L-arabinose Concentration Research Articles

Recombinant bromelain production in Escherichia coli: process optimization in shake flask culture by response surface methodology

Bromelain, a cysteine protease with various therapeutic and industrial applications, was expressed in Escherichia coli, BL21-AI clone, under different cultivation conditions (post-induction temperature, L-arabinose concentration and post-induction period). The optimized conditions by response surface methodology using face centered central composite design were 0.2% (w/v) L-arabinose, 8 hr and 25°C. The analysis of variance coupled with larger value of R2 (0.989) showed that the quadratic model used for the prediction was highly significant (p < 0.05). Under the optimized conditions, the model produced bromelain activity of 9.2 U/mg while validation experiments gave bromelain activity of 9.6 ± 0.02 U/mg at 0.15% (w/v) L-arabinose, 8 hr and 27°C. This study had innovatively developed cultivation conditions for better production of recombinant bromelain in shake flask culture.

Optimization of Arabitol Production by Karyoductant SP-K 7 of Saccharomyces cerevisiae V30 and Pichia stipitis CCY 39501 Using Response Surface Methodology

L-arabitol is used in the food and pharmaceutical industries. It can be secreted by genetically modified Saccharomyces cerevisiae carrying the genes responsible for pentose metabolism in yeast cells. The process of the biotransformation of L-arabinose to arabitol is highly dependent on culture conditions. The aim of this investigation was to use statistical response surface methodology (RSM) for optimization of biotransformation of L-arabinose to arabitol by a karyoductant of S. cerevisiae V30 and Pichia stipitis CCY 39501, named SP-K7. Batch cultures of yeast were performed according to a Plackett-Burman design, and three factors, rotation speed, L-arabinose concentration, and temperature, were chosen for a central composite design (CCD) applied in order to optimize the production of the polyol by the karyoductant. On the basis of results obtained using 20 combinations of batch cultures of karyoductant SP-K7, the optimal levels of the factors were determined as: rotation speed 150 rpm, concentration of L-arabinose 32.5 g/l, and temperature 28 degrees C. In such conditions, the predicted concentration of arabitol after two days of incubation of SP-K7 should be 18.367 g/l. The value of R2 = 0.93195 suggested that this model was well-fitted to the experimental data. A verification of the model in experimental conditions confirmed its usefulness.

Coexpression of chaperonin GroEL/GroES markedly enhanced soluble and functional expression of recombinant human interferon-gamma in Escherichia coli

Recombinant human interferon-gamma (rhIFN-γ) is a protein of great potential for clinical therapy due to its multiple biological activities. However, overexpressing rhIFN-γ in Escherichia coli was found to accumulate as cytoplasmic inclusion bodies. In this work, a system for soluble and active expression of rhIFN-γ was constructed by coexpressing chaperonin GroEL/GroES in E. coli. The rhIFN-γ gene was fused to a pET-28a expression vector, and rhIFN-γ was partially expressed as the soluble form following coexpression with a second vector producing chaperonin GroEL/GroES. The fermentation of recombinant E. coli harboring rhIFN-γ and GroEL/GroES plasmids was investigated, and the optimized conditions were as follows: culture temperature of 25°C, incubation time of 8 h, isopropyl-β-D-thio-galactoside concentration of 0.2 mM, and L-arabinose concentration of 0.5 g/L. As a result, the expression level of rhIFN-γ was improved accordingly by 2.2-fold than the control, while a significantly positive correlation was also found between the ratio of supernatant to precipitate of rhIFN-γ and the amount of chaperonin. Circular dichroism spectra, fluorescence spectra, size exclusion chromatography, and chemical crosslinking method were applied to characterize rhIFN-γ, indicating that the three-dimensional structure of rhIFN-γ was identical to that of the native rhIFN-γ. The enzyme-linked immunosorbent assay for active rhIFN-γ quantification showed that coexpression yielded 72.91 mg rhIFN-γ per liter fermentation broth. Finally, protein-protein interactions between rhIFN-γ and chaperonin were analyzed using the yeast two-hybrid system, which provided the direct evidence that chaperonin GroEL/GroES interacted with rhIFN-γ to increase the soluble expression and presented the potential in producing efficiently recombinant proteins.

Evaluation of promoters for gene expression in polyhydroxyalkanoate-producing Cupriavidus necator H16

Five kinds of promoters were evaluated as tools for regulated gene expression in the PHA-producing bacterium Cupriavidus necator. Several broad-host-range expression vectors were constructed by which expression of a reporter gene gfp was controlled by P(lac), P(tac), or P(BAD) derived from Escherichia coli, or promoter regions of phaC1 (P(phaC)) or phaP1 (P(phaP)) derived from C. necator. Then, the gfp-expression profiles were determined in C. necator strains harboring the constructed vectors when the cells were grown on fructose or soybean oil. P(lac), P(tac), P(phaC), and P(phaP ) mediated constitutive gene expression, among which P(tac) was the strongest promoter. lacI-P(tac) was not thoroughly functional even after addition of isopropyl-β-D-thiogalactopyranoside (IPTG), probably due to inability of C. necator to uptake IPTG. Gene expression by araC-P(BAD) could be regulated by varying L-arabinose concentration in the medium, although P(3HB) production rate was slightly decreased in the recombinant. phaR-P(phaP) exhibited an expression profile tightly coupled with P(3HB) accumulation, suggesting application of the vector harboring phaR-P(phaP ) for gene expression specific at the PHA-biosynthesis phase. The properties of these promoters were expected to be useful for effective engineering of PHA biosynthesis in C. necator.

Improving L-arabinose utilization of pentose fermenting Saccharomyces cerevisiae cells by heterologous expression of L-arabinose transporting sugar transporters

BackgroundHydrolysates of plant biomass used for the production of lignocellulosic biofuels typically contain sugar mixtures consisting mainly of D-glucose and D-xylose, and minor amounts of L-arabinose. The yeast Saccharomyces cerevisiae is the preferred microorganism for the fermentative production of ethanol but is not able to ferment pentose sugars. Although D-xylose and L-arabinose fermenting S. cerevisiae strains have been constructed recently, pentose uptake is still a limiting step in mixed sugar fermentations.ResultsHere we described the cloning and characterization of two sugar transporters, AraT from the yeast Scheffersomyces stipitis and Stp2 from the plant Arabidopsis thaliana, which mediate the uptake of L-arabinose but not of D-glucose into S. cerevisiae cells. A yeast strain lacking all of its endogenous hexose transporter genes and expressing a bacterial L-arabinose utilization pathway could no longer take up and grow with L-arabinose as the only carbon source. Expression of the heterologous transporters supported uptake and utilization of L-arabinose especially at low L-arabinose concentrations but did not, or only very weakly, support D-glucose uptake and utilization. In contrast, the S. cerevisiae D-galactose transporter, Gal2, mediated uptake of both L-arabinose and D-glucose, especially at high concentrations.ConclusionsUsing a newly developed screening system we have identified two heterologous sugar transporters from a yeast and a plant which can support uptake and utilization of L-arabinose in L-arabinose fermenting S. cerevisiae cells, especially at low L-arabinose concentrations.

Coexpression of molecular chaperones to enhance functional expression of anti-BNP scFv in the cytoplasm of Escherichia coli for the detection of B-type natriuretic peptide.

Molecular chaperones are a ubiquitous family of cellular proteins that mediate the correct folding of other target polypeptides. In our previous study, the recombinant anti-BNP scFv, which has promising applications for diagnostic, prognostic, and therapeutic monitoring of heart failure, was expressed in the cytoplasm of Escherichia coli. However, when the anti-BNP scFv was expressed, 73.4% of expressed antibodies formed insoluble inclusion bodies. In this study, molecular chaperones were coexpressed with anti-BNP scFv with the goal of improving the production of functional anti-BNP in the cytoplasm of E. coli. Five sets of molecular chaperones were assessed for their effects on the production of active anti-BNP scFv. These sets included the following: trigger factor (TF); groES/groEL; groES/groEL/TF; dnaK/dnaJ/grpE; groES/groEL/dnaK/dnaJ/grpE. Of these chaperones, the coexpression of anti-BNP scFv with the groES/groEL chaperones encoded in plasmid pGro7 exhibited the most efficient functional expression of anti-BNP scFv as an active form. Coexpressed with the groES/groEL chaperones, 64.9% of the total anti-BNP scFv was produced in soluble form, which is 2.4 times higher scFv than that of anti-BNP scFv expressed without molecular chaperones, and the relative binding activity was 1.5-fold higher. The optimal concentration of L-arabinose required for induction of the groES/groEL chaperone set was determined to be 1.0mM and relative binding activity was 3.5 times higher compared with that of no induction with L-arabinose. In addition, soluble anti-BNP scFv was increased from 11.5 to 31.4μg/ml with optimized inducer concentration (1.0mM L-arabinose) for the coexpression of the groES/groEL chaperones. These results demonstrate that the functional expression of anti-BNP scFv can be improved by coexpression of molecular chaperones, as molecular chaperones can identify and help to refold improperly folded anti-BNP scFv.

Gain of Function Mutations in Membrane Region M2C2 of KtrB Open a Gate Controlling K+ Transport by the KtrAB System from Vibrio alginolyticus

KtrB, the K(+)-translocating subunit of the Na(+)-dependent bacterial K(+) uptake system KtrAB, consists of four M(1)PM(2) domains, in which M(1) and M(2) are transmembrane helices and P indicates a p-loop that folds back from the external medium into the cell membrane. The transmembrane stretch M(2C) is, with its 40 residues, unusually long. It consists of three parts, the hydrophobic helices M(2C1) and M(2C3), which are connected by a nonhelical M(2C2) region, containing conserved glycine, alanine, serine, threonine, and lysine residues. Several point mutations in M(2C2) led to a huge gain of function of K(+) uptake by KtrB from the bacterium Vibrio alginolyticus. This effect was exclusively due to an increase in V(max) for K(+) transport. Na(+) translocation by KtrB was not affected. Partial to complete deletions of M(2C2) also led to enhanced V(max) values for K(+) uptake via KtrB. However, several deletion variants also exhibited higher K(m) values for K(+) uptake and at least one deletion variant, KtrB(Delta326-328), also transported Na(+) faster. The presence of KtrA did not suppress any of these effects. For the deletion variants, this was due to a diminished binding of KtrA to KtrB. PhoA studies indicated that M(2C2) forms a flexible structure within the membrane allowing M(2C3) to be directed either to the cytoplasm or (artificially) to the periplasm. These data are interpreted to mean (i) that region M(2C2) forms a flexible gate controlling K(+) translocation at the cytoplasmic side of KtrB, and (ii) that M(2C2) is required for the interaction between KtrA and KtrB.

Abstract A137: Chemotherapy molecule delivered by tumor-targeted salmonella enterica serovar typhimurium under the control of integrated modular gate

Abstract Several strictly or facultative anaerobic bacteria have attracted more and more attention in cancer therapeutics because of their targeting and accumulating abilities in solid tumors, and Salmonella enterica serovar Typhimurium is one of them. These bacteria were also adapted to deliver therapeutic factors and were applied alone or in combination with conventional therapeutics. The aim of this study is to test a new integrated gene expression system in S. Typhimurim SL1344, and the benefit of the new system in amplifying therapeutic molecule production during bacterium targeting and accumulating in solid tumor. We have constructed a synthetic genetic gate in S. Typhimurim SL1344 that integrates information from two promoters as inputs and actives a promoter output only when both input promoters are induced. The integration occurs via an interaction between L-arabinose concentration and bacterium cell density. When certain amount of L-arabinose were added in the system and bacterium population were higher enough, the tumor necrosis factor apoptosis inducing ligand(TRAIL) and fluorescent protein ZsGreen as the control were produced by bacteria. LS174T colon carcinoma cells were processed to form cylindroids between the bottom surface of a 96-well plate and the top surface of a set of polycarbonate cylindrical plugs attached to a polycarbonate lid to mimic solid tumor, and then cylindroid cultures were inoculated with S. typhimurium SL1344 carrying TRAIL or ZsGreen expression system. 0.2% (w/v) L-arabinose was added into the well after 12hr inoculation, and then washed out after 2hr induced. Extents of apoptosis in cylindroids were tested by caspase-3 activity assay. Results showed that the bacteria accumulated inside cylindroids indicated by ZsGreen expression, and the average normalized intensity of apoptotic cell was higher in the cylindroids inoculated by the bacteria carrying the TRAIL than those inoculated by the bacteria carrying the ZsGreen. It can be concluded that the constructed integrated modular gate could effectively control bacteria to produce functional chemotherapy molecule in solid tumor at desirable time and places. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A137.

Engineering of pentose transport in Corynebacterium glutamicum to improve simultaneous utilization of mixed sugars

Corynebacterium glutamicum strains CRA1 and CRX2 are able to grow on L-arabinose and D-xylose, respectively, as sole carbon sources. Nevertheless, they exhibit the major shortcoming that their sugar consumption appreciably declines at lower concentrations of these substrates. To address this, the C. glutamicum ATCC31831 L-arabinose transporter gene, araE, was independently integrated into both strains. Unlike its parental strain, resultant CRA1-araE was able to aerobically grow at low (3.6 g.l(-1)) L-arabinose concentrations. Interestingly, strain CRX2-araE grew 2.9-fold faster than parental CRX2 at low (3.6 g.l(-1)) D-xylose concentrations. The corresponding substrate consumption rates of CRA1-araE and CRX2-araE under oxygen-deprived conditions were 2.8- and 2.7-fold, respectively, higher than those of their respective parental strains. Moreover, CRA1-araE and CRX2-araE utilized their respective substrates simultaneously with D-glucose under both aerobic and oxygen-deprived conditions. Based on these observations, a platform strain, ACX-araE, for C. glutamicum-based mixed sugar utilization was designed. It harbored araBAD for L-arabinose metabolism, xylAB for D-xylose metabolism, D-cellobiose permease-encoding bglF317A, beta-glucosidase-encoding bglA and araE in its chromosomal DNA. In mineral medium containing a sugar mixture of D-glucose, D-xylose, L-arabinose, and D-cellobiose under oxygen-deprived conditions, strain ACX-araE simultaneously and completely consumed all sugars.

Identification and functional analysis of the gene cluster for L-arabinose utilization in Corynebacterium glutamicum.

Corynebacterium glutamicum ATCC 31831 grew on l-arabinose as the sole carbon source at a specific growth rate that was twice that on d-glucose. The gene cluster responsible for l-arabinose utilization comprised a six-cistron transcriptional unit with a total length of 7.8 kb. Three l-arabinose-catabolizing genes, araA (encoding l-arabinose isomerase), araB (l-ribulokinase), and araD (l-ribulose-5-phosphate 4-epimerase), comprised the araBDA operon, upstream of which three other genes, araR (LacI-type transcriptional regulator), araE (l-arabinose transporter), and galM (putative aldose 1-epimerase), were present in the opposite direction. Inactivation of the araA, araB, or araD gene eliminated growth on l-arabinose, and each of the gene products was functionally homologous to its Escherichia coli counterpart. Moreover, compared to the wild-type strain, an araE disruptant exhibited a >80% decrease in the growth rate at a lower concentration of l-arabinose (3.6 g liter(-1)) but not at a higher concentration of l-arabinose (40 g liter(-1)). The expression of the araBDA operon and the araE gene was l-arabinose inducible and negatively regulated by the transcriptional regulator AraR. Disruption of araR eliminated the repression in the absence of l-arabinose. Expression of the regulon was not repressed by d-glucose, and simultaneous utilization of l-arabinose and d-glucose was observed in aerobically growing wild-type and araR deletion mutant cells. The regulatory mechanism of the l-arabinose regulon is, therefore, distinct from the carbon catabolite repression mechanism in other bacteria.

YscU recognizes translocators as export substrates of the Yersinia injectisome

YscU is an essential component of the export apparatus of the Yersinia injectisome. It consists of an N-terminal transmembrane domain and a long cytoplasmic C-terminal domain, which undergoes auto-cleavage at a NPTH site. Substitutions N263A and P264A prevented cleavage of YscU and abolished export of LcrV, YopB and YopD but not of Yop effectors. As a consequence, yscU(N263A) mutant bacteria made needles without the LcrV tip complex and they could not form translocation pores. The graft of the export signal of the effector YopE, at the N-terminus of LcrV, restored LcrV export and assembly of the tip complex. Thus, YscU cleavage is required to acquire the conformation allowing recognition of translocators, which represent an individual category of substrates in the hierarchy of export. In addition, yscU(N263A) mutant bacteria exported reduced amounts of the YscP ruler and made longer needles. Increasing YscP export resulted in needles with normal size, depending on the length of the ruler. Hence, the effect of the yscU(N263A) mutation on needle length was the consequence of a reduced YscP export.

L-Arabinose metabolism in Candida arabinofermentans PYCC 5603T and Pichia guilliermondii PYCC 3012: influence of sugar and oxygen on product formation

L-Arabinose utilization by the yeasts Candida arabinofermentans PYCC 5603(T) and Pichia guilliermondii PYCC 3012 was investigated in aerobic batch cultures and compared, under similar conditions, to D-glucose and D-xylose metabolism. At high aeration levels, only biomass was formed from all the three sugars. When oxygen became limited, ethanol was produced from D-glucose, demonstrating a fermentative pathway in these yeasts. However, pentoses were essentially respired and, under oxygen limitation, the respective polyols accumulated--arabitol from L-arabinose and xylitol from D-xylose. Different L-arabinose concentrations and oxygen conditions were tested to better understand L-arabinose metabolism. P. guilliermondii PYCC 3012 excreted considerably more arabitol from L-arabinose (and also xylitol from D-xylose) than C. arabinofermentans PYCC 5603(T). In contrast to the latter, P. guilliermondii PYCC 3012 did not produce any traces of ethanol in complex L-arabinose (80 g/l) medium under oxygen-limited conditions. Neither sustained growth nor active metabolism was observed under anaerobiosis. This study demonstrates, for the first time, the oxygen dependence of metabolite and product formation in L-arabinose-assimilating yeasts.

대장균에서 분자 chaperone에 의한 alginate lyase의 가용성 발현 증대

When alginate lyase gene (aly) from Pseudoalteromonas elyakovii was expressed in E. coli, most of the gene product was produced as aggregated insoluble particles known as inclusion bodies. In order to produce a soluble and active form of alginate lyase, E. coli cells fore cotransformed with the plasmids designed to permit coexpression of aly together with molecular chaperones such as DnaK/DnaJ/GrpE or GroEL/ES chaperones. The results revealed that the coexpression of aly together with DnaK/DnaJ/GrpE chaperone had a marked effect on the production of this protein as a soluble and active form, presumably through facilitating correct folding of alginate lyase protein. The optimal concentration of L-arabinose for the induction of DnaK/DnaJ/GrpE chaperone was found to be 0.05 mg/ml. When DnaK/DnaJ/GrpE chaperone was coexpressed, about 34% in the total alginate lyase was produced in the soluble fraction. By addition of 10% cetylpyridinium chloride, a clear zone around the colony coexpressing aly and DnaK/DnaJ/GrpE chaperone was formed, indicating that the alginate in the medium was hydrolyzed by active alginate lyase enzyme.

Effect of molecular chaperones on the soluble expression of alginate lyase inE. coli

When the alginate lyase gene (aly) fromPseudoalteromonas elyakovii was expressed inE. coli, most of the gene product was organized as aggregated insoluble particles known as inclusion bodies. To examine the effects of chaperones on soluble and nonaggregated form of alginate lyase inE. coli, we constructed plasmids designed to permit the coexpression ofaly and the DnaK/DnaJ/GrpE or GroEL/ES chaperones. The results indicate that coexpression ofaly with the Dnak/DnaJ/GrpE chaperone together had a marked effect on the yield alginate lyase as a soluble and active form of the enzyme. It is speculated this result occurs through facilitation of the correct folding of the protein. The optimal concentration ofl-arabinose required for the induction of the DnaK/DnaJ/GrpE chaperone was found to be 0.05 mg/mL. An analysis of the protein bands on SDS-PAGE gel indicated that at least 37% of total alginate lyase was produced in the soluble fraction when the DnaK/DnaJ/GrpE chaperone was coexpressed.

Metabolic Control Analysis of Aspergillus nigerl‐Arabinose Catabolism

A mathematical model of the L-arabinose/D-xylose catabolic pathway of Aspergillus niger was constructed based on the kinetic properties of the enzymes. For this purpose L-arabinose reductase, L-arabitol dehydrogenase and D-xylose reductase were purified using dye-affinity chromatography, and their kinetic properties were characterized. For the other enzymes of the pathway the kinetic data were available from the literature. The metabolic model was used to analyze flux and metabolite concentration control of the L-arabinose catabolic pathway. The model demonstrated that flux control does not reside at the enzyme following the intermediate with the highest concentration, L-arabitol, but is distributed over the first three steps in the pathway, preceding and following L-arabitol. Flux control appeared to be strongly dependent on the intracellular L-arabinose concentration. At 5 mM intracellular L-arabinose, a level that resulted in realistic intermediate concentrations in the model, flux control coefficients for L-arabinose reductase, L-arabitol dehydrogenase and L-xylulose reductase were 0.68, 0.17 and 0.14, respectively. The analysis can be used as a guide to identify targets for metabolic engineering aiming at either flux or metabolite level optimization of the L-arabinose catabolic pathway of A. niger. Faster L-arabinose utilization may enhance utilization of readily available organic waste containing hemicelluloses to be converted into industrially interesting metabolites or valuable enzymes or proteins.

Bacterial expression system with tightly regulated gene expression and plasmid copy number

A new Escherichia coli host/vector system has been engineered to allow tight and uniform modulation of gene expression and γ origin ( ori) plasmid copy number. Regulation of γ ori plasmid copy number is achieved through arabinose-inducible expression of the necessary Rep protein, π, whose gene was integrated into the chromosome of the host strain under control of the P BAD promoter. γ ori replication can be uniformly modulated over 100-fold by changing the concentration of l-arabinose in the growth medium. This strain avoids the problem of all-or-nothing induction of P BAD because it is deficient in both arabinose uptake and degradation genes. Arabinose enters the cell by a mutant LacY transporter, LacYA177C, which is expressed from the host chromosome. Although this strain could be compatible with any γ ori plasmid, we describe the utility of a γ ori expression vector that allows especially tight regulation of gene expression. With this host/vector system, it is possible to independently modulate gene expression and gene dosage, facilitating the cloning and overproduction of toxic gene products. We describe the successful use of this system for cloning a highly potent toxin, Colicin E3, in the absence of its cognate immunity protein. This system could be useful for cloning genes encoding other potent toxins, screening libraries for potential toxins, and maintaining any γ ori vector at precise copy levels in a cell.

A Novel NADH-linked L-Xylulose Reductase in the L-Arabinose Catabolic Pathway of Yeast

An NADH-dependent l-xylulose reductase and the corresponding gene were identified from the yeast Ambrosiozyma monospora. The enzyme is part of the yeast pathway for l-arabinose catabolism. A fungal pathway for l-arabinose utilization has been described previously for molds. In this pathway l-arabinose is sequentially converted to l-arabinitol, l-xylulose, xylitol, and d-xylulose and enters the pentose phosphate pathway as d-xylulose 5-phosphate. In molds the reductions are NADPH-linked, and the oxidations are NAD(+)-linked. Here we show that in A. monospora the pathway is similar, i.e. it has the same two reduction and two oxidation reactions, but the reduction by l-xylulose reductase is not performed by a strictly NADPH-dependent enzyme as in molds but by a strictly NADH-dependent enzyme. The ALX1 gene encoding the NADH-dependent l-xylulose reductase is strongly expressed during growth on l-arabinose as shown by Northern analysis. The gene was functionally overexpressed in Saccharomyces cerevisiae and the purified His-tagged protein characterized. The reversible enzyme converts l-xylulose to xylitol. It also converts d-ribulose to d-arabinitol but has no activity with l-arabinitol or adonitol, i.e. it is specific for sugar alcohols where, in a Fischer projection, the hydroxyl group of the C-2 is in the l-configuration and the hydroxyl group of C-3 is in the d-configuration. It also has no activity with C-6 sugars or sugar alcohols. The K(m) values for l-xylulose and d-ribulose are 9.6 and 4.7 mm, respectively. To our knowledge this is the first report of an NADH-linked l-xylulose reductase.

E. coli에서 GroEL/ES chaperone 공발현에 의한 활성형 cyclodextrin glucanotransferase의 생산 증대

Chaperone 분자는 세포 내에서 새로 합성된 polypeptides의 misfolding을 보호하는 역할을 가진다. 이런 chaperone 분자와의 공발현은 활성형 재조합 단백질의 생산을 증가를 기대할 수 있다. 본 연구에서는 E. cozi에서 B. macerans 유래 cyclodextrin glucanotransferase (CGTase)의 활성형 생산에 GroEL/ES chaperone과의 공발현의 효과에 대해 조사하였다. cgt와 groEL/ES 유전자출 발현하는 pTCGT1과 pGro7은 각각 T7 promoter와 araB promoter에 의해 조절되고 이들을 E. coli cell에 co-transformation시켰다. 재조합 E. coli에서 IPTG와 L-arabinose의 최적 농도를 결정하기 위해 행한 결과 1 mM IPTG, 0.3 mg L-arabinose/<TEX>$m\ell$</TEX>에서 가장 높은 CGTase 활성을 나타내었다. 그리고 tube에서는 L-arabinose와 IPTG를 각각 0.4~0.5 <TEX>$OD_{600}$</TEX>과 0.8~l.0 <TEX>$OD_{600}$</TEX>에서 첨가하였을 때 활성형 CGTase의 생산이 증가되었다. GroEL/ES 공발현 조건에서는 가용성 CGTase 활성이 0.7~0.73 unit/<TEX>$m\ell$</TEX>로 단독 발현의 0.36~0.56 unit/<TEX>$m\ell$</TEX>에 비해 약 1.5 배 정도 증가함을 알 수 있었다. SDS-PAGE 분석에서는 GroEL/ES 공발현 조건에서 총 CGTase의 33.6%정도가 가용성 형태로 생산됨을 알 수 있었다. Molecular chaperones prevent the misfolding of newly synthesized polypeptides in the cell. The coexpression of molecular chaperones could be expected to improve the production of soluble and active recombinant proteins. In this study, the effect of coexpression of E. coli GroEL/ES chaperone on the active production of Bacillus macerans cyclodextrin glucanotransferase (CGTase) in E. coli was investigated. Two plasmids, pTCGT1 and pGro7 in which the cgt and the groEL/ES genes are under the control of 77 promoter and araB promoter, respectively, were co-transformed into E. coli. With a series of cultures of recombinant E. coli cells, the optimal concentrations of IPTG and L-arabinose were found be 1 mM and 0.3 mg/<TEX>$m\ell$</TEX>, respectively. When IPTG and L-arabinose were added at 0.8~1.0 <TEX>$OD_{600}$</TEX> and 0.4~0.5 <TEX>$OD_{600}$</TEX>, active CGTase production was increased significantly. This coexpression condition resulted in 1.5-fold increased level of soluble CGTase (0.7~0.73 unit/<TEX>$m\ell$</TEX>), compared to the level of CGTase in the single expression (0.36~0.56 unit/<TEX>$m\ell$</TEX>). An SDS-PACE analysis revealed that about 33.6% of CGTase in the total CGTase protein was found in the soluble fraction by coexpression of GroEL/ES chaperone.

Regulated expression of the Escherichia coli lepB gene as a tool for cellular testing of antimicrobial compounds that inhibit signal peptidase I in vitro.

Escherichia coli under-expressing lepB was utilized to test cellular inhibition of signal peptidase I (SPase). For the construction of a lepB regulatable strain, the E. coli lepB gene was cloned into pBAD, with expression dependent on L-arabinose. The chromosomal copy of lepB was replaced with a kanamycin resistance gene, which was subsequently removed. SPase production by the lepB regulatable strain in the presence of various concentrations of L-arabinose was monitored by Western blot analysis. At lower arabinose concentrations growth proceeded more slowly, possibly due to a decrease of SPase levels in the cells. A penem SPase inhibitor with little antimicrobial activity against E. coli when tested at 100 micro M was utilized to validate the cell-based system. Under-expression of lepB sensitized the cells to penem, with complete growth inhibition observed at 10 to 30 micro M. Growth was rescued by increasing the SPase levels. The cell-based assay was used to test cellular inhibition of SPase by compounds that inhibit the enzyme in vitro. MD1, MD2, and MD3 are SPase inhibitors with antimicrobial activity against Staphylococcus aureus, although they do not inhibit growth of E. coli. MD1 presented the best spectrum of antimicrobial activity. Both MD1 and MD2 prevented growth of E. coli under-expressing lepB in the presence of polymyxin B nonapeptide, with growth rescue observed when wild-type levels of SPase were produced. MD3 and MD4, a reactive analog of MD3, inhibited growth of E. coli under-expressing lepB. However, growth rescue in the presence of these compounds following increased lepB expression was observed only after prolonged incubation.

Internal response correction for fluorescent whole-cell biosensors.

Whole-cell biosensors based on reporter genes are finding a variety of applications in analytical chemistry. Despite their ability to selectively recognize the analyte in a complex mixture, few applications of such sensing devices to real sample analysis are reported. This is mainly due to nonspecific effects on the biosensor response caused by components of the sample matrix and by environmental changes. To overcome this problem, a bacterial biosensor with an internal correction mechanism of the analytical response was developed by introducing an additional reporter gene that provides a reference signal of the analytical performance of the biosensor. The first reporter (GFPuv), expressed in response to the concentration of L-arabinose, provides the analytical signal; the second reporter (EYFP), constitutively expressed if a constant amount of IPTG is added to each sample, was used as an internal reference. By inducing the biosensor with varying amounts of L-arabinose and a constant amount of IPTG, it was possible to obtain a dose-response curve for L-arabinose, together with a constant production of EYFP, which allowed for a dynamic evaluation of the metabolic activity of the cell. When tested in nonoptimal conditions (e.g., in the presence of either ethanol or deoxycholic acid at toxic concentrations), the presence of the internal reference system corrected the analytical response due to nonspecific interferences.