus trichosporium OB3b as the biocatalyst. M. trichosporium OB3b was cultured on NMS medium with a supply of 7:3 air/methane ratio at 30°C. The optimal concentrations of various methanol dehydrogenase inhibitors such as potassium phosphate and EDTA were determined to be 100 and 0.5 mM, respectively, for an efficient production of methanol. Sodium formate (40 mM) as a reducing power source was added to enhance the conversion efficiency. A productivity of 49.0 mg/l·h, titer of 0.393 g methanol/l, and conversion of 73.8% (mol methanol/mol methane) were obtained under the optimized batch condition.