• Journal of Microbiology and Biotechnology
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• v.23 no.3
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• pp.304-312
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• 2013

The thermotolerant methylotrophic yeast Hansenula polymorpha is attracting interest as a potential strain for the production of recombinant proteins and biofuels. However, only limited numbers of genome engineering tools are currently available for H. polymorpha. In the present study, we identified the HpPOL3 gene encoding the catalytic subunit of DNA polymerase ${\delta}$ of H. polymorpha and mutated the sequence encoding conserved amino acid residues that are important for its proofreading 3'${\rightarrow}$5' exonuclease activity. The resulting $HpPOL3^*$ gene encoding the error-prone proofreading-deficient DNA polymerase ${\delta}$ was cloned under a methanol oxidase promoter to construct the mutator plasmid pHIF8, which also contains additional elements for site-specific chromosomal integration, selection, and excision. In a H. polymorpha mutator strain chromosomally integrated with pHIF8, a $URA3^-$ mutant resistant to 5-fluoroorotic acid was generated at a 50-fold higher frequency than in the wild-type strain, due to the dominant negative expression of $HpPOL3^*$. Moreover, after obtaining the desired mutant, the mutator allele was readily removed from the chromosome by homologous recombination to avoid the uncontrolled accumulation of additional mutations. Our mutator system, which depends on the accumulation of random mutations that are incorporated during DNA replication, will be useful to generate strains with mutant phenotypes, especially those related to unknown or multiple genes on the chromosome.

• Applied Chemistry for Engineering
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• v.27 no.2
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• pp.171-178
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• 2016

Extensive efforts from all over the world have been made to solve energy problems, such as high oil prices, global warning due to the depletion of oil. Among them, biofuel has been drawing attention as a clean energy, which can replace fossil fuels. However, conventional biofuels were often converted from eatable biomass such as sugar cane, corn and soy which should be replaced with uneatable biomass. In this study, a techno-economical evaluation of the gasification of biomass waste with mixed alcohol synthesis process was performed. Considering available domestic biomass wastes, a 2000 ton/day conversion plant were assumed to produce 533000 L/day ethanol. Also, financial data from previous studies were evaluated and used and economical sensitivities with various operation conditions were established. Economic analysis were conducted by the payback period and internal rate of return (IRR) and net present value (NPV). Sensitivity analyses of raw material costs, initial investment, the major process cost, ethanol price changes and operating costs were all performed.

• Journal of Microbiology and Biotechnology
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• v.31 no.7
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• pp.1035-1043
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• 2021

Although engineered Saccharomyces cerevisiae fermenting cellobiose is useful for the production of biofuels from cellulosic biomass, cellodextrin accumulation is one of the main problems reducing ethanol yield and productivity in cellobiose fermentation with S. cerevisiae expressing cellodextrin transporter (CDT) and intracellular β-glucosidase (GH1-1). In this study, we investigated the reason for the cellodextrin accumulation and how to alleviate its formation during cellobiose fermentation using engineered S. cerevisiae fermenting cellobiose. From the series of cellobiose fermentation using S. cerevisiae expressing only GH1-1 under several culture conditions, it was discovered that small amounts of GH1-1 were secreted and cellodextrin was generated through trans-glycosylation activity of the secreted GH1-1. As GH1-1 does not have a secretion signal peptide, non-conventional protein secretion might facilitate the secretion of GH1-1. In cellobiose fermentations with S. cerevisiae expressing only GH1-1, knockout of TLG2 gene involved in non-conventional protein secretion pathway significantly delayed cellodextrin formation by reducing the secretion of GH1-1 by more than 50%. However, in cellobiose fermentations with S. cerevisiae expressing both GH1-1 and CDT-1, TLG2 knockout did not show a significant effect on cellodextrin formation, although secretion of GH1-1 was reduced by more than 40%. These results suggest that the development of new intracellular β-glucosidase, not influenced by non-conventional protein secretion, is required for better cellobiose fermentation performances of engineered S. cerevisiae fermenting cellobiose.

• Journal of Life Science
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• v.32 no.8
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• pp.659-665
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• 2022

Chlorella vulgaris is an important freshwater alga that is widely used as a food source by humans and animals. Recently, Chlorella has received considerable attention with regard to its potential application in aquaculture and the production of biofuels, nutrients, and therapeutic proteins. Recently, our laboratory acquired a new strain of C. vulgaris, PKVL7422, characterized by fast growth, ease of culture, and cultivability under dark conditions. However, the genes involved in its nitrogen assimilation are unknown. In this work, we identified the nitrate reductase (NR) gene of C. vulgaris PKVL7422 using rapid amplification of cDNA ends and genome walking. The NR gene of C. vulgaris PKVL7422 is approximately 8 kb long and composed of 18 introns and 19 exons, which encode 877 amino acids. An alignment analysis of the NR gene showed that it possesses the five domains and several invariant residues found in plant NRs. These results provide new insight into the molecular organization of the NR gene in algae.

• Journal of Microbiology and Biotechnology
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• v.33 no.11
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• pp.1403-1411
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• 2023

Carbon dioxide (CO₂) is the most abundant component of greenhouse gases (GHGs) and directly creates environmental issues such as global warming and climate change. Carbon capture and storage have been proposed mainly to solve the problem of increasing CO₂ concentration in the atmosphere; however, more emphasis has recently been placed on its use. Among the many methods of using CO₂, one of the key environmentally friendly technologies involves biologically converting CO₂ into other organic substances such as biofuels, chemicals, and biomass via various metabolic pathways. Although an efficient biocatalyst for industrial applications has not yet been developed, biological CO₂ conversion is the needed direction. To this end, this review briefly summarizes seven known natural CO₂ fixation pathways according to carbon number and describes recent studies in which natural CO₂ assimilation systems have been applied to heterogeneous in vivo and in vitro systems. In addition, studies on the production of methanol through the reduction of CO₂ are introduced. The importance of redox cofactors, which are often overlooked in the CO₂ assimilation reaction by enzymes, is presented; methods for their recycling are proposed. Although more research is needed, biological CO₂ conversion will play an important role in reducing GHG emissions and producing useful substances in terms of resource cycling.

• Journal of the Korean Applied Science and Technology
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• v.34 no.3
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• pp.683-693
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• 2017

Transport biofuels have been recognized as a promising means to resolve the following issues like global warming, oil depletion and environmental pollutions. Among various biofuels, biodiesel has several advantages such as less emission of air pollutants and higher cetane values compared to diesel oil. Demand for biodiesel in Korea is increasing that leads to higher dependence on the imported feedstocks. Therefore, it is important to utilize the waste materials collected domestically for biodiesel production. Food waste oil collected in waste treatment facility has not been used for biodiesel production due to high free fatty contents in the oil. In this work, biodiesel conversion of food waste oil by Amberlyst 15 was studied. Synthetic and actual food waste oils have been used in the study. First, the effects of the major operating parameters including reaction temperature, methanol to oil molar ratio and catalyst loading on the conversion rates and yields were determined with synthetic waste oil. Kinetic modelling work was also done to determine the activation energy of the reaction. From the work, optimization reaction conditions were determined to be 383K, 1: 26.1 for methanol molar ratio to oil, 10 wt.% for catalyst loading and 360 min for reaction time. Activation energy of the reaction is determined to be 29.75 kJ/mol, lower than those reported in the previous works. So the solid catalyst, Amberlyst 15, was more efficient for esterification than the solid catalysts employed in the other works. Agitation rates have the negligible effects on the conversion rates and yields. With the identified optimization conditions, conversion of the actual food waste oil was also carried out. The esterification yield of actual food waste oil in 60 min was 13% lower than that of synthetic waste oil but the final yields in 240 min were similar each other, 98.12% for synthetic oil and 97.62% for actual waste oil.

• Korean Chemical Engineering Research
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• v.55 no.5
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• pp.609-617
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• 2017

Lignocellulosic biomass is a renewable resource for production of biofuels and biochemicals. Furfural (FF) is an important platform chemical catalytically derived from the hemicellulose fraction of biomass. Tetrahydrofurfuryl alcohol (THFA) is a FF derivative and can be used as an eco-friendly solvent with thermal and chemical stability. Despite large numbers of experimental studies for catalytic conversion of FF to THFA, few research have conducted on the economic feasibility for large-scale THFA production from FF. At the stage of assessment of the potential for commercialization of conversion technology, a large-scale process study is required to identify technological bottleneck and to obtain information for solving scale-up problems. In this study, process simulation and technoeconomic evaluation for catalytic conversion of FF to THFA are performed, as the following three steps: integrated process design, heat integration, and economic evaluation. First, a large-scale process including conversion and separation processes is designed based on experimental results. When the FF processing rate is 255 tonnes per day, the FF-to-THFA yields are 63.2~67.9 mol%. After heat integration, the heating requirements are reduced by 14.4~16.4%. Finally, we analyze the cost drivers and calculate minimum selling price of THFA by economic evaluation. The minimum selling price of THFA for the developed process are $2,120~2,340 per tonne, which are close to the current THFA market price.

• Journal of Plant Biotechnology
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• v.37 no.3
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• pp.319-326
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• 2010

An interest in the production of seed-oil based fuel and raw materials, which comes from renewable plant sources, has been intrigued by the phenomenon of global warming and shortage of fossil fuels. Rapeseed (Brassica napus) is the most important oilseed crop, which produces seeds with 40% oil. It is desirable to develop genetically modified rapeseed producing oils, which can be easily converted to biodiesel. As an initial step for development of genetically modified rapeseed for the production of biofuels or bio-based materials, Korean rapeseed cultivars, Naehan, Youngsan, Tammi and Halla, were analyzed. Four Korean rapeseed cultivars produce 32 to 40% oil of seed dry weight, which is rich in oleic acid (more than 60 mole%). The cotyledonary petioles of rapeseed cultivar, Halla, were transformed using Agrobacterium tumefaciens strain GV3101, carrying the uidA gene encoding $\beta$-glucuronidase (GUS) as a reporter gene and the phosphinothricin acetyltransferase (PAT) gene as a selectable marker. The stable integration of PAT gene in the genome of transgenic rapeseeds was confirmed by PCR analysis. Expression of uidA gene in various rapeseed organs was determined by fluorometric assay and histochemical staining. Transformation efficiency of a Korean rapeseed Halla cultivar was 10.4%. Genetic inheritance of transgenes was confirmed in $T_2$ generation.

• Microbiology and Biotechnology Letters
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• v.44 no.3
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• pp.400-407
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• 2016

A unicellular green alga, Chlorella vulgaris KNUA027, was isolated from the roots of Panax ginseng seedlings and its biotechnological potential was investigated. The results of GC/MS analysis showed that C. vulgaris KNUA027 was rich in nutritionally important polyunsaturated fatty acids (PUFAs) such as alpha-linolenic acid (C_18:3 ω3, 45.8%, 50.8 mg/g) and hexadecatrienoic acid (C_16:3 ω3, 11.8%, 13.1 mg/g). Therefore, this Korean indigenous microalga may have potential as a source of omega-3 PUFAs. It was also found that the saturated palmitic acid (C_16:0, 37.1%, 41.2 mg/g), which is suitable for biodiesel production, was one of the major fatty acids produced by strain KNUA027. The proximate analysis showed that the volatile matter content was 88.5%, and the ultimate analysis indicated that the higher heating value was 19.8 MJ/kg. Therefore, the results from this research with C. vulgaris KNUA027 may provide the basis for the production of microalgae-based biofuels and biomass feedstock.

• Journal of Life Science
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• v.19 no.12
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• pp.1836-1840
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• 2009

Recently, hydroxymethylfurfrual (HMF) has been highlighted as a key intermediate for the production of liquid biofuels and other valuable compounds. We used sesame roots as a biocatalyst to synthesize HMF using flask cultures. The synthesis of HMF was identified by GC-mass analysis. The highest root growth was observed in cultures with 1.0 mg/l NAA at $30^{\circ}C$, while root growth was not found in those without NAA treatment. When silver nitrate ($AgNO_3$) was added, the root growth was greatest in those treated with 0.5 mg/l $AgNO_3$ and cultured at $30^{\circ}C$. In the case of HMF synthesis, its highest yield was obtained in those treated with 0.5 mg/l NAA at $25^{\circ}C$, but low HMF was detected in those treated without naphthaleneacetic acid (NAA). The addition of $AgNO_$ to the culture medium showed a 8-10% reduction in HMF yield compared to that of the control, indicating its inhibitory effect on the synthesis of HMF. On the whole, an optimal culture temperature for HMF synthesis seemed to be between $25-30^{\circ}C$.