Jeong-Woo Seo

Efficient production of ethanol from crude glycerol by a Klebsiella pneumoniae mutant strain.

A mutant strain of Klebsiella pneumoniae, termed GEM167, was obtained by γ irradiation, in which glycerol metabolism was dramatically affected on exposure to γ rays. Levels of metabolites of the glycerol reductive pathway, 1,3-propanediol (1,3-PD) and 3-hydroxypropionic acid (3-HP), were decreased in the GEM167 strain compared to a control strain, whereas the levels of metabolites derived from the oxidative pathway, 2,3-butanediol (2,3-BD), ethanol, lactate, and succinate, were increased. Notably, ethanol production from glycerol was greatly enhanced upon fermentation by the mutant strain, to a maximum production level of 21.5 g/l, with a productivity of 0.93 g/l/h. Ethanol production level was further improved to 25.0 g/l upon overexpression of Zymomonas mobilis pdc and adhII genes encoding pyruvate decarboxylase (Pdc) and aldehyde dehydrogenase (Adh), respectively in the mutant strain GEM167.

Sequential acid-/alkali-pretreatment of empty palm fruit bunch fiber.

Pretreatment processes are key technologies for generating fermentable sugars based on lignocellulosic biomass. In this study, we developed a novel method for empty palm fruit bunch fiber (EPFBF) using sequential pretreatment with dilute acid and then alkali. Dilute sulfuric acid was used in the first step, which removed 90% of the hemicellulose and 32% of the lignin, but left most of the cellulose under the optimum pretreatment condition. Sodium hydroxide was then applied in the second step, which extracted lignin effectively with a 70% delignification yield, partially disrupting the ordered fibrils of the EPFBF and thus enhancing the enzyme digestibility of the cellulose. The sequentially pretreated biomass consisted of 82% cellulose, less than 1% hemicellulose, and 30% lignin content afterward. The pretreated biomasses morphologically revealed rough, porous, and irregularly ordered surfaces for enhancing enzyme digestibility. These results indicate that the sequentially acid/alkali-pretreated EPFBF could be broadly useful as a novel biomass.

Production of 3-hydroxypropionic acid through propionaldehyde dehydrogenase PduP mediated biosynthetic pathway in Klebsiella pneumoniae

The pduP gene encodes a propionaldehyde dehydrogenase (PduP) was investigated for the role in 3-hydroxypropionic acid (3-HP) glycerol metabolism in Klebsiella pneumoniae. The enzyme assay showed that cell extracts from a pduP mutant strain lacked measurable dehydrogenase activity. Additionally, the mutant strain accumulated the cytotoxic intermediate metabolite 3-hydroxypropionaldehyde (3-HPA), causing both cell death and a lower final 3-HP titer. Ectopic expression of pduP restored normal cell growth to mutant. The enzymatic property of recombinant protein from Escherichia coli was examined, exhibiting a broad substrate specificity, being active on 3-HPA. The present work is thus the first to demonstrate the role of PduP in glycerol metabolism and biosynthesis of 3-HP.

Improved ethanol tolerance in Escherichia coli by changing the cellular fatty acids composition through genetic manipulation

To investigate the effect of cellular fatty acids composition on ethanol tolerance in Escherichia coli, we overexpressed either des, encoding fatty acid desaturase from Bacillus subtilis, or fabA, encoding β-hydroxydecanoyl thio-ester dehydrase from E. coli, or both genes together, into E. coli. Recombinant E. coli harboring fabA had elevated tolerance against ethanol compared to wild type strain. In contrast, des decreased resistance to ethanol. Co-expression of both genes together complemented ethanol tolerance of E. coli. This result indicates how to engineer bacterial strains to be resistant to higher concentrations of ethanol.

Identification and utilization of a 1,3-propanediol oxidoreductase isoenzyme for production of 1,3-propanediol from glycerol in Klebsiella pneumoniae

In a previous study, we showed that 1,3-propanediol (1,3-PD) was still produced from glycerol by the Klebsiella pneumoniae mutant strain defective in 1,3-PD oxidoreductase (DhaT), although the production level was lower compared to the parent strain. As a potential candidate for another putative 1,3-PD oxidoreductase, we identified and characterized a homolog of Escherichia coli yqhD (88% homology in amino acid sequence), which encodes an alcohol dehydrogenase and is well known to replace the function of DhaT in E. coli. Introduction of multiple copies of the yqhD homolog restored 1,3-PD production in the mutant K. pneumoniae strain defective in DhaT. In addition, by-product formation was still eliminated in the recombinant strain due to the elimination of the glycerol oxidative pathway. An increase in NADP-dependent 1,3-PD oxidoreductase activity was observed in the recombinant strain harboring multiple copies of the yqhD homolog. The level of 1,3-PD production during batch fermentation in the recombinant strain was comparable to that of the parent strain; further engineering can generate an industrial strain producing 1,3-propanediol.

Production and purification of human papillomavirus type 33 L1 virus-like particles from Spodoptera frugiperda 9 cells using two-step column chromatography

The major capsid protein L1 of human papillomavirus (HPV) is essential in construction of recombinant antigen vaccines against cervical cancer. HPV type 33 accounts for about 10% of all HPV infections in Asia. The gene encoding the major capsid protein L1 of the high-risk HPV type 33 was isolated from a Korean patient and expressed in Sf-9 insect cells using a baculovirus expression system. HPV33 L1 protein was isolated by two-step chromatographic purification using strong-cation exchange and ceramic hydroxyapatite chromatography. Strong-cation-exchange chromatography was performed to achieve initial purification of HPV33 L1 and to remove most contaminating proteins, and secondary ceramic hydroxyapatite chromatography yielded pure HPV33 L1 virus-like particles (VLPs). Ceramic hydroxyapatite columns are particularly useful in the purification of antibodies, antigens, human viruses, and VLPs, and we thus used this system. The expression of HPV L1 protein in Sf-9 cells was examined by SDS-PAGE, Western-blotting, and ELISA analyses, and the data showed that HPV33 L1 VLPs were determined to > 98% purity and 58.7% recovery by a quantitative immuno-ELISA assay. Transmission electron microscopy analysis revealed that the HPV VLPs were approximately 50-60 nm in diameter and created by self-assembly of HPV L1 protein. The efficient and simple purification process described here should be useful in production of a cervical cancer vaccine.

Eicosapentaenoic acid (EPA) biosynthetic gene cluster ofShewanella oneidensis MR-1: Cloning, heterologous expression, and effects of temperature and glucose on the production of EPA inEscherichia coli

The putative EPA synthesis gene cluster was mined from the entire genome sequence ofShewanella oneidensis MR-1. The gene cluster encodes a PKS-like pathway that consists of six open reading frames (ORFs): ORFSO1602 (multi-domain beta-ketoacyl synthase, KS-MAT-4ACPs-KR), ORFSO 1600 (acyl transferase, AT), ORFSO 1599 (multi-domain beta-ketoacyl synthase, KS-CLF-DH-DH), ORFSO 1597 (enoyl reductase, ER), ORFSO 1604 (phosphopentetheine transferase, PPT), and ORFSO1603 (transcriptional regulator). In order to prove involvement of the PKS-like machinery in EPA synthesis, a 20.195-kb DNA fragment containing the genes was amplified fromS. oneidensis MR-1 by the long-PCR method. Its identity was confirmed by the methods of restriction enzyme site mapping and nested PCR of internal genesorfSO1597 andorfSO1604. The DNA fragment was cloned intoEscherichia coli using cosmid vector SuperCos1 to form pCosEPA. Synthesis of EPA was observed in fourE. coli clones harboring pCosEPA, of which the maximum yield was 0.689% of the total fatty acids in a clone designated 9704-23. The production yield of EPA in theE. coli clone was affected by cultivation temperature, showing maximum yield at 20°C and no production at 30°C or higher. In addition, production yield was inversely proportional to glucose concentration of the cultivation medium. From the above results, it was concluded that the PKS-like modules catalyze the synthesis of EPA. The synthetic process appears to be subject to regulatory mechanisms triggered by various environmental factors. This most likely occurs via the control of gene expression, protein stability, or enzyme activity.

The production of 1,3-propanediol from mixtures of glycerol and glucose by a Klebsiella pneumoniae mutant deficient in carbon catabolite repression

In the present study, mutant strain of Klebsiella pneumoniae with deletion of the crr gene encoding EIIA(Glc) (a component of the glucose-specific phosphoenolpyruvate-dependent transferase system [PTS]) was prepared. This eliminated the ability of the strain to mediate carbon catabolite repression (CCR). Production of 1,3-propanediol (1,3-PD) from glycerol by the crr mutant strain was enhanced (compared to that of the parent) in the presence of glucose. Using molasses as a co-substrate of glycerol, the maximum yield of 1,3-PD was 60.4% greater (81.2g/l) than that obtained when glycerol was used alone, under optimum fermentation conditions.

Growth of the oleaginous microalga Aurantiochytrium sp. KRS101 on cellulosic biomass and the production of lipids containing high levels of docosahexaenoic acid

We examined the growth of a novel oleaginous microalga, Aurantiochytrium sp. KRS101, using cellulosic materials as nutrients, and the resultant production of lipids containing high levels of docosahexaenoic acid (DHA). The microalgal strain could grow using either carboxymethylcellulose or cellobiose as a carbon source, and produced lipids containing high levels of DHA (49–58% of total fatty acids). In line with this growth behavior, carboxymethylcellulase and cellobiohydrolase activities were evident in both cell-free lysates and culture broths. Additionally, an industrial cellulosic biomass, palm oil empty fruit bunches (POEFB), a by-product of the palm oil industry, were utilized by the microalgal strain for cell growth and lipid production.

Lipase-catalyzed in-situ biosynthesis of glycerol-free biodiesel from heterotrophic microalgae, Aurantiochytrium sp. KRS101 biomass

Heterotrophic microalgae, Aurantiochytrium sp. KRS101 had a large amount of lipid (56.8% total lipids). The cells in the culture medium were easily ruptured due to thin cell wall of Aurantiochytrium sp., which facilitated in-situ fatty acid methyl esters (FAMEs) production directly from biomass. The harvested biomass had a high content of free fatty acids (FFAs), which was advantageous for glycerol-free FAMEs production. FAMEs were directly produced from Aurantiochytrium sp. KRS101 biomass (48.4% saponifiable lipids) using Novozyme 435-catalyzed in-situ esterification in dimethyl carbonate (DMC). DMC was used as a lipid extraction reagent, acyl acceptor and reaction medium. A 433.09mg FAMEs/g biomass was obtained with 89.5% conversion under the optimal condition: DMC to biomass ratio of 5:1 (v/w) and enzyme to biomass ratio of 30% (w/w) at 50°C for 12h. Glycerol could not be detected in the produced FAMEs.