Sun-Ki Kim

Enhanced production of 3-hydroxypropionic acid from glycerol by modulation of glycerol metabolism in recombinant Escherichia coli

3-Hydroxypropionic acid (3-HP) is a valuable biochemical with high potential for bioplastic manufacturing. The endogenous glycerol metabolism and by-product formation pathway in Escherichia coli were modulated to enhance 3-HP production from glycerol. Double deletion of glpK and yqhD directed the glycerol flux to 3-HP biosynthesis and reduced the formation of 1,3-propanediol. Since 3-hydroxypropionaldehyde (3-HPA), a precursor of 3-HP, is toxic to cell growth, the gene encoding Pseudomonas aeruginosa semialdehyde dehydrogenase (PSALDH) highly active on 3-HPA was expressed in E. coli. Finally, fed-batch culture of recombinant E. coli BL21star(DE3) without glpK and yqhD, and expressing Lactobacillus brevis DhaB-DhaR, and P. aeruginosa PSALDH resulted in 57.3g/L 3-HP concentration, 1.59g/L-h productivity and 0.88g/g yield. In conclusion, modulation of the glycerol metabolism in combination with enhanced activity of 3-HPA dehydrogenation improved the production of 3-HP from glycerol.

A multifunctional protein EWS regulates the expression of Drosha and microRNAs

EWS (Ewing’s Sarcoma) gene encodes an RNA/DNA-binding protein that is ubiquitously expressed and involved in various cellular processes. EWS deficiency leads to impaired development and early senescence through unknown mechanisms. We found that EWS regulates the expression of Drosha and microRNAs (miRNAs). EWS deficiency resulted in increased expression of Drosha, a well-known microprocessor, and increased levels of miR-29b and miR-18b. Importantly, miR-29b and miR-18b were directly involved in the post-transcriptional regulation of collagen IV alpha 1 (Col4a1) and connective tissue growth factor (CTGF) in EWS knock-out (KO) mouse embryonic fibroblast cells. The upregulation of Drosha, miR-29b and miR-18b and the sequential downregulation of Col4a1 and CTGF contributed to the deregulation of dermal development in EWS KO mice. Otherwise, knockdown of Drosha rescued miRNA-dependent downregulation of Col4a1 and CTGF proteins. Taken together, our data indicate that EWS is involved in post-transcriptional regulation of Col4a1 and CTGF via a Drosha–miRNA-dependent pathway. This finding suggests that EWS has a novel role in dermal morphogenesis through the modulation of miRNA biogenesis.

Simple amino acid tags improve both expression and secretion of Candida antarctica lipase B in recombinant Escherichia coli

Escherichia coli is the best‐established microbial host strain for production of proteins and chemicals, but has a weakness for not secreting high amounts of active heterologous proteins to the extracellular culture medium, of which origins belong to whether prokaryotes or eukaryotes. In this study, Candida antarctica lipase B (CalB), a popular eukaryotic enzyme which catalyzes a number of biochemical reactions and barely secreted extracellularly, was expressed functionally at a gram scale in culture medium by using a simple amino acid‐tag system of E. coli. New fusion tag systems consisting of a pelB signal sequence and various anion amino acid tags facilitated both intracellular expression and extracellular secretion of CalB. Among them, the N‐terminal five aspartate tag changed the quaternary structure of the dimeric CalB and allowed production of 1.9 g/L active CalB with 65 U/mL activity in culture medium, which exhibited the same enzymatic properties as the commercial CalB. This PelB‐anion amino acid tag‐based expression system for CalB can be extended to production of other industrial proteins hardly expressed and exported from E. coli, thereby increasing target protein concentrations and minimizing purification steps. Biotechnol. Bioeng. 2015;112: 346–355.

EXPRESSION OF HEAT SHOCK PROTEIN 70 BLOCKS THYMIC DIFFERENTIATION OF T CELLS IN TRANSGENIC MICE

Heat shock protein 70 (HSP70) is involved not only in protein folding, but also in processes of differentiation and cell‐cycle progression. Recently, HSP70 has been implicated in mediation of functions of some immunosuppressive agents. To study the role of HSP70 in differentiation of haematopoietic cells, we generated transgenic mice using the human inducible hsp70 gene fused to the mouse H‐2K promoter. These mice develop a T‐cell deficiency that is characterized by thymic hypoplasia and a significant reduction in peripheral T cells. The total number of thymocytes is about 100‐fold less than that in normal mice. The majority of the thymocytes are immature T cells that express neither CD4 nor CD8 molecules, indicating that T cells are affected at an early stage of thymic differentiation. Expression of the transgenic HSP70 was detected both in bone marrow cells and in thymocytes. Furthermore, injection of normal bone marrow cells into the T‐cell deficient mice led to the generation of mature T cells indicating that the T‐cell deficiency was caused by the action of HSP70 in T cells. The blockage of differentiation occurred only in T cells, both αβ‐ and γδ‐T‐cell receptor (TCR)‐bearing cells, but not in B cells, granulocytes, and monocytes. The observations suggest that HSP70 may inhibit a cellular process that is essential for the differentiation of early stage T cells. Further experiments using this model system will widen our understanding of HSP70 and its function on a molecular level.

Application of repeated aspartate tags to improving extracellular production of Escherichia coli l-asparaginase isozyme II

Asparaginase isozyme II from Escherichia coli is a popular enzyme that has been used as a therapeutic agent against acute lymphoblastic leukemia. Here, fusion tag systems consisting of the pelB signal sequence and various lengths of repeated aspartate tags were devised to highly express and to release active asparaginase isozyme II extracellularly in E. coli. Among several constructs, recombinant asparaginase isozyme II fused with the pelB signal sequence and five aspartate tag was secreted efficiently into culture medium at 34.6 U/mg cell of specific activity. By batch fermentation, recombinant E. coli produced 40.8 U/ml asparaginase isozyme II in the medium. In addition, deletion of the gspDE gene reduced extracellular production of asparaginase isozyme II, indicating that secretion of recombinant asparaginase isozyme II was partially ascribed to the recognition by the general secretion machinery. This tag system composed of the pelB signal peptide, and repeated aspartates can be applied to extracellular production of other recombinant proteins.