Production of capsaicinoid nonivamide from plant oil and vanillylamine via whole-cell biotransformation.

Capsaicinoids are mostly derived from chili peppers and have widespread applications in food, feed, and pharmacology. Compared with plant extraction, the use of microbial cell factories for capsaicinoids production is considered as a more efficient approach. Here, the biotransformation of renewable plant oil and vanillylamine into capsaicinoid nonivamide was investigated. Nonivamide biosynthesis using nonanoic acid and vanillylamine as substrates was achieved in Escherichia coli by heterologous expression of genes encoding amide-forming N-acyltransferase and CoA-ligase. Through increasing nonanoic acid tolerance of chassis cell, screening key enzymes involved in nonivamide biosynthesis and optimizing biotransformation conditions, the nonivamide titer reached 0.5 g/L. By further integrating a route for conversion of oleic acid to nonanoic acid, nonivamide biosynthesis was finally achieved using olive oil and vanillylamine as substrates, yielding a titer of approximately 10.7 mg/L. Results from this study provide valuable information for constructing highly efficient cell factories for the production of capsaicinoid compounds.

Engineering Escherichia coli for efficient assembly of heme proteins.

BACKGROUND: Heme proteins, such as hemoglobin, horseradish peroxidase and cytochrome P450 (CYP) enzyme, are highly versatile and have widespread applications in the fields of food, healthcare, medical and biological analysis. As a cofactor, heme availability plays a pivotal role in proper folding and function of heme proteins. However, the functional production of heme proteins is usually challenging mainly due to the insufficient supply of intracellular heme. RESULTS: Here, a versatile high-heme-producing Escherichia coli chassis was constructed for the efficient production of various high-value heme proteins. Initially, a heme-producing Komagataella phaffii strain was developed by reinforcing the C4 pathway-based heme synthetic route. Nevertheless, the analytical results revealed that most of the red compounds generated by the engineered K. phaffii strain were intermediates of heme synthesis which were unable to activate heme proteins. Subsequently, E. coli strain was selected as the host to develop heme-producing chassis. To fine-tune the C5 pathway-based heme synthetic route in E. coli, fifty-two recombinant strains harboring different combinations of heme synthesis genes were constructed. A high-heme-producing mutant Ec-M13 was obtained with negligible accumulation of intermediates. Then, the functional expression of three types of heme proteins including one dye-decolorizing peroxidase (Dyp), six oxygen-transport proteins (hemoglobin, myoglobin and leghemoglobin) and three CYP153A subfamily CYP enzymes was evaluated in Ec-M13. As expected, the assembly efficiencies of heme-bound Dyp and oxygen-transport proteins expressed in Ec-M13 were increased by 42.3-107.0% compared to those expressed in wild-type strain. The activities of Dyp and CYP enzymes were also significantly improved when expressed in Ec-M13. Finally, the whole-cell biocatalysts harboring three CYP enzymes were employed for nonanedioic acid production. High supply of intracellular heme could enhance the nonanedioic acid production by 1.8- to 6.5-fold. CONCLUSION: High intracellular heme production was achieved in engineered E. coli without significant accumulation of heme synthesis intermediates. Functional expression of Dyp, hemoglobin, myoglobin, leghemoglobin and CYP enzymes was confirmed. Enhanced assembly efficiencies and activities of these heme proteins were observed. This work provides valuable guidance for constructing high-heme-producing cell factories. The developed mutant Ec-M13 could be employed as a versatile platform for the functional production of difficult-to-express heme proteins.

Reconstruction of the main phytoplankton population off the Changjiang Estuary in the East China Sea and its assemblage shift in recent decades: From observations to simulation.

Under eutrophication background, the increasing dinoflagellates blooms relative to diatoms blooms off the Changjiang Estuary has caused much concern. We have provided sediment evidence for the first time that the time window of diatoms-to-dinoflagellates shift off the Changjiang Estuary in the East China Sea is early 1990s. Investigations to the water column revealed different surface-bottom concentration matchup patterns between peridinin (dinoflagellates) and fucoxanthin (diatoms), which suggests that the diatoms-dinoflagellates shift recorded in the sediment may have come from more dinoflagellate blooms since 1990s. Physical-biogeochemical 3D numerical simulations for the past decades suggest that the effect of increasing spring sea surface temperature and increasing N/P ratio on the diatoms-dinoflagellates shift is dominant and recessive, respectively.

Overlooked contribution of water column to nitrogen removal in estuarine turbidity maximum zone (TMZ).

Estuarine systems are important sites of eliminating reactive nitrogen (N) delivered from land to sea. Numerous studies have focused on N cycling in estuarine sediment. However, the N elimination role of suspended sediments in estuarine turbid water column, which might provide anaerobic microenvironment for N loss, is rarely considered. This study examined the community dynamics and activities of denitrifying and anaerobic ammonium oxidation (anammox) bacteria in the water column of the turbidity maximum zone (TMZ) of the Yangtze Estuary, using molecular and 15N isotope-tracing techniques. Results showed that the anammox bacterial community was dominated by Candidatus Kuenenia and Candidatus Brocadia in the TMZ water column, while the main nirS-harboring denitrifiers were affiliated with Rhodobacterales. The denitrifying nirS gene was two orders of magnitude more abundant than anammox bacterial 16S rRNA gene, ranging from 1.77 × 105 to 1.42 × 108 copies l-1 and from 7.68 × 104 to 4.27 × 106 copies l-1, respectively. Compared with anammox, denitrification, with rates of 0.88 to 20.83 µmol N l-1 d-1, overwhelmingly dominated the N removal in the TMZ water column and was significantly correlated to suspended sediment concentrations (SSC). Based on the measured N removal rates, it was estimated that about 2.5 × 105 ton N was annually removed from the TMZ water column, accounting for approximately 18.5% of the total inorganic N (TIN) discharged from the Yangtze River. Overall, this study implies the importance of estuarine turbid water column in controlling N budget, and also improves the understanding of N loss mechanisms in estuarine TMZ systems.

Characterization, stability improvement, and bread baking applications of a novel cold-adapted glucose oxidase from Cladosporium neopsychrotolerans SL16.

Glucose oxidases are widely used in various industrial processes, including bread baking. In this study, a novel glucose oxidase gene, CngoxA, from Cladosporium neopsychrotolerans SL16, was cloned and expressed in Pichia pastoris. Recombinant CnGOXA exhibited maximal activity at 20 °C and pH 7.0, and was stable at 30 °C and pH 6.0-9.0 for 1 h, with a half-life of 15 min at 40 °C. Compared with CnGOXA, the half-life of its mutant CnGOXA-M1 (Y169C-A211C), at 40 °C increased approximately 48-fold, and was stable at 30 °C and pH 3.0-12.0 for 1 h. The kcat and catalytic efficiency of CnGOXA-M1 were enhanced 0.7- and 1.6-fold, respectively. Both enzymes were cold-adapted and highly resistant to SDS. Furthermore, CnGOXA-M1 had a more significant effect on bread volume than that of GOX from Aspergillus niger. These favorable enzymatic properties of CnGOXA-M1 make it a potentially useful enzyme for many industrial applications.

Thermostability improvement of a Talaromyces leycettanus xylanase by rational protein engineering.

Thermophilic xylanases with high catalytic efficiency are of great interest in the biofuel, food and feed industries. This study identified a GH11 xylanase gene, Tlxyn11B, in Talaromyces leycettanus JCM12802. Recombinant TlXyn11B produced in Pichia pastoris is distinguished by high specific activity (8259 ± 32 U/mg with beechwood xylan as substrate) and excellent pH stability (from 1.0 to 10.5). The beechwood xylan hydrolysates consisted mainly of xylobiose, xylotriose and xylotetraose, thus TlXyn11B could be used for the production of prebiotic xylooligosaccharide. By using the structure-based rational approach, the N-terminal sequence of TlXyn11B was modified for thermostability improvement. Mutants S3F and S3F/D35V/I/Q/M had elevated T m values of 60.01 to 67.84 °C, with S3F/D35I the greatest. Homology modeling and molecular dynamics (MD) simulation analysis revealed that the substituted F3 and I35 formed a sandwich structure with S45 and T47, which may enhance the overall structure rigidity with lowered RMSD values. This study verifies the efficiency of rational approach in thermostability improvement and provides a xylanase candidate of GH11 with great commercialization potential.

[Clinical application of advanced percutaneous vertebroplasty in treating of senile osteoporotic vertebral compression fracture].

OBJECTIVE: To compare the differences between advanced percutaneous vertebroplasty and routine percutaneous vertebroplasty,and evaluate the clinical application of advanced percutaneous vertebroplasty in treating senile osteoporotic vertebral compression fracture. METHODS: From January 2010 to January 2011,28 patients with senile osteoporotic vertebral compression fracture were concealed random allocated to advanced percutaneous vertebroplasty and routine percutaneous vertebroplasty group according to whether using advanced percutaneous pedicle puncture. Of them,there were 5 males and 23 females with an average age of (70.50 +/- 9.33) years old (ranged, 53 to 85). Course of disease was from 2 to 28 days with an average of (7.62 +/- 4.52). The fracture involved 34 vertebral bodies (19 segments of thoracic vertebrae and 15 segments of lumbar vertebra), 3 day's antibiotics were applied to prevent infections. The opreation time, X-ray fluoroscopy times were compared, and pain relief effect were evaluated by VAS (visual analogue scales) score. RESULTS: For operation time, there were significant meaning between routine group (49.00 +/- 7.74) min and advanced group (32.36 +/- 4.81) min (t = 6.828,P = 0.000 < 0.05); X-ray fluoroscopy times in advanced group (4.28 +/- 1.38) times was shorter than routine group (8.78 +/- 2.33) times, and had statistical significance (t = 6.222, P = 0.000 < 0.05); while there were no meaning in VAS scores between routine group (2.85 +/- 0.94) and advanced group (2.57 +/- 1.08) (t = 0.740, P = 0.456 > 0.05). CONCLUSION: Compared with routine group, advanced group can shorten opreration time, reduce radiological hazard, further reduce image monitoring costs and surgical risk, and benefit from the application of advanced percutaneous vertebroplasty in treating senile osteoporotic vertebral compression fracture. Furthmore, it can provide reference to vertebral body biopsy and minimally invasive of pedicle screw fixation.

[Combined procedure for the treatment of ankle and foot deformities secondary to spina bifida].

OBJECTIVE: To study surgical strategies for ankle and foot deformities secondary to spina bifida and treatment methods for different types of deformities. METHODS: From January 1990 and July 2009, 107 patients with ankle and foot deformities secondary to spina bifida were retrospectively analyzed. There were 44 males and 63 females, with an average age of 17.7 years (rangd from 1.3 to 52 years). Among the patients, 58 patients had double ankle deformities, 49 patients had unilateral deformities (22 cases on the left side, 27 cases on the right). Ninety-nine patients with equinus deformities were treated by achilles tendon lengthening and tendon transfering; 25 patients with talipes were treated by release of anterior tendon of ankle and tendon transfer; 17 patients with valgus and varus deformities were treated by tendon transfer and calcaneal osteotomy; 15 patients with flail deformities were managed treated by bone fusion between calcaneus and talus and shortening of achilles tendon; 9 patients with claw toe deformities were treated by bone fusion of interphalangeal joint or Ilizarovs distraction. AOFAS (American Orthopaedic Foot & Ankle Society) comprehensive scoring system was used to evaluate subjective pain and objective functional. RESULTS: Seventy-nine (127 feet) of 107 patients were followed up, and the duration ranged from 48 to 180 months (averaged, 64 months). According to AOFAS scoring system, the results were rated as excellent in 89 feet, good in 26 feet, moderate in 9 feet and poor in 3 feet. CONCLUSION: Treatment strategies for ankle-foot deformities mainly included four principles, deformity correction, muscular balance, joint stability and reservation of foot elasticity. Different combined procedure was applied for different foot deformities and received good therapeutic effects.

[Clinical analysis of CT guided unilateral PVP for the treatment of osteoporotic vertebral compression fracture in senile patients].

OBJECTIVE: To evaluate the therapeutic effect and security of CT guided unilateral percutaneous vertebroplasty (PVP) for the treatment of osteoporotic vertebral compression fracture (OVCF) in senile patients. METHODS: From April 2009 to June 2010, 26 patients undergoing CT guided unilateral percutaneous vertebroplasty were analyzed retrospectively. There were 9 males and 17 females,ranging in age from 60 to 85 years with an average of (67.50+/-6.76) years, ranging in course of disease from 2 to 30 days with an average of (8.92+/-4.36) d. The affected segments involved 35 vertebras. The major clinical manifestations of OVCF were lumbar-back pain (especially when turning over or stooping down) and unable to bear. The needle was punctured into vertebral of lesions through unilateral puncture under the CT guidance; and then 3-5 ml bone cement was injected into vertebral. Antibiotic was used 3 days to prevent postoperative infections. Postoperative complications were observed after operation, such as local leakage of bone cement, penetrating spinal cord and/or segmental spinal nerve injuries and pulmonary embolism. X-ray was used to measure the height of anterior, middle and exterior of vertebral before and after treatment. A visual analog scale (VAS) scoring was applied to evaluate pain score preoperative, 48 hours postoperative and the terminal follow-up. RESULTS: Twenty-six patients achieved success in punctuation without serious complications. Local leakage of bone cement occurred in 6 cases, but without clinical symptoms or signs. One patient suffered from acute intraoperative reactions to bone cement and relieved by 5 mg dexamethasone and oxygen. All patients were followed up for 6 to 12 months [averaged (8.4+/-1.6) months]. The postoperative vertebrae height was higher than preoperative,but there was no statistical difference between postoperative and preoperative (P>0.05). Preoperative VAS scores was 7.63+/-0.92, postoperative score was 3.00+/-1.09, the final follow-up score was 2.38+/-1.17; there was significant difference between preoperative and postoperative at 48 hours (P<0.05), but there was no statistical difference between final follow-up and postoperative at 48 hours (P>0.05). CONCLUSION: Unilateral PVP under CT guided can increase the vertebral strength and stabilize vertebral body,and the procedure is a safe and effective method for OVCF in elderly patients.

[Clinical analysis of 107 patients with foot and ankle deformities caused by spinal bifida].

OBJECTIVE: To analyze the incidence, clinical features, deformity categories and orthopedic treatment of foot and ankle deformities caused by spinal bifida. METHODS: The charts of the patients received surgical treatment between January 1990 and July 2009 were studied retrospectively, and the data were analyzed. RESULTS: One hundred and seven cases of foot and ankle deformities caused by spinal bifida received surgical treatment and were included. There were 44 male and 63 female patients. The average age was 17.7 years (range, 1.3 - 52.0 years). And 50.5% (54/107) of cases were over 18 years old and had spinal bifida occulta, and the other 49.5% had spinal bifida manifesta. There was only one case of thoracic spinal bifida (T(3-8)), while the other 106 cases had lumbosacral vertebrae cleft (mainly L(3) to Sacrum). Among a total of 165 feet, unilateral involvement was found in 49 cases (22 cases on the left side, 27 cases on the right side), bilateral involvement in 58 cases. Combined ankle-foot deformities included 76 varus talipes, 23 talipes valgus, 15 flail feet, and 51 other foot deformities. Other site deformities, as a result of spinal bifida, included knee flexion or hyperextension deformity in 4 cases, hip deformity (hip adduction, flexion, or hip dislocation, pelvic tilt, lower extremity discrepancy, etc.) in 17 cases, and urinatory dysfunction and defecation in 30 cases. Twenty-nine of 54 cases with spinal bifida occulta failed to be diagnosed in other hospitals and the misdiagnosed rate reached 53.7% (29/54). Corrective surgery was performed in only 26 patients. And 50.5% (54/107) of patients (over 18 years old) had severe foot and ankle deformities due to a failure of prior surgical treatment. CONCLUSIONS: Spinal bifida is the most commonly found in the lumbosacral vertebrae. Although the main pathogenesis is developmental abnormalities of spinal cord and nerve, the secondary deformity is usually located on the foot and ankle. Some young orthopedic surgeons may not have enough awareness and treatment experience about this disease due to over-specialty of the orthopaedics, so the delay of early diagnosis and treatment is often found and many severe foot and ankle deformities occur.

[Application of traditional Chinese drug soaking, continual irrigation and focus elimination for the treatment of foot osteomyelitis complicated with massive skin defect and denervation].

OBJECTIVE: To evaluate therapeutic effects of traditional Chinese drug soaking,continual irrigation and focus elimination for foot osteomyelitis complicated with massive skin defect and denervation. METHODS: There were 25 males and 28 females in the study. All the patients suffering from foot osteomyelitis complicated with massive skin defect and denervation were treated with comprehensive therapy including preoperative traditional Chinese drug soaking, skin release, focus elimination,continual irrigation, and two-stage suture if necessary. Among the patients, there were 42 cases of left foot and 11 cases of right one. Fifty-one patients complicated with skin defect and sinus tract,only 2 patients complicated with skin defect. There were 48 patients with denervation caused by spina bifida, 5 patients by other causes. RESULTS: The follow-up duration was from 1 to 12 years,the mean time was about 5 years. Osteomyelitis recurred in 3 cases and skin defect reoccurred in original position in 9 cases. CONCLUSION: The methods of traditional Chinese drug soaking, continual irrigation and elimination of focus have good effects for foot osteomyelitis complicated with massive skin defect and denervation. To some degree,this therapy may avoid skin flap transplantation, bone graft and amputation. It deserves further studies and promotion.

Cohort study on the effect of a combined treatment of traditional Chinese medicine and Western medicine on the relapse and metastasis of 222 patients with stage II and III colorectal cancer after radical operation.

OBJECTIVE: To evaluate the effectiveness of a comprehensive therapy of traditional Chinese medicine (TCM) in reducing the relapse and metastasis of stage II and III colorectal cancer based on conventional Western medicine (WM) therapy. METHODS: Two hundred and twenty-two patients in total, diagnosed as stage II and III colorectal cancer from February 2000 to March 2006, were recruited from Xiyuan Hospital, China Academy of Chinese Medical Sciences and the General Hospital of Beijing Military Area. They were followed-up once every 3-6 months. Twenty cases dropped out from the cohort. The remaining 202 patients were all treated with routine WM treatment [including R0 radical operation, or chemotherapy or/and radiotherapy according to national comprehensive cancer network (NCCN) clinical guidelines]. These patients were assigned to two groups based on whether or not they were additionally treated with TCM comprehensive therapy (orally administered with a decoction according to syndrome differentiation, combined with a traditional patent drug over one year). Ninety-eight patients from Xiyuan Hospital were treated with WM and TCM (combined group), and 104 patients from the General Hospital of Beijing Military Area were treated with WM alone (WM group). The demographic data at baseline were comparable, including the operation times, age, sex, TNM staging, and pathological types. The patients were followed-up for one to five years. Up to now, there are 98, 98, 77, 64, and 47 patients with 1, 2, 3, 4, and 5 years of follow-up in the combined group, respectively; and 104, 104, 97, 81, and 55 patients in the WM group, respectively. The results of the 5-year follow-up of all the patients will be available in 2011. RESULTS: The relapse/metastasis rate of 1-, 2-, 3-, 4-, and 5-year were 0 (0/98), 2.04% (2/98), 11.69% (9/77), 14.06% (9/64), and 21.28% (10/47) in the combined group, and were 4.80%(5/104), 16.35% (17/104), 21.65% (21/97), 25.93% (21/81), and 38.18%(21/55) in the WM group, respectively. A significant difference was found in the second year between the two groups (chi (2)=12.117, P=0.000). Median relapse/metastasis time was 26.5 months in the combined group and 16.0 months in the WM group. CONCLUSION: The combined therapy of TCM and WM may have great clinical value and a potential for decreasing the relapse or metastasis rate in stage II and III colorectal cancer after conventional WM therapy.