Novel cyanobacterial biosensor for detection of herbicides

The aim of this work was to generate a cyanobacterial biosensor that could be used to detect herbicides and other environmental pollutants. A representative freshwater cyanobacterium, Synechocystis sp. strain PCC6803, was chromosomally marked with the luciferase gene luc (from the firefly Photinus pyralis) to create a novel bioluminescent cyanobacterial strain. Successful expression of the luc gene during growth of Synechocystis sp. strain PCC6803 cultures was characterized by measuring optical density and bioluminescence. Bioluminescence was optimized with regard to uptake of the luciferase substrate, luciferin, and the physiology of the cyanobacterium. Bioassays demonstrated that a novel luminescent cyanobacterial biosensor has been developed which responded to a range of compounds including different herbicide types and other toxins. This biosensor is expected to provide new opportunities for the rapid screening of environmental samples or for the investigation of potential environmental damage.     

The styrene-responsive StyS/StyR regulation system controls expression of an auxiliary phenylacetyl-coenzyme A ligase: implications for rapid metabolic coupling of the styrene upper- and lower-degradative pathways

Pseudomonas sp. strain Y2 degrades styrene through oxidation to phenylacetic acid via the styABCD operon-encoded enzymes, whose expression is induced in response to styrene by the StyS/StyR two-component regulatory system. Further transformation of phenylacetic acid to tricarboxylic acid cycle intermediates is mediated by the enzymes of paa catabolic genes, whose expression is regulated by the PaaX repressor. The first step of this paa degradation pathway is catalysed by paaF-encoded phenylacetyl-coenzyme A ligases that produce phenylacetyl-coenzyme A. This metabolic intermediate, upon being bound by PaaX, inactivates PaaX-mediated repression of both the paa genes and the styABCD operon. Strain Y2 is unique in having three paaF genes located within two complete copies of the paa gene clusters. Expression of both paaF and paaF3 is controlled by the PaaX repressor. Here we use specific mutants in combination with in vivo and in vitro assays to demonstrate that paaF2, adjacent to the StyS/StyR regulatory genes, belongs to the StyR regulon and is not subject to repression by PaaX. We propose that this unexpected styrene-responsive regulatory strategy for the otherwise metabolically redundant PaaF2 auxiliary enzyme provides a system for rapid co-ordinate de-repression of the two sets of catabolic genes required for styrene degradation.     

Novel Cyanobacterial Biosensor for Detection of Herbicides

The aim of this work was to generate a cyanobacterial biosensor that could be used to detect herbicides and other environmental pollutants. A representative freshwater cyanobacterium, Synechocystis sp. strain PCC6803, was chromosomally marked with the luciferase gene luc (from the firefly Photinus pyralis) to create a novel bioluminescent cyanobacterial strain. Successful expression of the luc gene during growth of Synechocystis sp. strain PCC6803 cultures was characterized by measuring optical density and bioluminescence. Bioluminescence was optimized with regard to uptake of the luciferase substrate, luciferin, and the physiology of the cyanobacterium. Bioassays demonstrated that a novel luminescent cyanobacterial biosensor has been developed which responded to a range of compounds including different herbicide types and other toxins. This biosensor is expected to provide new opportunities for the rapid screening of environmental samples or for the investigation of potential environmental damage.     

Novel organization of catechol <Emphasis Type="Italic">meta</Emphasis> pathway genes in the nitrobenzene degrader <Emphasis Type="Italic">Comamonas</Emphasis> sp. JS765 and its evolutionary implication

The catechol meta cleavage pathway is one of the central metabolic pathways for the degradation of aromatic compounds. A novel organization of the pathway genes, different from that of classical soil microorganisms, has been observed in Sphingomonas sp HV3 and Pseudomonas sp. DJ77. In a Comamonas sp. JS765, cdoE encoding catechol 2,3-dioxygenase shares a common ancestry only with tdnC of a Pseudomonas putida strain, while codG encoding 2-hydroxymuconic semialdehyde dehydrogenase shows a higher degree of similarity to those genes in classical bacteria. Located between cdoE and cdoG are several putative genes, whose functions are unknown. These genes are not found in meta pathway operons of other microorganisms with the exception of cdoX2, which is similar to cmpX in strain HV3. Therefore, the gene cluster in JS765 reveals a third type of gene organization of the meta pathway.     

Carbon metabolism and product inhibition determine the epoxidation efficiency of solvent-tolerant Pseudomonas sp. strain VLB120DeltaC

Utilization of solvent tolerant bacteria as biocatalysts has been suggested to enable or improve bioprocesses for the production of toxic compounds. Here, we studied the relevance of solvent (product) tolerance and inhibition, carbon metabolism, and the stability of biocatalytic activity in such a bioprocess. Styrene degrading Pseudomonas sp. strain VLB120 is shown to be solvent tolerant and was engineered to produce enantiopure (S)-styrene oxide from styrene. Whereas glucose as sole source for carbon and energy allowed efficient styrene epoxidation at rates up to 97 micromol/min/(g cell dry weight), citrate was found to repress epoxidation by the engineered Pseudomonas sp. strain VLB120DeltaC emphasizing that carbon source selection and control is critical. In comparison to recombinant Escherichia coli, the VLB120DeltaC-strain tolerated higher toxic product levels but showed less stable activities during fed-batch cultivation in a two-liquid phase system. Epoxidation activities of the VLB120DeltaC-strain decreased at product concentrations above 130 mM in the organic phase. During continuous two-liquid phase cultivations at organic-phase product concentrations of up to 85 mM, the VLB120DeltaC-strain showed stable activities and, as compared to recombinant E. coli, a more efficient glucose metabolism resulting in a 22% higher volumetric productivity. Kinetic analyses indicated that activities were limited by the styrene concentration and not by other factors such as NADH availability or catabolite repression. In conclusion, the stability of activity of the solvent tolerant VLB120DeltaC-strain can be considered critical at elevated toxic product levels, whereas the efficient carbon and energy metabolism of this Pseudomonas strain augurs well for productive continuous processing.     

藤黄绿脓菌素的自诱导及假单胞菌M18抗生物质代谢相关性初步分析

假单胞菌M18的生防功能归功于其分泌吩嗪-1-羧酸和藤黄绿脓菌素。为了研究抗生物质合成代谢相关性及调控机制,分别构建了两种抗生物质合成基因簇插入突变株M18T和M18Z1。用翻译融合表达载体pMEAZ(pltA′-′lacZ)分别转化野生株和突变株M18T、发酵培养并测定β-半乳糖苷酶活性,结果显示,添加藤黄绿脓菌素使突变株M18T(pMEAZ)的β-半乳糖苷酶活性比野生株M18(pMEAZ)增加约6倍,表明藤黄绿脓菌素对自身基因簇具正向自诱导作用。抗生物质的测定结果显示,突变株M18T无藤黄绿脓菌素合成,而吩嗪-1-羧酸的合成量与野生株相同;突变株M18Z1与野生株相比,吩嗪-1-羧酸明显减少,藤黄绿脓菌素却显著提高。过量的吩嗪-1-羧酸又抑制藤黄绿脓菌素的合成。表明,假单胞菌M18中独有的代谢相关方式为:藤黄绿脓菌素不影响吩嗪-1-羧酸,但吩嗪-1-羧酸负调控藤黄绿脓菌素。     

Lipase and its modulator from Pseudomonas sp. strain KFCC 10818: proline-to-glutamine substitution at position 112 induces formation of enzymatically active lipase in the absence of the modulator

A lipase gene, lipK, and a lipase modulator gene, limK, of Pseudomonas sp. strain KFCC 10818 have been cloned, sequenced, and expressed in Escherichia coli. The limK gene is located immediately downstream of the lipK gene. Enzymatically active lipase was produced only in the presence of the limK gene. The effect of the lipase modulator LimK on the expression of active lipase was similar to those of the Pseudomonas subfamily I.1 and I.2 lipase-specific foldases (Lifs). The deduced amino acid sequence of LimK shares low homology (17 to 19%) with the known Pseudomonas Lifs, suggesting that Pseudomonas sp. strain KFCC 10818 is only distantly related to the subfamily I.1 and I.2 Pseudomonas species. Surprisingly, a lipase variant that does not require LimK for its correct folding was isolated in the study to investigate the functional interaction between LipK and LimK. When expressed in the absence of LimK, the P112Q variant of LipK formed an active enzyme and displayed 63% of the activity of wild-type LipK expressed in the presence of LimK. These results suggest that the Pro(112) residue of LipK is involved in a key step of lipase folding. We expect that the novel finding of this study may contribute to future research on efficient expression or refolding of industrially important lipases and on the mechanism of lipase folding.     

Phenol/cresol degradation by the thermophilic Bacillus thermoglucosidasius A7: cloning and sequence analysis of five genes involved in the pathway

Bacillus thermoglucosidasius A7 degraded phenol at 65 degrees C via the meta cleavage pathway. Five enzymes used in the metabolism of phenol were cloned from B. thermoglucosidasius A7 into pUC18. Nine open reading frames were present on the 8.1kb insert, six of which could be assigned a function in phenol degradation using database homologies and enzyme activities. The phenol hydroxylase is a two-component enzyme encoded by pheA1 and pheA2. The larger component (50kDa) has 49% amino acid identity with the 4-hydroxyphenylacetate hydroxylase of Escherichia coli, while the smaller component (19kDa) is most related (30% amino acid identity) to the styrene monoxygenase component B from Pseudomonas fluorescens. Both components were neccessary for activity. The catechol 2, 3-dioxygenase encoded by pheB has 45% amino acid identity with dmpB of Pseudomonas sp. CF600 and could be assigned to superfamily I, family 2 and a new subfamily of the Eltis and Bolin grouping. The 2-hydroxymuconic acid semialdehyde hydrolase (2HMSH), encoded by pheC, revealed the highest amino acid identity (36%) to the equivalent enzyme from Pseudomonas sp. strain CF600, encoded by dmpD. Based on sequence identity, pheD and pheE were deduced to encode the 2-hydroxypenta-2,4-dienoate hydratase (2HDH), demonstrating 45% amino acid identity to the gene product of cumE from Pseudomonas fluorescens and the acetaldehyde dehydrogenase (acylating) demonstrating 57% amino acid identity to the gene product of bphJ from Pseudomonas LB400.     

Production host selection for asymmetric styrene epoxidation: Escherichia coli vs. solvent-tolerant Pseudomonas

Selection of the ideal microbe is crucial for whole-cell biotransformations, especially if the target reaction intensively interacts with host cell functions. Asymmetric styrene epoxidation is an example of a reaction which is strongly dependent on the host cell owing to its requirement for efficient cofactor regeneration and stable expression of the styrene monooxygenase genes styAB. On the other hand, styrene epoxidation affects the whole-cell biocatalyst, because it involves toxic substrate and products besides the burden of additional (recombinant) enzyme synthesis. With the aim to compare two fundamentally different strain engineering strategies, asymmetric styrene epoxidation by StyAB was investigated using the engineered wild-type strain Pseudomonas sp. strain VLB120ΔC, a styrene oxide isomerase (StyC) knockout strain able to accumulate (S)-styrene oxide, and recombinant E. coli JM101 carrying styAB on the plasmid pSPZ10. Their performance was analyzed during fed-batch cultivation in two-liquid phase biotransformations with respect to specific activity, volumetric productivity, product titer, tolerance of toxic substrate and products, by-product formation, and product yield on glucose. Thereby, Pseudomonas sp. strain VLB120ΔC proved its great potential by tolerating high styrene oxide concentrations and by the absence of by-product formation. The E. coli-based catalyst, however, showed higher specific activities and better yields on glucose. The results not only show the importance but also the complexity of host cell selection and engineering. Finding the optimal strain engineering strategy requires profound understanding of bioprocess and biocatalyst operation. In this respect, a possible negative influence of solvent tolerance on yield and activity is discussed.     

Expression and characterization of styrene monooxygenases of Rhodococcus sp. ST-5 and ST-10 for synthesizing enantiopure (S)-epoxides

Styrene monooxygenase (StyA, SMOA)- and flavin oxidoreductase (StyB, SMOB)-coding genes of styrene-assimilating bacteria Rhodococcus sp. ST-5 and ST-10 were successfully expressed in Escherichia coli. Determined amino acid sequences of StyAs and StyBs of ST-5 and ST-10 showed more similarity with those of Pseudomonas than with self-sufficient styrene monooxygenase (StyA2B) of Rhodococcus. Recombinant enzymes were purified from E. coli cells as functional proteins, and their properties were characterized in detail. StyBs (flavin oxidoreductase) of strains ST-5 and ST-10 have similar enzymatic properties to those of Pseudomonas, but StyB of strain ST-10 exhibited higher temperature stability than that of strain ST-5. StyAs of strains ST-5 and ST-10 catalyzed the epoxidation of vinyl side-chain of styrene and its derivatives and produced (S)-epoxides from styrene derivatives and showed high stereoselectivity. Both StyAs showed higher specific activity on halogenated styrene derivatives than on styrene itself. Additionally, the enzymes could catalyze the epoxidation of short-chain 1-alkenes to the corresponding (S)-epoxides. Aromatic compounds including styrene, 3-chlorostyrene, styrene oxide, and benzene exhibited marked inhibition of SMO reaction, although linear 1-alkene showed no inhibition of SMO activity at any concentration.     

假单胞菌M18株pltZ基因转录阻抑藤黄绿菌素ABC转运系统

假单胞菌(Pseudomonassp .)M18株的藤黄绿菌素(Pyoluteorin ,Plt )生物合成基因簇下游存在一个Plt生物合成负调控基因pltZ和一个负责Plt分泌及自身抗性的ABC(ATP_bindingcassette)转运系统基因簇。利用启动子探针载体pME6 0 15和pME6 5 2 2分别构建ABC转运基因pltH与lacZ的翻译和转录融合表达质粒pHZLF和pHZCF ,分别引入野生型假单胞菌M18株和pltZ突变菌株M18Z。半乳糖苷酶活性的测定结果表明:在pltZ突变株M18Z中,pltH’-‘lacZ翻译融合表达水平约比野生型提高3 7～8 4倍,pltH’‘lacZ转录融合表达水平显著提高了2 8～7 4倍,表明pltZ能在转录水平上阻抑PltABC转运系统的表达,pltZ很可能通过阻抑PltABC转运系统的表达,间接地负调控Plt的生物合成     

RpoS as an intermediate in RsmA-dependent regulation of secondary antifungal metabolites biosynthesis in Pseudomonas sp. M18

To investigate the regulatory mechanism governing antifungal metabolite biosynthesis, two kinds of global regulator genes in Pseudomonas sp. M18, an rpoS and an rsmA gene, were cloned and mutated by inserting with an aacC1 cassette, respectively. Two mutants showed the same regulatory mode with repression of phenazine-1-carboxylic acid and remarkable enhancement of pyoluteorin. In the rpoS-mutant, a translational rsmA'-'lacZ fusion was expressed at the same level corresponding to that in the wild-type strain. In the rsmA-mutant, however, expression of the translational rpoS'-'lacZ fusion was only about 30% of that in the wild-type strain. The results indicated that the absence of RsmA leads to repression of the rpoS gene expression, which has further been confirmed with construction of chromosomal rpoS-lacZ fusion in the rsmA-mutant and the wild-type strain, respectively. The findings showed a new regulatory cascade controlling antifungal metabolite production in Pseudomonas sp. M18, suggesting that RpoS may serve as a mediator in RsmA-dependent regulation of secondary metabolite biosynthesis.