Enhancing yield of S-adenosylmethionine in Pichia pastoris by controlling NH4 + concentration

Zhang, Jianguo; Wang, Xuedong; Zheng, Yu; Fang, Guochen; Wei, Dongzhi

doi:10.1007/s00449-007-0146-8

Cited by 18 publications

(6 citation statements)

References 12 publications

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“…[18] DCC was chosen for encapsulation and coating because of previous studies citing cartilage matrix as a potentially chondroinductive material. [13,15,16,31,32] The use of decellularized cartilage instead of native or devitalized cartilage is advantageous from clinical and commercial standpoints because of decreased immunogenicity and long-term storage of the material. Successful decellularization of tissues, i.e., complete removal of residual DNA and immunogenic antigens, may also eventually lead to safer xenogeneic tissue implants for all tissue types.…”

Section: Discussionmentioning

confidence: 99%

“…[8][9][10][11] Previous studies have reported adipose derived stem cell (ASC) and bone marrow-derived mesenchymal stem cell (BMSC) differentiation in the presence of DCC. [3,12,13] One challenge with DCC-based scaffolds is that the mechanical function of the scaffolds may be compromised during the decellularization process. [4,11,14] To help fabricate a material with a compressive strength suitable for articular cartilage repair, combining DCC with a polymeric scaffold has previously been shown to achieve greater mechanical performance than DCC scaffolds alone.…”

Section: Introductionmentioning

confidence: 99%

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Bioactive Microsphere‐Based Scaffolds Containing Decellularized Cartilage

Sutherland

Detamore

2015

Macromolecular Bioscience

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The aim of this study was to fabricate mechanically functional microsphere-based scaffolds containing decellularized cartilage (DCC), with the hypothesis that this approach would induce chondrogenesis of rat bone marrow-derived mesenchymal stem cells (rBMSCs) in vitro. The DCC was derived from porcine articular cartilage and decellularized using a combination of physical and chemical methods. Four types of scaffolds were fabricated: Poly(D,L-lactic-co-glycolic acid) (PLGA) only (negative control), TGF-β encapsulated (positive control), PLGA surface coated with DCC, and DCC-encapsulated. These scaffolds were seeded with rBMSCs and cultured up to 6 weeks. The compressive modulus of the DCC-coated scaffolds prior to cell seeding was significantly lower than all other scaffold types. Gene expression was comparable between DCC-encapsulated and TGF-β encapsulated groups. Notably, DCC-encapsulated scaffolds contained 70% higher glycosaminoglyan (GAG) content and 85% more hydroxyproline compared to the TGF-β group at week 3 (with baseline levels subtracted out from acellular DCC scaffolds). Certainly bioactivity was demonstrated in eliciting a biosynthetic response from the cells with DCC, although true demonstration of chondrogenesis remained elusive under the prescribed conditions. Encapsulation of DCC appeared to lead to improved cell performance relative to coating with DCC, although this finding may be a dose-dependent observation. Overall, DCC introduced via microsphere-based scaffolds appears to be promising as a bioactive approach to cartilage regeneration, although additional studies will be required to conclusively demonstrate chondroinductivity.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Bioactive Microsphere‐Based Scaffolds Containing Decellularized Cartilage

Sutherland

Detamore

2015

Macromolecular Bioscience

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“…Hematoxylin and eosin staining (H&E), immunohistochemistry (IHC), SEM imaging, mass spectrometry, ELISA, and quantitative DNA assays have confirmed the reduction of cells, cell fragments, cell‐associated proteins, and nucleic acids in chemically decellularized cartilage. [10a,c,d],, Chemical decellularization methods such as 2% SDS treatment for 2 h or tritonX‐100, EDTA and nuclease treatment have also shown that the DCC can retain collagen II and GAGs in the material and has been confirmed by immunofluorescent staining, histological staining, 1‐9‐dimethylmethylene blue (DMMB) sulfated GAG assay, and chloramine‐T hydroxyproline assay . The amount of GAG retained in DCC, however, significantly decreased with increasing chemical decellularization, while collagen II levels did not significantly decrease .…”

Section: Native Cartilage Matrixmentioning

confidence: 99%

“… Various formulations of DNases and RNases are also commonly used to remove nucleic acids from the material. [10d], Many chemical decellularization protocols encompass some combination of these chemicals. Because of the dense nature of articular cartilage, to improve the efficiency of chemical decellularization the native macrostructure must often be disrupted, which allows the material to be more effectively exposed to the chemical decellularization agents for shorter amounts of time .…”

Section: Native Cartilage Matrixmentioning

confidence: 99%

The Bioactivity of Cartilage Extracellular Matrix in Articular Cartilage Regeneration

Sutherland

Converse

Hopkins

et al. 2014

Adv Healthcare Materials

153

173

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Cartilage matrix is a particularly promising acellular material for cartilage regeneration given the evidence supporting its chondroinductive character. The ‘raw materials’ of cartilage matrix can serve as building blocks and signals for enhanced tissue regeneration. These matrices can be created by chemical or physical methods: physical methods disrupt cellular membranes and nuclei but may not fully remove all cell components and DNA, whereas chemical methods when combined with physical methods are particularly effective in fully decellularizing such materials. Critical endpoints include no detectable residual DNA or immunogenic antigens. It is important to first delineate between the sources of the cartilage matrix, i.e., derived from matrix produced by cells in vitro or from native tissue, and then to further characterize the cartilage matrix based on the processing method, i.e., decellularization or devitalization. With these distinctions, four types of cartilage matrices exist: decellularized native cartilage (DCC), devitalized native cartilage (DVC), decellularized cell derived matrix (DCCM), and devitalized cell derived matrix (DVCM). Delivery of cartilage matrix may be a straightforward approach without the need for additional cells or growth factors. Without additional biological additives, cartilage matrix may be attractive from a regulatory and commercialization standpoint. Source and delivery method are important considerations for clinical translation. Only one currently marketed cartilage matrix medical device is decellularized, although trends in filed patents suggest additional decellularized products may be available in the future. To choose the most relevant source and processing for cartilage matrix, qualifying testing needs to include targeting the desired application, optimizing delivery of the material, identify relevant FDA regulations, assess availability of raw materials, and immunogenic properties of the product.

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“…SAM2 gene was expressed and cystathione-β-synthase gene was knocked out from P. pastoris for production of SAM (He et al 2006; Yu and Shen 2012). Enhanced accumulation of SAM was achieved by manipulating the culture conditions like increased oxygen levels and nitrogen source (Chen et al 2007; Zhang et al 2008a; Zhang et al 2008b; Chu et al 2013). Earlier studies reported elevated intracellular production of SAM using recombinant P. pastoris; however, these reports did not confirm the expression of the enzyme by SDS-PAGE analyses, and molecular characterization of the accumulated SAM using MS/MS method.…”

Section: Introductionmentioning

confidence: 99%

Engineered Pichia pastoris for enhanced production of S-adenosylmethionine

et al. 2013

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A genetically engineered strain of Pichia pastoris expressing S-adenosylmethionine synthetase gene from Saccharomyces cerevisiae under the control of AOX 1 promoter was developed. Induction of recombinant strain with 1% methanol resulted in the expression of SAM2 protein of ~ 42 kDa, whereas control GS115 showed no such band. Further, the recombinant strain showed 17-fold higher enzyme activity over control. Shake flask cultivation of engineered P. pastoris in BMGY medium supplemented with 1% L-methionine yielded 28 g/L wet cell weight and 0.6 g/L S-adenosylmethionine, whereas control (transformants with vector alone) with similar wet cell weight under identical conditions accumulated 0.018 g/L. The clone cultured in the bioreactor containing enriched methionine medium showed increased WCW (117 g/L) as compared to shake flask cultures and yielded 2.4 g/L S-adenosylmethionine. In spite of expression of SAM 2 gene up to 90 h, S-adenosylmethionine accumulation tended to plateau after 72 h, presumably because of the limited ATP available in the cells at stationery phase. The recombinant P pastoris seems promising as potential source for industrial production of S-adenosylmethionine.

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Enhancing yield of S-adenosylmethionine in Pichia pastoris by controlling NH4 + concentration

Cited by 18 publications

References 12 publications

Bioactive Microsphere‐Based Scaffolds Containing Decellularized Cartilage

Bioactive Microsphere‐Based Scaffolds Containing Decellularized Cartilage

The Bioactivity of Cartilage Extracellular Matrix in Articular Cartilage Regeneration

Engineered Pichia pastoris for enhanced production of S-adenosylmethionine

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