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  • The recombinant Scl collagen system has

    2020-03-20

    The recombinant Scl2 collagen system has shown capability as a biomaterial as well because of its adaptability and scalability. Scl2 was functionalized to crosslink into a hydrogel without disrupting its triple helix [130]. The Scl2–hydrogel crosslinking also did not disrupt cell adhesion and integrin binding when the α1 and α2 integrin binding motifs of collagen were inserted into the Scl2 [130]. This Scl2–hydrogel has been incorporated in the development of a vascular graft with suitable biomechanical properties [144] and in the development of an injectable medicine to stimulate chondrogenesis [145]. Additionally, a high-throughput batch purification methodology for Scl2 recombinant collagen has been developed [146]. This methodology has shown to be very scalable and produce a high percentage (>95%) of pure protein [146,147]. As our understanding of DDR–collagen interactions advances, it shall be possible to engineer biomaterials with DDR-activating or inhibiting properties.
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    Acknowledgements We thank Prof. Barbara Brodsky for valuable comments and discussion. We thank the support of the Tufts start-up fund and the Knez Family Faculty Investment Fund for Y.-S. L, and the Tufts Summer Scholar program for E.C.
    -4-Hydroxy--proline (Hyp) is a valuable amino gw501516 acted as a constituent of pharmaceutical intermediates such as echinocandins , etamycin and actinomycins . It has traditionally been produced by acid hydrolysis of animal collagen, a complex process which has a low recovery rate and generates a large volume of waste water . To overcome these problems, microbial production of Hyp is regarded as a promising method due to its economic advantages and environmentally-benign features . Using this method, -proline is hydroxylated to Hyp by proline-4-hydroxylase (P4H), which was initially discovered in the etamycin biosynthesis pathway of P8648 . The P4H gene from sp. RH1 has since been cloned and expressed in strain W1485 , as well as , . P4H catalyzes hydroxylation of proline at the -4-position, with the co-substrate of α-ketoglutarate undergoing oxidative decarboxylation to succinate. WD3 lacking α-ketoglutarate dehydrogenase activity cannot grow in minimal medium due to blockage of the tricarboxylic acid (TCA) cycle during succinate synthesis. P4H activity in recombinant was able to restart the blocked TCA cycle, thereby coupling -proline hydroxylation with cell growth. Due to the catalytic characteristics of P4H, production of Hyp is a high-oxygen-demand process. In addition, fermentation broths for Hyp production will exhibit high viscosity during culture , , which can further hinder oxygen transfer. Therefore, oxygen supply is generally regarded as one of the main limiting factors in the Hyp bioproduction process by recombinant whole cells . Conventionally, oxygen transfer for microbial fermentation can be improved by increasing the agitation and aeration rates , but this will lead to high energy consumption and cause physical damage to the cells . hemoglobin (VHb) is an oxygen-binding protein with an oxygen dissociation rate constant of 5600 s, which is hundreds of times higher than other hemoglobins. VHb enhances respiration and energy metabolism by promoting oxygen transfer to the intracellular terminal oxidases under oxygen-limited conditions in high cell density fermentation processes , , . Since the VHb gene () was first cloned in 1988 and its native oxygen-dependent promoter was characterized in in 1989 , it has been widely used in recombinant strains to improve growth and production of target compounds by inducing expression of , , , , , , , , , , , , , (). However, to the best of our knowledge, there are no reports on the application of VHb together with P4H for Hyp production in recombinant . In this study, the gene under the control of the native promoter was integrated into the chromosome of expressing P4H to improve Hyp production. The effect of VHb expression on Hyp production in the recombinant strain was examined in shaker flask culture with different loading volumes. The results showed that VHb expression helped to increase cell growth and Hyp production by alleviating oxygen limitation. The P4H-expressing strain with expression of VHb was further cultivated in a 1.4 L bioreactor to validate the effectiveness of VHb for improving Hyp production.