Enhanced biodegradation of toxic organophosphate compounds using recombinant Escherichia coli with sec pathway-driven periplasmic secretion of organophosphorus hydrolase

Although Escherichia coli can be genetically engineered to degrade environmental toxic organophosphate compounds (OPs) to nontoxic materials, a critical problem in such whole cell systems is limited substrate diffusion. The present work examined whether periplasmic expression of organophosphorus hydrolase (OPH) resulted in better whole cell enzymatic activity compared to standard cytosolic expression. Recombinant OPH periplasmic expression was achieved using the general secretory (sec) pathway with the pelB signal sequence. We found that while total OPH activity in periplasmic-expressing cell lysates was lower compared to that in cytosolic-expressing cell lysates whole cell OPH activity was 1.8-fold greater at 12 h post-induction in the periplasmic-expressing cells as a result of OPH translocation into the periplasmic space ( approximately 67% of whole cell OPH activity was found in the periplasmic fraction). These data suggest that E. coli engineered to periplasmically secrete OPH via the sec pathway may provide an improved whole cell biodegradation system for destruction of environmental toxic OPs.     

Coexpression of molecular chaperone enhances activity and export of organophosphorus hydrolase in Escherichia coli

Periplasmic secretion has been used in attempts to construct an efficient whole‐cell biocatalyst with greatly reduced diffusion limitations. Previously, we developed recombinant Escherichia coli that express organophosphorus hydrolase (OPH) in the periplasmic space using the twin‐arginine translocation (Tat) pathway to degrade environmental toxic organophosphate compounds. This system has the advantage of secreting protein into the periplasm after folding in the cytoplasm. However, when OPH was expressed with a Tat signal sequence in E. coli, we found that the predominant OPH was an insoluble premature form in the cytoplasm, and thus, the whole‐cell OPH activity was significantly lower than its cell lysate activity. In this work, we, for the first time, used a molecular chaperone coexpression strategy to enhance whole‐cell OPH activity by improving the periplasmic translocation of soluble OPH. We found that the effect of GroEL‐GroES (GroEL/ES) assistance on the periplasmic localization of OPH was secretory pathway dependent. We observed a significant increase in the amount of soluble mature OPH when cytoplasmic GroEL/ES was expressed; this increase in the amount of mature OPH might be due to enhanced OPH folding in the cytoplasm. Importantly, the whole‐cell OPH activity of the chaperone–coexpressing cells was ～5.5‐fold greater at 12 h after induction than that of cells that did not express the chaperone as a result of significant Tat‐based periplasmic translocation of OPH in the chaperone–coexpressing cells. Collectively, these data suggest that molecular chaperones significantly enhance the whole‐cell activity of periplasmic OPH‐secreting cells, yielding an effective whole‐cell biocatalyst system with highly reduced diffusion limitations. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 925–930, 2012     

Versatile signal peptide of Flavobacterium‐originated organophosphorus hydrolase for efficient periplasmic translocation of heterologous proteins in Escherichia coli

Organophosphorus hydrolase (OPH) from Flavobacterium species is a membrane‐associated homodimeric metalloenzyme and has its own signal peptide in its N‐terminus. We found that OPH was translocated into the periplasmic space when the original signal peptide‐containing OPH was expressed in recombinant Escherichia coli even though its translocation efficiency was relatively low. To investigate the usability of this OPH signal peptide for periplasmic expression of heterologous proteins in an E. coli system, we employed green fluorescent protein (GFP) as a cytoplasmic folding reporter and alkaline phosphatase (ALP) as a periplasmic folding reporter. We found that the OPH signal peptide was able to use both twin‐arginine translocation (Tat) and general secretory (Sec) machineries by switching translocation pathways according to the nature of target proteins in E. coli. These results might be due to the lack of Sec‐avoidance sequence in the c‐region and a moderate hydrophobicity of the OPH signal peptide. Interestingly, the OPH signal peptide considerably enhanced the translocation efficiencies for both GFP and ALP compared with commonly used TorA and PelB signal peptides that have Tat and Sec pathway dependences, respectively. Therefore, this OPH signal peptide could be successfully used in recombinant E. coli system for efficient periplasmic production of target protein regardless of the subcellular localization where functional folding of the protein occurs. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:848–854, 2016     

Selection of a whole-cell biocatalyst for methyl parathion biodegradation

Whole-cell biocatalyst has the potential to become a cost-effective alternative to conventional enzyme methods for solving ecological and energy issues. However, cytosolic-expressing biocatalyst systems are critically disadvantaged due to the low permeability of the cell membrane. To overcome substrate transport barrier, periplasmic secretion and surface display biocatalysts were developed by expressing signal peptides or anchor proteins in Escherichia coli. In this work, six carriers were compared in regard to whole-cell activity of methyl parathion hydrolase (MPH). Our results indicate that the surface display systems yielded one to three times whole-cell activity than the periplasmic secretion systems. Although periplasmic secretion systems showed generally more stable than surface display systems, surface display appeared more suitable for whole-cell biocatalyst. It should note that the applicability of the DsbA/PhoA/AIDA-I leader to MPH expression is shown here for the first time. In addition, the result provided a useful reference for other whole-cell biocatalyst selection.     

Extracellular recombinant protein production from an Escherichia coli lpp deletion mutant

E. coli is one of the most commonly used host strains for recombinant protein production. However, recombinant proteins are usually found intracellularly, in either cytoplasm or periplasmic space. Inadequate secretion to the extracellular environment is one of its limitations. This study addresses the outer membrane barrier for the translocation of recombinant protein directed to the periplasmic space. Specifically, using recombinant maltose binding protein (MalE), xylanase, and cellulase as model proteins, we investigated whether the lpp deletion could render the outer membrane permeable enough to allow extracellular protein production. In each case, significantly higher excretion of recombinant protein was observed with the lpp deletion mutant. Up to 90% of the recombinant xylanase activity and 70% of recombinant cellulase activity were found in the culture medium with the deletion mutant, whereas only 40-50% of the xylanase and cellulase activities were extracellular for the control strain. Despite the weakened outer membrane in the mutant strain, cell lysis did not occur, and increased excretion of periplasmic protein was not due to cell lysis. The lpp deletion is a simple method to generate an E. coli strain to effect significant extracellular protein production. The phenotype of extracellular protein production without cell lysis is useful in many biotechnological applications, such as bioremediation and plant biomass conversion.     

Effect of culture conditions on the whole cell activity of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> expressing periplasmic organophosphorus hydrolase and cytosolic GroEL/ES chaperone

Specific whole cell activity strongly affects sensitivity and detection limit of whole cell-based biosensors. Previously, we developed recombinant Escherichia coli coexpressing periplasmic organophosphorus hydrolase (OPH) and cytosolic chaperone GroEL-GroES (GroEL/ES). In present work, we investigated the effect of culture conditions on whole cell OPH activity. Especially, the whole cell OPH activity was significantly affected by the concentration of tetracycline that is an inducer for chaperone GroEL/ES. When cultured at 20°C for 31 h in M9 medium containing 1 mM IPTG, 50 ng/mL tetracycline, and 500 µM CoCl₂, the recombinant E. coli exhibited a specific whole cell OPH activity (U/OD₆₀₀) of ~0.55, which is 2.6-fold higher than that of recombinant E. coli cultured as previously described conditions. In addition, recombinant cells showed adequate storage stability for 1 week with 100% of original response. Finally, the improved activity and adequate stability in the whole cell biocatalyst will contribute to sensitivity, detection time, and stability of a whole cell-based biosensor for the detection of toxic organophosphates.     

Simultaneous degradation of organophosphorus pesticides and p-nitrophenol by a genetically engineered Moraxella sp. with surface-expressed organophosphorus hydrolase.

Moraxella sp., a native soil organism that grows on p-nitrophenol (PNP), was genetically engineered for the simultaneous degradation of organophosphorus (OP) pesticides and p-nitrophenol (PNP). The truncated ice nucleation protein (INPNC) anchor was used to target the pesticide-hydrolyzing enzyme, organophosphorus hydrolase (OPH), onto the surface of Moraxella sp., alleviating the potential substrate uptake limitation. A shuttle vector, pPNCO33, coding for INPNC-OPH was constructed and the translocation, surface display, and functionality of OPH were demonstrated in both E. coli and Moraxella sp. However, whole cell activity was 70-fold higher in Moraxella sp. than E. coli. The resulting Moraxella sp. degraded organophosphates as well as PNP rapidly, all within 10 h. The initial hydrolysis rate was 0.6 micromol/h/mg dry weight, 1.5 micromol/h/mg dry weight, and 9.0 micromol/h/mg dry weight for methyl parathion, parathion, and paraoxon, respectively. The possibility of rapidly degrading OP pesticides and their byproducts should open up new opportunities for improved remediation of OP nerve agents in the future.     

IL-1023-57-PE40分泌表达的初步研究

将IL-1023-57-PE40基因与pelB信号肽融合置于pET-20b构建分泌表达质粒pET-20b-IL-1023-57-PE40,然后将pET-20b-IL-1023-57-PE40分别转化至BL21(DE3),BL21(DE3)pLysS,Rosetta(DE3),E·coliK12TB1,ER2566中。无论是在37℃或是在26℃,亦或在培养基中添加葡萄糖的情况下,IPTG诱导后,IL-1023-57-PE40蛋白只在BL21(DE3)pLysS菌中以可溶分泌形式表达,其中以37℃时培养基中不添加葡萄糖表达量为最高,占菌体蛋白总量的15%,说明蛋白的分泌表达与菌种的选择有关。表达产物经免疫印记检测可被抗PE40的特异抗体识别。通过质粒稳定性实验证明,pET-20b-IL-1023-57-PE40在BL21(DE3)中不稳定,导致蛋白的不表达,在Rosetta(DE3)BL21,E·coliK12TB1,ER2566中稳定但不表达,因此,以Rosetta(DE3)BL21为例,通过SDS-PAGE、DNAStar和ANThewin蛋白分析软件对本室构建的几种PE重组毒素进行比较分析,我们发现:并不是所有PE重组毒素融合信号肽序列后,就能分泌表达,PE重组毒素分泌表达还可能与导向部分的性质有关。     

Processing of DNase domain during translocation of colicin E7 across the membrane of Escherichia coli

Translocation of colicin across the membrane of sensitive cells has been studied extensively. However, processing of the toxicity domain of colicin during translocation has been the subject of much controversy. To investigate the final translocation product of colicin across the membrane of Escherichia coli, an endogenously expressed His-tagged Im7 protein was constructed to detect any translocation product containing the DNase domain traversed the inner membrane into cytoplasm of the E. coli cells. As a result, a final processed DNase domain of ColE7 was identified in the intracellular space of the cells treated with Col-Im complex. In the presence of periplasmic extracts, in vitro processing of DNase domain of ColE7 was also observed. These results suggest that the processing of ColE7 has occurred for translocation of the DNase-type colicin across the membrane and the process is probably taking place in the periplasmic space of the membrane.     

Flexibility in the receptor-binding domain of the enzymatic colicin E9 is required for toxicity against Escherichia coli cells

The events that occur after the binding of the enzymatic E colicins to Escherichia coli BtuB receptors that lead to translocation of the cytotoxic domain into the periplasmic space and, ultimately, cell killing are poorly understood. It has been suggested that unfolding of the coiled-coil BtuB receptor binding domain of the E colicins may be an essential step that leads to the loss of immunity protein from the colicin and immunity protein complex and then triggers the events of translocation. We introduced pairs of cysteine mutations into the receptor binding domain of colicin E9 (ColE9) that resulted in the formation of a disulfide bond located near the middle or the top of the R domain. After dithiothreitol reduction, the ColE9 protein with the mutations L359C and F412C (ColE9 L359C-F412C) and the ColE9 protein with the mutations Y324C and L447C (ColE9 Y324C-L447C) were slightly less active than equivalent concentrations of ColE9. On oxidation with diamide, no significant biological activity was seen with the ColE9 L359C-F412C and the ColE9 Y324C-L447C mutant proteins; however diamide had no effect on the activity of ColE9. The presence of a disulfide bond was confirmed in both of the oxidized, mutant proteins by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The loss of biological activity of the disulfide-containing mutant proteins was not due to an indirect effect on the properties of the translocation or DNase domains of the mutant colicins. The data are consistent with a requirement for the flexibility of the coiled-coil R domain after binding to BtuB.     

Defective export in Escherichia coli caused by DsbA'-PhoA hybrid proteins whose DsbA' domain cannot fold into a conformation resistant to periplasmic proteases.

The disulfide bond-forming factor DsbA and the alkaline phosphatase are stable in the Escherichia coli periplasmic space and can be overproduced without significant perturbation of the cell's physiology. By contrast, DsbA'-PhoA hybrid proteins resulting from TnphoA insertions into different regions of a plasmid-borne dsbA gene could become toxic (lethal) to bacteria. Toxicity was concomitant with an impairment of some step of the export mechanism and depended on at least three parameters, i.e., (i) the rate of expression of the hybrid protein, (ii) the ability of the amino-terminal DsbA' domain of the hybrid protein to fold into a protease-resistant conformation in the periplasmic space, and (iii) the activity of the DegP periplasmic protease. Even under viable conditions of low expression, DsbA' folding-deficient hybrid proteins accumulated more than the folding-proficient ones in the insoluble material and this was aggravated in a strain lacking the DegP protease. When production was more elevated, the folding-deficient hybrid proteins became lethal, but only in strains lacking the DegP activity, while the folding-proficient ones were not. Under conditions of very high production by degP+ or degP strains, both types of hybrid proteins accumulated as insoluble preproteins. Meanwhile, the export machinery was dramatically handicapped and the cells lost viability. However, the folding-deficient hybrid proteins had a higher killing efficiency than the folding-proficient ones. Free DsbA'-truncated polypeptides, although not toxic, were processed more slowly when they could not fold into a protease-resistant form in the periplasmic space. This provides indications in E. coli for a direct or indirect influence of the folding of a protein in the periplasmic environment on export efficiency.     

Escherichia coli rna gene encoding RNase I: cloning, overexpression, subcellular distribution of the enzyme, and use of an rna deletion to identify additional RNases.

The cloning and overexpression of the Escherichia coli rna gene encoding RNase I are described. Only a single copy of the rna gene is present on the E. coli chromosome. Although cells with as much as a 100-fold increase in RNase I activity were constructed, little effect on cell growth was observed. Overexpressed RNase I was found in the periplasmic space to the same degree (approximately 85%) as wild-type enzyme, suggesting no limitation in RNase I transport. The rna clone was used to identify a deletion strain totally lacking the rna gene. The normal growth of this strain showed that RNase I is not essential for cell viability. Extracts from the RNase I deletion strain still retained a low level of RNase activity in the presence of EDTA, conclusively demonstrating the existence of additional EDTA-active RNases in E. coli. The possibility of a RNase I inhibitor is also discussed.     

Killing of E coli cells by E group nuclease colicins

The process by which the endonuclease domain of colicin E9 is translocated across the outer membrane, the periplasmic space and the cytoplasmic membrane to reach the cytoplasm of E. coli cells, resulting in DNA degradation and cell death, is a unique event in prokaryotic biology. Although considerable information is known about the role of the BtuB outer membrane receptor, as well as the mostly periplasmic Tol proteins that are essential for the translocation process, the precise nature of the interactions between colicin E9 and these proteins remains to be elucidated. In this review, we consider our current understanding of the key events in this process, concentrating on recent findings concerning receptor-binding, translocation and the mechanism of cytotoxicity.     

Identification of an essential cleavage site in ColE7 required for import and killing of cells

Colicin E7 (ColE7), a nuclease toxin released from Escherichia coli, kills susceptible bacteria under environmental stress. Nuclease colicins are processed during translocation with only the cytotoxic nuclease domains traversing the inner membrane to cleave tRNA, rRNA, or DNA in the cytoplasm of target cells. In this study, we show that the E. coli periplasmic extract cleaves ColE7 between Lys(446) and Arg(447) in the presence or absence of its inhibitor Im7 protein. Several residues near cleavage sites were mutated, but only mutants of Arg(447) completely lost in vivo cell-killing activity. Both the full-length and the nuclease domain of Arg(447) mutants retained their nuclease activities, indicating that failure to kill cells was not a consequence of damage to the endonuclease activity of the enzyme. Moreover, the R447E ColE7 mutant was not cleaved at its 447 site by periplasmic extracts or transported into the cytoplasm of target cells. Collectively, these results suggest that ColE7 is cleaved at Arg(447) during translocation and that cleavage is an essential step for ColE7 import into the cytoplasm of target cells and its cell-killing activity. Conserved basic residues aligned with Arg(447) have also been found in other nuclease colicins, implying that the processing at this position may be common to other colicins during translocation.     

Novel synthetic signal peptides for the periplasmic secretion of green fluorescent protein in Escherichia coli

New secretion vectors containing synthetic signal peptides were constructed to study the periplasmic translocation of green fluorescent protein (GFP) in Escherichia coli. These constructs encode synthetic signal peptides spA and spD fused to the amino terminal end of GFP, and expressed from T7/lac promoter in the BL21DE3 strain by induction with IPTG. The recombinant protein was detected in both the cytoplasmic and periplasmic fractions. Fluorescence analysis revealed that recombinant proteins with signal peptides were not fluorescent, indicating translocation to periplasmic space. In contrast, recombinant proteins without signal peptide were fluorescent. These results indicate that the expressed recombinant proteins were translocated into the periplasm. Therefore, the synthetic signal peptides derived from signal peptides of Bacillus sp. could efficiently secrete the heterologous proteins to the periplasmic space of E. coli.     

Bacillus licheniformis6816碱性蛋白酶基因在E.coli中的克隆和表达

用PCR方法从地衣芽孢杆菌6816中扩增了碱性蛋白酶基因（apr),扩增的1.14kb的DNA片段插入到大肠杆菌载体pET-20b中,构建成重组分泌型表达载体pAPR1。pAPR1中碱性蛋白酶基因在大肠杆菌宿主JM109（DE3）中得到表达,SDS-PAGE分析显示融合表达产物的分子量为30kD,同核酸序列测定所推导的值相符,表达产物占细胞总蛋白的7.5%,重组菌的酶活比出发菌株提高了3.3倍,研究发现,重组的碱性蛋白酶在进入大肠杆菌周质空间时存在前肽自动脱落的现象。     

Cytochemical Localization of Certain Phosphatases in Escherichia coli

Cytochemical studies of Escherichia coli at the light and electron microscopic levels have revealed alkaline phosphatase, hexose monophosphatase, and cyclic phosphodiesterase reaction products in the periplasmic space and at the cell surface. In preparations for both light and electron microscopy, reaction product filled polar caplike enlargements of the periplasmic space, such as those described in plasmolyzed cells, indicating significant terminal concentrations of these enzymes; dense substance was often seen within these polar caps in morphological specimens. Staining of the bacterial surface was commonly encountered, but could represent artifactual accumulation of precipitate along the cell wall. Alkaline phosphatase was demonstrated with several substrates (ethanolamine phosphate, glycerophosphate, p-nitrophenylphosphate, and glucose-6-phosphate) over a wide pH range in a bacterial strain (C-90) known to be constitutive for this enzyme, whereas strains deficient in this enzyme (U-7, repressed K-37), showed no activity with these substrates. Hexose monophosphatase and cyclic phosphodiesterase activities were characterized by reaction-product deposition with specific substrates at acid or neutral, but not at alkaline, pH in strains of E. coli lacking alkaline phosphatase (U-7 and repressed K-37). Fixation in Formalin or the use of calcium as a capture reagent seemed to interfere with periplasmic staining in cells prepared for electron microscopy. Formalin fixation had little effect on biochemical assays of the phosphatase activity of intact cells in suspension, but partially reduced the activity evident in sonically treated extracts or in suspensions of dispersed cryostat sections. Glutaraldehyde treatment impaired enzyme activity more drastically.     

Membrane targeting of a folded and cofactor-containing protein.

Targeting of proteins to and translocation across the membranes is a fundamental biological process in all organisms. In bacteria, the twin arginine translocation (Tat) system can transport folded proteins. Here, we demonstrate in vivo that the high potential iron-sulfur protein (HiPIP) from Allochromatium vinosum is translocated into the periplasmic space by the Tat system of Escherichia coli. In vitro, reconstituted HiPIP precursor (preHoloHiPIP) was targeted to inverted membrane vesicles from E. coli by a process requiring ATP when the Tat substrate was properly folded. During membrane targeting, the protein retained its cofactor, indicating that it was targeted in a folded state. Membrane targeting did not require a twin arginine motif and known Tat system components. On the basis of these findings, we propose that a pathway exists for the insertion of folded cofactor-containing proteins such as HiPIP into the bacterial cytoplasmic membrane.