Kohsuke Honda

In vitro production of n-butanol from glucose

The heat treatment of recombinant mesophiles having heterologous thermotolerant enzymes results in the one-step preparation of highly selective biocatalytic modules. The assembly of these modules enables us to readily construct an artificial metabolic pathway in vitro. In this work, we constructed a non-natural, cofactor-balanced, and oxygen-insensitive pathway for n-butanol production using 16 thermotolerant enzymes. The whole pathway was divided into 7 parts, in each of which NAD(H)-dependent enzymes were assigned to be the last step, and the fluxes through each part were spectrophotometrically determined. This real-time monitoring technique enabled the experimental optimization of enzyme level to achieve a desired production rate. Through the optimized pathway, n-butanol could be produced from glucose with a molar yield of 82% at a rate of 8.2 µmol l(-1) min(-1). Our approach would be widely applicable to the rational optimization of artificial metabolic pathways as well as to the in vitro production of value-added biomolecules.

Bacteria Interface Pickering Emulsions Stabilized by Self-assembled Bacteria–Chitosan Network

An oil-in-water Pickering emulsion stabilized by biobased material based on a bacteria-chitosan network (BCN) was developed for the first time in this study. The formation of self-assembled BCN was possible due to the electrostatic interaction between negatively charged bacterial cells and polycationic chitosan. The BCN was proven to stabilize the tetradecane/water interface, promoting formation of highly stable oil-in-water emulsion (o/w emulsion). We characterized and visualized the BCN stabilized o/w emulsions by scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM). Due to the sustainability and low environmental impact of chitosan, the BCN-based emulsions open up opportunities for the development of an environmental friendly new interface material as well as the novel type of microreactor utilizing bacterial cells network.

Synthetic metabolic engineering-a novel, simple technology for designing a chimeric metabolic pathway.

BackgroundThe integration of biotechnology into chemical manufacturing has been recognized as a key technology to build a sustainable society. However, the practical applications of biocatalytic chemical conversions are often restricted due to their complexities involving the unpredictability of product yield and the troublesome controls in fermentation processes. One of the possible strategies to overcome these limitations is to eliminate the use of living microorganisms and to use only enzymes involved in the metabolic pathway. Use of recombinant mesophiles producing thermophilic enzymes at high temperature results in denaturation of indigenous proteins and elimination of undesired side reactions; consequently, highly selective and stable biocatalytic modules can be readily prepared. By rationally combining those modules together, artificial synthetic pathways specialized for chemical manufacturing could be designed and constructed.ResultsA chimeric Embden-Meyerhof (EM) pathway with balanced consumption and regeneration of ATP and ADP was constructed by using nine recombinant E. coli strains overproducing either one of the seven glycolytic enzymes of Thermus thermophilus, the cofactor-independent phosphoglycerate mutase of Pyrococcus horikoshii, or the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase of Thermococcus kodakarensis. By coupling this pathway with the Thermus malate/lactate dehydrogenase, a stoichiometric amount of lactate was produced from glucose with an overall ATP turnover number of 31.ConclusionsIn this study, a novel and simple technology for flexible design of a bespoke metabolic pathway was developed. The concept has been testified via a non-ATP-forming chimeric EM pathway. We designated this technology as “synthetic metabolic engineering”. Our technology is, in principle, applicable to all thermophilic enzymes as long as they can be functionally expressed in the host, and thus would be potentially applicable to the biocatalytic manufacture of any chemicals or materials on demand.

Construction of BAC‐based physical map and analysis of chromosome rearrangement in chinese hamster ovary cell lines

Chinese hamster ovary (CHO) cells have frequently been used in biotechnology for many years as a mammalian host cell platform for cloning and expressing genes of interest. A detailed physical chromosomal map of the CHO DG44 cell line was constructed by fluorescence in situ hybridization (FISH) imaging using randomly selected 303 BAC clones as hybridization probes (BAC‐FISH). The two longest chromosomes were completely paired chromosomes; other chromosomes were partly deleted or rearranged. The end sequences of 624 BAC clones, including 287 mapped BAC clones, were analyzed and 1,119 informative BAC end sequences were obtained. Among 303 mapped BAC clones, 185 clones were used for BAC‐FISH analysis of CHO K1 chromosomes and 94 clones for primary Chinese hamster lung cells. Based on this constructed physical map and end sequences, the chromosome rearrangements between CHO DG44, CHO K1, and primary Chinese hamster cells were investigated. Among 20 CHO chromosomes, eight were conserved without large rearrangement in CHO DG44, CHO K1, and primary Chinese hamster cells. This result suggested that these chromosomes were stable and essential in CHO cells and supposedly conserved in other CHO cell lines. Biotechnol. Bioeng. 2012; 109:1357–1367.

Overexpression of GADD34 Enhances Production of Recombinant Human Antithrombin III in Chinese Hamster Ovary Cells

To improve the production of recombinant human antithrombin III (AT-III) in Chinese hamster ovary (CHO) cells, the gene encoding growth arrest and DNA damage inducible protein 34 (GADD34), which is a transcription factor involved in the unfolded protein response (UPR), was cloned from CHO-K1 cells. Overexpression of GADD34 significantly enhanced the production of recombinant AT-III in CHO 13D-35D cells. The specific rate of AT-III production in the GADD34-overexpressing CHO 13D-35D cells reached approximately 28 pg/cell/d. After 144 h of incubation, the AT-III concentration in the culture supernatant was approximately 40% higher than that observed in the case of the parental CHO 13D-35D cells. The mRNA expression, specific activity, and fucosylation of AT-III were not affected by GADD34 overexpression. Overexpression of GADD34 is a promising method of improving the production of secreted protein pharmaceuticals in CHO cells.

Enhancement of sialylation on humanized IgG-like bispecific antibody by overexpression of α2,6-sialyltransferase derived from Chinese hamster ovary cells

Improvement of glycosylation is one of the most important topics in the industrial production of therapeutic antibodies. We have focused on terminal sialylation with alpha-2,6 linkage, which is crucial for anti-inflammatory activity. In the present study, we have successfully cloned cDNA of beta-galactosyl alpha-2,6 sialyltransferase (ST6Gal I) derived from Chinese hamster ovary (CHO) cells regardless of reports that stated this was not endogenously expressed in CHO cells. After expressing cloned ST6Gal I in Escherichia coli, the transferase activity was confirmed by HPLC and lectin binding assay. Then, we applied ST6Gal I to alpha-2,6 sialylation of the recombinant antibody; the ST6Gal I expression vector was transfected into the CHO cell line producing a bispecific antibody. The N-glycosylation pattern of the antibody was estimated by HPLC and sialidase digestion. About 70% of the total N-linked oligosaccharide was alpha-2,6 sialylated in the transfected cell line whereas no sialylation was observed in the non-transfected cell line. The improvement of sialylation would be of practical importance for the industrial production of therapeutic antibodies.

Assembly and multiple gene expression of thermophilic enzymes in Escherichia coli for in vitro metabolic engineering

In vitro reconstitution of an artificial metabolic pathway is an emerging approach for the biocatalytic production of industrial chemicals. However, several enzymes have to be separately prepared (and purified) for the construction of an in vitro metabolic pathway, thereby limiting the practical applicability of this approach. In this study, genes encoding the nine thermophilic enzymes involved in a non‐ATP‐forming chimeric glycolytic pathway were assembled in an artificial operon and co‐expressed in a single recombinant Escherichia coli strain. Gene expression levels of the thermophilic enzymes were controlled by their sequential order in the artificial operon. The specific activities of the recombinant enzymes in the cell‐free extract of the multiple‐gene‐expression E. coli were 5.0–1,370 times higher than those in an enzyme cocktail prepared from a mixture of single‐gene‐expression strains, in each of which a single one of the nine thermophilic enzymes was overproduced. Heat treatment of a crude extract of the multiple‐gene‐expression cells led to the denaturation of indigenous proteins and one‐step preparation of an in vitro synthetic pathway comprising only a limited number of thermotolerant enzymes. Coupling this in vitro pathway with other thermophilic enzymes including the H2O‐forming NADH oxidase or the malate/lactate dehydrogenase facilitated one‐pot conversion of glucose to pyruvate or lactate, respectively. Biotechnol. Bioeng. 2015;112: 189–196.

A Comparison of Various Methods to Predict Bacterial Predilection for Organic Solvents Used as Reaction Media

Bacterial predilection for organic solvents is important in whole-cell biocatalysis in organic media. Although various methods of measuring bacterial hydrophobicity have been proposed, it is not fully determined whether they are applicable to the assessment of bacterial predilection for organic solvents in whole-cell biocatalytic processes. In this study, bacterial predilection for organic solvents was assessed by bacterial adhesion to hydrocarbon (BATH), contact angle measurement (CAM), hydrophobic interaction chromatography (HIC), and glass adhesion test (GAT). These methods were applied to the cultures of four bacterial species of industrial importance, namely, Rhodococcus opacus B-4, R. erythropolis PR4, Pseudomonas putida T-57, and Escherichia coli JM109, in organic media. Experimental results revealed that CAM assays could be used to predict the dispersibility of bacterial cells in anhydrous organic solvents. However, when bacteria were suspended in aqueous-organic (A/O) two-phase media, the results of BATH assays provided the most reliable assessment of bacterial predilection for organic solvents. This discrepancy noted between CAM and BATH assays was attributed to the effect of electrostatic interaction between bacteria and oil droplets. In A/O two-phase media, the accessibility of a water-immiscible dye, nile red, to the bacterial cell surface, correlated well with BATH assay results.

Glycosylation pattern of humanized IgG-like bispecific antibody produced by recombinant CHO cells

The glycosylation pattern of a humanized anti-EGFR×anti-CD3 bispecific single-chain diabody with an Fc portion (hEx3-scDb-Fc) produced by recombinant Chinese hamster ovary cells was evaluated and compared with those of a recombinant humanized anti-IL-8 antibody (IgG1) and human serum IgG. N-Linked oligosaccharide structures were estimated by two-dimensional high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. No sialylation was observed with purified hEx3-scDb-Fc and the anti-IL-8 antibody. From the analysis of neutral oligosaccharides, approximately more than 90% of the N-linked oligosaccharides of hEx3-scDb-Fc and the anti-IL-8 antibody were alpha-1,6-fucosylated. The galactosylated biantennary oligosaccharides comprise over 40% of the total N-linked oligosaccharides in both hEx3-scDb-Fc and the anti-IL-8 antibody. The fully galactosylated biantennary oligosaccharides from hEx3-scDb-Fc and the anti-IL-8 antibody accounted for only 10% of the N-linked; however, more than 20% of the N-linked oligosaccharides were fully galactosylated biantennary oligosaccharides in human serum IgG. The glycosylation pattern of hEx3-scDb-Fc was quite similar to that of the anti-IL-8 antibody.

Expression of Rhodococcus opacus alkB Genes in Anhydrous Organic Solvents

Rhodococcus opacus B-4 is a benzene-tolerant bacterium which was isolated from a gasoline-contaminated soil sample. We previously demonstrated that this organism was able to survive and exhibit biocatalytic activity in anhydrous organic solvents for at least 5 d. In the present study, we cloned the alkB1 and alkB2 genes encoding alkane hydroxylases from R. opacus B-4. Heterologous expression of the alkB1 and alkB2 genes in Escherichia coli JM109 showed that they encode functional alkane hydroxylases with a substrate range of C(5)-C(16). Promoters of the alkB1 and alkB2 genes, designated P(alkB1) and P(alkB2), respectively, were examined for activity in anhydrous bis (2-ethylhexyl) phthalate (BEHP) containing C(5)-C(16)n-alkanes. Two recombinant plasmids, pP(alkB1)EGFP and pP(alkB2)EGFP, were constructed by inserting the egfp gene downstream of P(alkB1) and P(alkB2), respectively and transformed into R. opacus B-4. Resting cells of R. opacus B-4 (pP(alkB1)EGFP) showed greater levels of EGFP fluorescence in anhydrous BEHP than in 0.85% NaCl, when C(8)-C(16)n-alkanes were supplied as an inducer. Furthermore, n-alkane inducibility of P(alkB1) activity in anhydrous BEHP was noticeably different from that in 0.85% NaCl. This paper presents the first evidence that bacteria can express their genes in essentially anhydrous organic solvents.