• Biotechnology and Bioprocess Engineering:BBE
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• 제9권3호
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• pp.184-190
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• 2004

The influences of impeller types on morphology and protein expression were investigated in a submerged culture of Aspergillus oryzae. The impeller types strongly affected mycelial morphology and protein production in batch and fed-batch fermentations. Cells that were cultured by propeller agitation grew in the form of a pellet, whereas cells that were cultured by turbine agitation grew in a freely dispersed-hyphal manner and in a clumped form. Pellet-grown cells showed high levels of protein production for both the intracellularly heterologous protein (${\beta}$-glucuronidase) and the extracellularly homologous protein (${\alpha}$-amylase). The feeding mode of the carbon source also influenced the morphological distribution and protein expression in fed-batch fermentation of A. oryzae. Pulsed-feeding mainly showed high protein expression and homogeneous distribution of pellet whereas continuous feeding resulted in less protein expression and heterogeneous distribution with pellet and dispersed-hyphae. The pellet growth with propeller agitation paralleling with the pulsed-feeding of carbon source showed a high level of protein production in the submerged fed-batch fermentation of recombinant A. oryzae.

• Biotechnology and Bioprocess Engineering:BBE
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• 제9권4호
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• pp.252-255
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• 2004

A pfl ldhA double mutant Escherichia coli strain NZN 111 was used to produce succinic acid by overexpressing the E. coli malic enzyme gene (sfcA). This strain, however, produced a large amount of malic acid as well as succinic acid. After the analyses of the metabolic pathways, the fumB gene encoding the anaerobic fumarase of E. coli was co-amplified to solve the problem of malic acid accumulation. A plasmid, pTrcMLFu, was constructed, which contains an artificial operon (sfcA-fumB) under the control of the inducible trc promoter. From the batch culture of recombinant E. coli NZN 111 harboring pTrcMLFu, 7 g/L of succinic acid was produced from 20 g/L of glucose, with no accumulation of malic acid. From the metabolic flux analysis the strain was found under reducing power limiting conditions by severe reorientation of metabolic fluxes.

• Journal of Microbiology and Biotechnology
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• 제24권4호
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• pp.483-488
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• 2014

In order to improve the expression of heat-resistant xylanase XYNB from Aspergillus niger SCTCC 400264, XynB has been cloned into Pichia pastoris secretary vector pPIC9K. The XynB production of recombinant P. pastoris was four times that of E. coli, and the $V_{max}$ and specific activity of XynB reached $2,547.7{\mu}mol/mg$ and 4,757 U/mg, respectively. XynB still had 74% residual enzyme activity after 30 min of heat treatment at $80^{\circ}C$. From the van der Waals force analysis of XYNB (ACN89393 and AAS67299), there is one more oxygen radical in AAS67299 in their catalytic site, indicating that the local cavity is much more free, and it is more optimal for substrate binding, affinity reaction, and proton transfer, etc, and eventually increasing enzyme activity. The H-bonds analysis of XYNB indicated that there are two more H-bonds in the 33rd Ser of XYNB (AAS67299) than in the 33rd Ala(ACN89393 ), and two H-bonds between Ser70 and Asp67.

• Journal of Microbiology and Biotechnology
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• 제10권4호
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• pp.496-501
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• 2000

Escherichia cole NZN111, which is known as a pfl ldhA double mutant strin, was metabolically engineered to produce succinic acid by overexpressing malic enzyme into the E. coli controlled by a trc promoter. Fermentation studies were carried out in a LB medium by first growing cells aerobically to an $OD_{600}$ of 5. At this point, 0.01 mM IPTG was added to induce the overexpression of malic enzyme and the agitation speed was gradually lowered. When the culture $OD_{600}$ reached 11, a complete anaerobic condition was achieved by flushing with a $CO_3-H_2$ gas mixture. When NZN111(pTrcML) was cultured at $37^{\circ}C$, the final succinic acid concentration of 2.8 g/l could be obtained after 30 h of anaerobic cultivation. The fermentation results were analyzed by the calculation of metabolic fluxes. Metaolic flux analysis showed that about 85% of phosphoenolpyruvate (PEP) was converted to pyruvate, and further converted to malic acid by malic enzyme.

• Journal of Microbiology and Biotechnology
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• 제28권11호
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• pp.1916-1927
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• 2018

Corynebacterium glutamicum is an excellent platform for the production of amino acids, and is widely used in the fermentation industry. Most industrial strains are traditionally obtained by repeated processes of random mutation and selection, but the genotype of these strains is often unclear owing to the absence of genomic information. As such, it is difficult to improve the growth and amino acid production of these strains via metabolic engineering. In this study, we generated a complete genome map of an industrial L-valine-producing strain, C. glutamicum XV. In order to establish the relationship between genotypes and physiological characteristics, a comparative genomic analysis was performed to explore the core genome, structural variations, and gene mutations referring to an industrial L-leucine-producing strain, C. glutamicum CP, and the widely used C. glutamicum ATCC 13032. The results indicate that a 36,349 bp repeat sequence in the CP genome contained an additional copy each of lrp and brnFE genes, which benefited the export of L-leucine. However, in XV, the kgd and panB genes were disrupted by nucleotide insertion, which increase the availability of precursors to synthesize L-valine. Moreover, the specific amino acid substitutions in key enzymes increased their activities. Additionally, a novel strategy is proposed to remodel central carbon metabolism and reduce pyruvate consumption without having a negative impact on cell growth by introducing the CP-derived mutant $H^+$/citrate symporter. These results further our understanding regarding the metabolic networks in these strains and help to elucidate the influence of different genotypes on these processes.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVI)
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• pp.424-424
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• 2005

• 한국생물정보학회:학술대회논문집
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• 한국생물정보시스템생물학회 2000년도 International Symposium on Bioinformatics
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• pp.13-16
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• 2000

Microorganisms have been widely employed for the production of useful bioproducts including primary metabolites such as ethanol, succinic acid, acetone and butanol, secondary metabolites represented by antibiotics, proteins, polysaccharides, lipids and many others. Since these products can be obtained in small quantities under natural condition, mutation and selection processes have been employed for the improvement of strains. Recently, metabolic engineering strategies have been employed for more efficient production of these bioproducts. Metabolic engineering can be defined as purposeful modification of cellular metabolic pathways by introducing new pathways, deleting or modifying the existing pathways for the enhanced production of a desired product or modified/new product, degradation of xenobiotics, and utilization of inexpensive raw materials. Metabolic flux analysis and metabolic control analysis along with recombinant DNA techniques are three important components in designing optimized metabolic pathways, This powerful technology is being further improved by the genomics, proteomics, metabolomics and bioinformatics. Complete genome sequences are providing us with the possibility of addressing complex biological questions including metabolic control, regulation and flux. In silico analysis of microbial metabolic pathways is possible from the completed genome sequences. Transcriptome analysis by employing ONA chip allows us to examine the global pattern of gene expression at mRNA level. Two dimensional gel electrophoresis of cellular proteins can be used to examine the global proteome content, which provides us with the information on gene expression at protein level. Bioinformatics can help us to understand the results obtained with these new techniques, and further provides us with a wide range of information contained in the genome sequences. The strategies taken in our lab for the production of pharmaceutical proteins, polyhydroxyalkanoate (a family of completely biodegradable polymer), succinic acid and me chemicals by employing metabolic engineering powered by genomics, proteomics, metabolomics and bioinformatics will be presented.

• Journal of Microbiology and Biotechnology
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• 제16권1호
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• pp.64-67
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• 2006

To enhance the stability of ascorbic acid, the glycosylation of ascorbic acid was studied using the transfructosylation activity of levan fructotransferase. When levan was used as glycosyl donor, a novel fructoside (ascorbic acid 2-ffuctoside) was formed by the transfructosylation activity of the levan fructotransferase. The production of ascorbic acid 2-fructoside was highly affected by the concentration of the fructosyl acceptor (ascorbic acid). When $35\%$ of ascorbic acid and $2\%$ of levan were incubated with LFTase of 0.5 unit/glevan at $37^{\circ}C$ for 85 h, a maximum 52 g/l of AA-2F was produced.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2004년도 Annual Meeting BioExibition International Symposium
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• pp.236-241
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• 2004

In Escherichia coli, L-lysine biosynthetic pathway is composed of nine enzymatic reactions. It has been well established that most of the lysine biosynthetic genes are regulated by the lysine availability, even though they are all scattered around the chromosome without forming any multigenic operon structure. However, no transcriptional regulatory mechanism has been identified except for the activation of lysA gene by the LysR. In this study, changes in transcriptome profiles of wild type cells and lysR deletion mutant cells grown in the absence or presence of lysine were investigated by using DNA microarray technique. Microarray data analysis revealed three groups of genes whose expression varies depending on the availability of lysine or LysR or both. To further examine the regulatory patterns of lysine biosynthetic genes, lacZ operon fusions were constructed and their expression was measured under various conditions. Obtained results strongly suggest that there is an additional regulatory mechanism which senses the lysine availability and coordinates gene expression.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVI)
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• pp.431-432
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• 2005