Integrated pathway engineering and transcriptome analysis for improved astaxanthin biosynthesis in Yarrowia lipolytica.

Astaxanthin is a high value carotenoid with a broad range of commercial applications due to its superior antioxidant properties. In this study, ß-carotene-producing Yarrowia lipolytica XK17 constructed in the lab was employed for astaxanthin biosynthesis. The catalytic effects of ß-carotene ketolase CrtW and ß-carotene hydroxylase CrtZ from various species were investigated. The PspCrtW from Paracoccus sp. and HpCrtZ# from Haematococcus pluvialis were confirmed to be the best combination in converting ß-carotene. Several key bottlenecks in biomass and astaxanthin biosynthesis were effectively eliminated by optimizing the expression of the above enzymes and restoring uracil/leucine biosynthesis. In addition, the effects of astaxanthin biosynthesis on cell metabolism were investigated by integrated analysis of pathway modification and transcriptome information. After further optimization, strain DN30 was able to synthesize up to 730.3 mg/L astaxanthin in laboratory 5-L fermenter. This study provides a good metabolic strategy and a sustainable development platform for high-value carotenoid production.

Chemical and Biological Study of Novel Aplysiatoxin Derivatives from the Marine Cyanobacterium Lyngbya sp.

Since 1970s, aplysiatoxins (ATXs), a class of biologically active dermatoxins, were identified from the marine mollusk Stylocheilus longicauda, whilst further research indicated that ATXs were originally metabolized by cyanobacteria. So far, there have been 45 aplysiatoxin derivatives discovered from marine cyanobacteria with various geographies. Recently, we isolated two neo-debromoaplysiatoxins, neo-debromoaplysiatoxin G (1) and neo-debromoaplysiatoxin H (2) from the cyanobacterium Lyngbya sp. collected from the South China Sea. The freeze-dried cyanobacterium was extracted with liquid-liquid extraction of organic solvents, and then was subjected to multiple chromatographies to yield neo-debromoaplysiatoxin G (1) (3.6 mg) and neo-debromoaplysiatoxin H (2) (4.3 mg). They were elucidated with spectroscopic methods. Moreover, the brine shrimp toxicity of the aplysiatoxin derivatives representing differential structural classifications indicated that the debromoaplysiatoxin was the most toxic compound (half inhibitory concentration (IC50) value = 0.34 ± 0.036 µM). While neo-aplysiatoxins (neo-ATXs) did not exhibit apparent brine shrimp toxicity, but showed potent blocking action against potassium channel Kv1.5, likewise, compounds 1 and 2 with IC50 values of 1.79 ± 0.22 µM and 1.46 ± 0.14 µM, respectively. Therefore, much of the current knowledge suggests the ATXs with different structure modifications may modulate multiple cellular signaling processes in animal systems leading to the harmful effects on public health.

Metabolic engineering of ß-carotene biosynthesis in Yarrowia lipolytica.

OBJECTIVE: Carotenoids, as potent antioxidant compounds, have gained extensive attention, especially in human health. In this study, the combination of CRISPR/Cas9 integration strategy and fermenter cultivation was utilized to obtain efficient ß-carotene-producing Yarrowia lipolytica cell factories for potential industrial application. RESULTS: The introduction of the genes of Mucor circinelloides, encoding phytoene dehydrogenase (carB) and bifunctional phytoene synthase/lycopene cyclase (carRP), contributed to the heterologous production of ß-carotene in Y. lipolytica XK2. Furthermore, ß-carotene production was efficiently enhanced by increasing the copy numbers of the carB and carRP genes and overexpressing of GGS1, ERG13, and HMG, the genes related to the mevalonate (MVA) pathway. Thus, the optimized strain overexpressed a total of eight genes, including three copies of carRP, two copies of carB, and single copies of GGS1, HMG, and ERG13. As a consequence, strain Y. lipolytica XK19 accumulated approximately 408 mg/L ß-carotene in shake flask cultures, a twenty-four-fold increase compared to the parental strain Y. lipolytica XK2. CONCLUSIONS: 4.5 g/L ß-carotene was obtained in a 5-L fermenter through a combination of genetic engineering and culture optimization, suggesting a great capacity and flexibility of Y. lipolytica in the production of carotenoids.

Multicopy integrants of crt genes and co-expression of AMP deaminase improve lycopene production in Yarrowia lipolytica.

Lycopene has been broadly studied in recent decades due to its health benefits including cancer prevention, anti-atherogenic and anti-obesity effects, and modulation of the immune system. To obtain efficient synthesis of lycopene, extensive researches have been conducted in various microbial cells, including Yarrowia lipolytica, to heterologously produce lycopene using various genetic and metabolic engineering methods. In this study, the effects of copy numbers of lycopene synthesis genes, a variety of key central metabolic genes (especially AMP deaminase-encoding gene AMPD), and 5-L fermenter cultivation on lycopene production in Y. lipolytica were investigated and the engineered strains with significantly enhanced lycopene content (46-60 mg/g DCW) were achieved. It is therefore possible to make use of the obtained strains to meet the industrial demand of lycopene production on the basis of further genetic and process optimization.

Complications and clinical outcomes of minimally invasive transforaminal lumbar interbody fusion for the treatment of one- or two-level degenerative disc diseases of the lumbar spine in patients older than 65 years.

BACKGROUND: Minimally invasive transforaminal lumbar interbody fusion (MIS-TLIF) has been successfully used to treat degenerative diseases of the lumbar spine. There are few reports comparing the complications and clinical outcomes in older patients who have undergone one- or two-level MIS-TLIF with those of younger patients. The aim of this study was to investigate the clinical outcomes of MIS-TLIF in the treatment of degenerative disc disease of lumbar spine of the patients older than 65 years, with an emphasis on perioperative complications compared to the younger patients. METHODS: One hundred and fifty-one consecutive cases of one- or two-level degenerative disc disease of lumbar spine treated with MIS-TLIF were reviewed for the radiological and clinical outcomes. They were divided into elderly group (age ≥ 65 years old) and younger group (age < 65 years old), and were followed for at least 6 months. Radiographs were obtained before and after surgery, 3 months postoperatively, and at the final follow-up to determine the presence of fusion, hardware-related problems. The clinical outcomes were evaluated using the Oswestry Disability Index (ODI) before and after surgery, and at the final follow-up. The visual analogue scale (VAS) score of back and leg pain were evaluated as well. The intra-operative data and peri-operative complications were recorded. RESULTS: The mean age of these patients at operation was (57.7 ± 14.2) years (range 26 - 82 years). Of 151 patients, 62 were 65 years or older. The elderly patients had more comorbidities and more porportion of lumbar canal stenosis. The overall fusion rate was 88.4% at the final follow-up, with no significant difference between younger and elderly patients. The ODI, the VAS of back pain and radicular pain of both young and elderly group were significantly improved after surgery and at the final follow-up, without significant difference between two groups. There were 16 complications with an incidence of 10.6%, including 7 major complications and 9 minor complications. There was no significant difference of the incidence of complications between two groups. The incidence of dura tear was significantly related to bilateral deompression. CONCLUSIONS: The clinical and radiological outcomes of MIS-TLIF in the treatment of one- or two-level degenerative disc diseases of lumbar spine in the elderly patients were satisfactory. Though there are more pre-operative comorbidities, with proper patient selection, the elderly patients are not at increased risk of perioperative complications compared to younger patients. Screw malposition and dura tear, which are the most frequent complications, were more related to the surgical technique and should be avoided.

Therapeutic strategy for traumatic instability of subaxial cervical spine.

BACKGROUND: A simple, safe and effective therapeutic strategy for traumatic instability of the subaxial cervical spine, as well as its prognostic assessment, is still controversial. METHODS: The therapeutic options for 83 patients of traumatic instability of the subaxial cervical spine, whose average age was 35 years, were determined, according to the Allen-Ferguson classification, general health and concomitant traumatic conditions, neurological function, position of compression materials, concomitant traumatic disc herniation/damage, concomitant locked-facet dislocation, the involved numbers and position, and the patients' economic conditions. An anterior, posterior or combination approach was used to decompress and reconstruct the cervical spine. No operations with an anterior-posterior-anterior approach were performed. RESULTS: The average follow-up was three years and nine months. Distraction-flexion and compression-flexion were the most frequent injury subtypes. There were 46, 28 and 9 cases of anterior, posterior and combination operations, respectively. The average score of the Japanese Orthopaedics Association, visual analog scale and American Spinal Cord Injury Association (ASIA) motor index improved from 11.2, 7.8 and 53.5, respectively, before operation, to 15.3, 2.6 and 67.8, respectively, at final follow-up. For incomplete spinal cord injury (SCI), the average ASIA neurological function scale was improved by 1-2 levels. Patients with complete SCI had no neurological recovery, but recovery of nerve root function occurred to different extents. After surgery, radiological parameters improved to different extents. Fusion was achieved in all patients and 12 developed complications. CONCLUSIONS: The best surgical strategy should be determined by the type of subaxial cervical injury, patients' general health, local pathological anatomy and neurological function.