DNA chip을 이용한 대장균의 고농도 배양시 transcriptome 분석

윤성호1, 이상엽1, 임근배2*

대전광역시 유성구 구성동 373-1 한국과학기술원 화학공학과 및 생물공정연구센터 1, 삼성종합기술원 마이크로시스템 실험실 2*

Transcriptome analysis of Escherichia coli during high cell density cultivation using DNA chip

Sung Ho Yoon 1, Sang Yup Lee 1, and Geunbae Lim 2*

Department of Chemical Engineering and BioProcess Engineering Research Center, KAIST 373-1 Kusong-dong, Yusong-gu, Taejon 305-701 1, MEMS Lab SAITECH, KiHeung 2*

Introduction

The study of gene expression on a genomic scale is the most obvious opportunity to be made possible by a complete genome sequences of the model organisms. Recently developed DNA chip has emerged as a prime candidate for the performance of such analyses. DNA chip makes it possible to measure the transcription levels of all genes at the same time. High cell-density culture (HCDC) techniques for culturing E. coli have been developed for high-level production of various proteins and non-protein products such as amino acids and poly(3-hydroxybutyrate). Fed-batch cultures have most often been used to obtain high cell density. The most difficulty of process optimization by fed-batch culture is little knowledge on the physiology and metabolism during high cell density. Compared with low cell density, the cells are assumed to undergo large metabolic and physiological changes under high cell density (>50g DCW/L) culture condition. In this study, we focused on the effect of the high cell density on variation in metabolism and physiology of cell during fed-batch culture.

MATERIALS AND METHODS

Bacterial strain and culture condition

Escherichia coli W3110 was used in this work. 200 mL of cell culture was grown at 37 ℃ and 250 rpm before it was used to inoculate a BioFlo3000 bioreactor containing 1.8 L of modified R-medium (per L: glucose 10g; (NH4)2HPO4, 3g; KH2PO4, 7g; citric acid, 0.8g; MgSO4·7H2O, 1g; trace metal solution, 3 mL).  Cells were cultured at 37 ℃ and pH 6.8 (automatic feeding of 25% NH4OH).  Dissolved oxygen level was maintained above 40% of air saturation by supplying air (1 vvm) and by automatically controlling the agitation speed up to 1000 rpm.  Pure oxygen was supplemented when required. During the fed-batch cultures, nutrients were fed after batch culture and intermittently upon observing an increase in pH (0.1 above set point in pH). The composition of the nutrient feed was (per L): glucose, 700g; MgSO4·7H2O, 20g.

Preparation of E. coli gene and DNA chip

DNA chip containing 207 different genes of E. coli was made using microarrayer which was developed by our lab. The spotted E. coli genes can be grouped according to their metabolic roles: glycolysis (37 ea), TCA cycle (29), fermentation (22), chaperones (10), central intermediary metabolism (32), pyruvate dissimilation (6), secretion-related (20), beta-oxidation (10), sigma factor (9), cell division genes (26), and others. Information regarding E. coli gene sequence was obtained from E. coli genome project home page at Wisconsin University (http://www.genetics.wisc.edu) and the open reading frames (ORFs) were amplified by the polymerase chain reaction (PCR). Primers for each coding sequence were designed by the primer design program that we developed. For immobilizing DNA on a solid support, a surface of the slide glass is treated with poly-L-lysine. Onto this modified slide glass, DNA samples are arrayed by using microarrayer we developed. Genes were spotted with intervals of 320 □m and each gene was duplicated on the same slide.

Isolation of total RNA and labeling

Total RNA was isolated from 1.5×109 cells by Qiagen Rneasy columns as manufacturer's protocol. The resulting RNA was resuspended in a 300 □L of DEPC-treated water and consecutively treated for 30 min with 15 units RNase-free DNase followed by 15 min with 40 □g of Proteinase K. Three times of phenol extraction was performed before ethanol precipitation and purified RNA was resuspended in 20 □L of DEPC-treated water. Fluorescence labeled DNA was made during reverse transcription of total RNA (25 □g) by using a random hexamer (10 □g) in 40 L reaction solution containing 0.5 mM dATP, dCTP, dGTP, 0.2 mM dTTP, 10 mM DTT, 0.1 mM Cy3 or Cy5 dUTP, 40 U RNAsin. 200 U Superscript II reverse transcriptase was added after incubation for 5 min at 65 ℃ and probe was synthesized for 2 h at 42 ℃. The reaction was stopped with 5 L of 0.5 M EDTA for 10 min and template RNA was degraded by incubation at 60 ℃ for 1 h with 10 □L of 1 N NaOH. Labeled cDNA was purified and concentrated to about 20 □L using Microcon 30 concentrators.

Hybridization and data analysis

The E. coli chip was hybridized with 13 □L labeled cDNA solution containing 3 ×SSC, 8 □g of pd(A)40-60, 4 □g of yeast tRNA (Sigma), and 0.2 % SDS. Prior to hybridization, the solution was boiled for 2 minutes and then cooled at room temperature. Hybridization was performed for 10 hrs in a 65 ℃ water bath. After hybridization, DNA chips were first washed in a 0.2 ×SSC with 0.1% (w/v) SDS and subsequently rinsed with 0.2×SSC. Excess liquid on the surface was removed by centrifugation for 5 min at 500 rpm. The DNA chip was scanned by ScanArray 5000 (GSI-Lumonics). Signal intensities and local background were determined by Image-Quant (GSI-Lumonics). Following background subtraction, signal intensities were calculated as the percent of total signal as a means of normalization. Hierarchical clustering and self organizing map (SOM) method was applied to clustering genes using Stanford Software.

RESULTS AND DISCUSSION

Bacterial cell growth was shown in Fig. 1. The sampling time points were when the cell density reached 0.8 (S1), 4.7 (S2), 9.2 (S3), 30 (S4), 64 (S5), 110 (S6), 140 (S7), 153 (S8), 161 (S9) in OD600. The cDNA made from purified mRNA from each sample (S2 to S6) was labeled with the fluorescent dye Cy5 (red) and mixed with a reference probe consisting of cDNA made from purified mRNA from cell culture (control, S1) labeled with a second fluorescent dye, Cy3 (green). During course of the fed-batch cultivation, the cells were exposed to different environmental conditions. At the initial phase with glucose excess in culture, cell growth rate reached max of 0.57 hr-1, and after log phase, cells enters exponential growth phase. When cell concentrations reached 140 in OD600 in 19 hrs after inoculation, growth rate slowed down and reached stationary phase. During fed-batch culture, most metabolic pathway related genes were thought to be stable with expression levels varying less then 2-fold. 96 out of 207 genes contained in E. coli chip showed negligible expression level and hardly detected, and 111 genes were grouped by functional role with cell density and applied to clustering with hierarchical method and SOM.

Fig.1. Time profile of bacterial growth

Most gene involved in glycolysis and TCA cycle was quite stable, with expression varying under 1.5-fold. The fructose regulon, FruR, showed nearly constant gene expression level during growth phase, and down-regulated by 0.7 at the stationary phase (S8, S9). FruR  regulates expression of all of these genes, thus repressing synthesis of key enzymes constituting the former pathways while activating synthesis of the latter three pathway. During high cell density culture, fruR was down-regulated by 0.65-fold at glucose depletion time (S3), and the activity was recovered during exponential growth phase (S4, S5, S6). And cell growth entered stationary phase, the activity decreased to 0.47-fold. Positively regulated genes by pfuR, pckA (PEP carboxykinase), icd (isocitrate dehydrogenase) showed similar expression pattern with fruR. The known negatively regulated genes by fruR, edd (6-phosphogluconate dehydrase), eda (2-keto-3-deoxy-6-phosphogluconate aldolase), pykF (pyruvate kinase isoenzymes) showed constant expression activity throughout cultivation (Fig. 2a).

Fermentation is a condition under which growth occurs without any exogenous electron acceptor. Three genes out of 22 fermentation genes (ldhA, aldH, hycE) was up-regulated by about 1.5 fold only at the glucose starvation time (S3), and others showed nearly constant or depressed expression level compared to reference (Fig. 2b).

For achieving high cell density and overproduction of recombinant protein by microorganism, cell division has been studied in bacteria. Cell division genes have been identified in E. coli on the basis of conditional lethal mutations that result in a filamenting  temperature-sensitive phenotype (fts). The ftsZ gene in E. coli is well known to be an essential cell division gene. Most of fts genes (ftsZ, ftsW, ftsA, ftsQ, murF) investigated showed down-regulated by about 0.7-fold at glucose starvation time (S3), up-regulated during exponential growth phase, and slowly down-regulated when entered stationary phase (Fig. 2c).

The most interesting gene investigated during high cell culture was ffs encoding 4.5S RNA. 4.5S RNA is a 114-nucleotide RNA essential for the growth of the E. coli, which is not a structural component of the ribosome, but binds to the ribosomal particle during translation. 4.5S RNA is known to be metabolically stable. The E. coli in which synthesis of the 4.5S RNA turned off by removing an inducer of the lac operon, resulted in cell death. In our study, the increase of gene expression of ffs was quite matched with cell density, from 1.7 to 6.0 fold. Even when the cell growth was stopped, ffs showed high level of gene activity by 4.4-fold (Fig. 2d).

In clustering analysis by SOM, there were little particular characteristics between 14 nodes for genes. This is because most genes investigated were metabolic cycle involved genes, and showed little variation between growth time points. The interesting result from SOM was clustering of time points. We used 3 nodes for 8 different arrays, and the time points were clustered into two group. One involved glucose depletion time (S3), and time points when cell growth rate slowed down (S7, S8, S10). The lag phase (S2) and exponential phase (S4, S5, S6) were grouped in the other node.

Acknowledgement

This work was supported by the Ministry of Commerce, Industry and Energy through the Korea Institute of Industrial Technology Evaluation and Planning.

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Fig. 2. Expression monitoring during high cell density culture of E. coli (a) fruR (b) fermentative genes (c) cell division related genes (d) 4.5 S RNA (ffs)