Ki Jun Jeong

Isolation of fully synthetic promoters for high-level gene expression in Corynebacterium glutamicum

Corynebacterium glutamicum is an important industrial organism that is widely used in the production of amino acids, nucleotides and vitamins. To extend its product spectrum and improve productivity, C. glutamicum needs to undergo further engineering, including the development of applicable promoter system. Here, we isolated new promoters from the fully synthetic promoter library consisting of 70‐bp random sequences in C. glutamicum. Using green fluorescent protein (GFP) as a reporter, highly fluorescent cells were screened from the library by fluorescent activated cell sorting (FACS). Twenty potential promoters of various strengths were isolated and characterized through extensive analysis of DNA sequences and mRNA transcripts. Among 20 promoters, 6 promoters which have different strengths were selected and their activities were successfully demonstrated using two model proteins (antibody fragment and endoxylanase). Finally, the strongest promoter (PH36) was employed for the secretory production of endoxylanase in fed‐batch cultivation, achieving production levels of 746 mg/L in culture supernatant. This is the first report of synthetic promoters constructed in C. glutamicum, and our screening strategy together with the use of synthetic promoters of various strengths will contribute to the future engineering of C. glutamicum. Biotechnol. Bioeng. 2013;110: 2959–2969.

Secretory production of human leptin in Escherichia coli

Human leptin is a 16 kDa (146 amino acids) protein secreted from adipocytes and influences body weight homeostasis. In this study, human leptin was produced and secreted efficiently in Escherichia coli using a novel Bacillus sp. endoxylanase signal peptide. The endoxylanase signal sequence consisted of 28 amino acids (84 bp) was fused to the leptin structural gene. The fused gene was expressed using an inducible promoter (T7 or Trc) by adding 1 mM IPTG. Using T7 promoter in E. coli BL21(DE3), most of protein produced was in a premature form. Using the Trc promoter, which is weaker than T7, leptin was efficiently produced and secreted as a mature form (40% of total proteins) at 37 degrees C. However, most of leptin (about 90%) formed the inclusion bodies in the periplasmic space of E. coli. At 30 degrees C, ca. 90% of leptin was produced in a soluble form, but the total amount of leptin produced was 40% less than that obtained at 37 degrees C. When the periplasmic oxidoreductase of E. coli, DsbA, was co-expressed, 69% of the secreted leptin (26% of total proteins) was produced as soluble form at 37 degrees C without the decrease of the amount of leptin produced.

Engineering Escherichia coli for Increased Productivity of Serine-Rich Proteins Based on Proteome Profiling

ABSTRACT Variations in proteome profiles of Escherichia coli in response to the overproduction of human leptin, a serine-rich (11.6% of total amino acids) protein, were examined by two-dimensional gel electrophoresis. The levels of heat shock proteins increased, while those of protein elongation factors, 30S ribosomal protein, and some enzymes involved in amino acid biosynthesis decreased, after leptin overproduction. Most notably, the levels of enzymes involved in the biosynthesis of serine family amino acids significantly decreased. Based on this information, we designed a strategy to enhance the leptin productivity by manipulating the cysK gene, encoding cysteine synthase A. By coexpression of the cysK gene, we were able to increase the cell growth rate by approximately twofold. Also, the specific leptin productivity could be increased by fourfold. In addition, we found that cysK coexpression can improve the production of another serine-rich protein, interleukin-12 β chain, suggesting that this strategy may be useful for the production of other serine-rich proteins as well. The approach taken in this study should be useful in designing a strategy for improving recombinant protein production.

Beyond toothpicks: new methods for isolating mutant bacteria

Over the past 50 years genetic analysis in microbiology has relied predominantly on selections and plate assays using chromogenic enzyme substrates — for example, X-gal assays for the detection of β-galactosidase activity. Recent advances in fluorescent assays and high throughput screening technologies have paved the way for the rapid isolation of mutants that confer complex phenotypes and for the quantitative analysis of the evolution of new traits in bacterial populations. This Review highlights the power of novel single-cell screening technologies and their applications to genetics, evolution and the biotechnological uses of bacteria.

Excretion of Human β-Endorphin into Culture Medium by Using Outer Membrane Protein F as a Fusion Partner in Recombinant Escherichia coli

ABSTRACT Escherichia coli BL21 strains were found to excrete a large amount of outer membrane protein F (OmpF) into culture medium during high-cell-density cultivation. From this interesting phenomenon, a novel and efficient OmpF fusion system was developed for the excretion of recombinant proteins by E. coli. The ompF gene of E. coli BL21(DE3) was first knocked out by using the red operon of bacteriophage λ to construct E. coli MBEL-BL101. For the excretion of human β-endorphin as a model protein, the β-endorphin gene was fused to the C terminus of the E. coli ompF gene by using a linker containing the Factor Xa recognition site. To develop a fed-batch culture condition that allows efficient production of OmpF-β-endorphin fusion protein, three different feeding strategies, an exponential feeding strategy and two pH-stat strategies with defined and complex nutrient feeding solutions, were examined. Among these, the pH-stat feeding strategy with the complex nutrient feeding solution resulted in the highest productivity (0.33 g of protein per liter per h). Under this condition, up to 5.6 g of OmpF-β-endorphin fusion protein per liter was excreted into culture medium. The fusion protein was purified by anion-exchange chromatography and cleaved by Factor Xa to yield β-endorphin, which was finally purified by reverse-phase chromatography. From 2.7 liters of culture supernatant, 545.4 mg of β-endorphin was obtained.

Recombinant antibodies: engineering and production in yeast and bacterial hosts.

After the appearance of the first FDA‐approved antibody 25 years ago, antibodies have become major therapeutic agents in the treatment of many human diseases, including cancer and infectious diseases, and the use of antibodies as therapeutic/diagnostic agents is expected to increase in the future. So far, a variety of strategies have been devised for engineering of these fascinating molecules to develop superior properties and functions. Recent progress in systems biology has provided more information about the structures and cellular networks of antibodies, and, in addition, recent development of biotechnology tools, particularly in regard to high‐throughput screening, has made it possible to perform more intensive engineering on these substances. Based on a sound understanding and new technologies, antibodies are now being developed as more powerful drugs. In this review, we highlight the recent, significant progress that has been made in antibody engineering, with a particular focus on Fc engineering and glycoengineering for improved functions, and cellular engineering for enhanced production of antibodies in yeast and bacterial hosts.

APEx 2-hybrid, a quantitative protein–protein interaction assay for antibody discovery and engineering

We have developed a bacterial system for the discovery of interacting proteins that, unlike other two-hybrid technologies, allows for the selection of protein pairs on the basis of affinity or expression. This technology relies on the anchored periplasmic expression (APEx) of one protein (bait) on the periplasmic side of the inner membrane of Escherichia coli and its interacting partner (prey) as a soluble, epitope-tagged, periplasmic protein. Upon removal of the outer membrane by spheroplasting, periplasmic proteins, including any unbound epitope-tagged prey, are released into the extracellular fluid. However, if the epitope-tagged prey can bind to the membrane-anchored bait, it remains associated with the cell and can be detected quantitatively by using fluorescent anti-epitope tag antibodies. Cells expressing prey:bait pairs exhibiting different affinities can be readily distinguished by flow cytometry. The utility of this technology, called APEx two-hybrid, was demonstrated in two demanding antibody engineering applications: First, single-chain variable fragment (scFvs) with increased affinity to the protective antigen of Bacillus anthracis were isolated from cells coexpressing libraries of scFv random mutants, together with endogenously expressed antigen. Second, APEx two-hybrid coupled with multicolor FACS analysis to account for protein expression was used for the selection of mutant Fab antibody fragments exhibiting improved expression in the bacterial periplasm.

Enhanced production of recombinant proteins in Escherichia coli by filamentation suppression

ABSTRACT During growth of high-cell-density cultures of Escherichia coli, overproduction of recombinant proteins often results in increased stress response, cell filamentation, and growth cessation. Filamentation of cells consequently lowers final achievable cell concentration and productivity of the target protein. Reported here is a methodology that should prove useful for the enhancement of cell growth and protein productivity by the suppression of cell filamentation. By the coexpression of the E. coli ftsA and ftsZ genes, which encode key proteins in cell division, growth of recombinant strains as well as production of human leptin and human insulin-like growth factor I was improved. Observation of cell morphology revealed that the coexpression of the ftsA and ftsZ genes successfully suppressed filamentation caused by the accumulation of recombinant proteins.

Evaluation of intracellular lipid bodies in Chlamydomonas reinhardtii strains by flow cytometry.

A comparative study of Chlamydomonas reinhardtii wild type CC124 and a cell wall-less mutant sta6-1 is described using FACS in conjunction with two different lipophilic fluorescent dyes, Nile Red and BODIPY 505/515. The results indicate that BODIPY 505/515 is more effective for the vital staining of intracellular lipid bodies and single cell sorting than Nile Red. While BODIPY 505/515 stained cells continued to grow after single cell sorting using FACS, Nile Red stained cells failed to recover from sorting. In addition, a comprehensive study was performed to establish a quantitative baseline for future studies for either lipid accumulation and/or microalgal growth by measuring various parameters such as cell count, size, fatty acid contents/composition, and optical/confocal images of the wild type and mutant.

Molecular Cloning and Characterization of an Endoxylanase Gene of Bacillus sp. in Escherichia coli

A gene encoding an endoxylanase of Bacillus sp. was cloned and expressed in Escherichia coli. The entire nucleotide sequence of a 1,620 bp SmaI fragment containing the endoxylanase gene was determined. The endoxylanase gene was 639 bp long and encoded 213 amino acids which showed up to 96% amino acid homology with other endoxylanases. The endoxylanase produced by E. coli harboring pKJX4 was purified by ion-exchange chromatography (DE-52 and CM-52) and its N-terminal sequence was determined to be Ala-Gly-Thr-Asp-Tyr-Trp-Gln-Asn-Trp-Thr-Asp-Gly-Gly-Gly-Thr. The endoxylanase expressed in E. coli was identical to that of the original Bacillus sp. whose molecular weight was approximately 20,400. Most of the produced endoxylanase was localized in the periplasmic space of E. coli. When the endoxylanase was reacted with 2% oat spelts xylan (w/v) at 40 degrees C for 10 h, the major product was xylobiose which is known to be a selective growth stimulant to one of the healthy intestinal microflora, Bifidobacteria.