Xyl Operon Research Articles

High Production of γ-Aminobutyric Acid by Activating the xyl Operon of Lactobacillus brevis.

γ-Aminobutyric acid (GABA) is an inhibitory neurotransmitter with important physiological functions such as sleep assistance and anti-depression. In this study, we developed a fermentation process for the high-efficiency production of GABA by Lactobacillus brevis (Lb. brevis) CE701. First, xylose was found as the optimal carbon source that could improve the GABA production and OD600 in shake flasks to 40.35 g/L and 8.64, respectively, which were 1.78-fold and 1.67-fold of the glucose. Subsequently, the analysis of the carbon source metabolic pathway indicated that xylose activated the expression of the xyl operon, and xylose metabolism produced more ATP and organic acids than glucose, which significantly promoted the growth and GABA production of Lb. brevis CE701. Then, an efficient GABA fermentation process was developed by optimizing the medium components using response surface methodology. Finally, the production of GABA reached 176.04 g/L in a 5 L fermenter, which was 336% higher than that in a shake flask. This work enables the efficient synthesis of GABA using xylose, which will provide guidance for the industrial production of GABA.

Efficient anaerobic consumption of D-xylose by E. coli BL21(DE3) via xylR adaptive mutation

BackgroundMicroorganisms can prioritize the uptake of different sugars depending on their metabolic needs and preferences. When both D-glucose and D-xylose are present in growth media, E. coli cells typically consume D-glucose first and then D-xylose. Similarly, when E. coli BL21(DE3) is provided with both D-glucose and D-xylose under anaerobic conditions, glucose is consumed first, whereas D-xylose is consumed very slowly.ResultsWhen BL21(DE3) was adaptively evolved via subculture, the consumption rate of D-xylose increased gradually. Strains JH001 and JH019, whose D-xylose consumption rate was faster, were isolated after subculture. Genome analysis of the JH001 and JH019 strains revealed that C91A (Q31K) and C740T (A247V) missense mutations in the xylR gene (which encodes the XylR transcriptional activator), respectively, controlled the expression of the xyl operon. RT-qPCR analyses demonstrated that the XylR mutation caused a 10.9-fold and 3.5-fold increase in the expression of the xylA (xylose isomerase) and xylF (xylose transporter) genes, respectively, in the adaptively evolved JH001 and JH019 strains. A C91A adaptive mutation was introduced into a new BL21(DE3) background via single-base genome editing, resulting in immediate and efficient D-xylose consumption.ConclusionsAnaerobically-adapted BL21(DE3) cells were obtained through short-term adaptive evolution and xylR mutations responsible for faster D-xylose consumption were identified, which may aid in the improvement of microbial fermentation technology.

Production of poly-γ-glutamic acid (γ-PGA) from xylose-glucose mixtures by Bacillus amyloliquefaciens C1.

Due to the promising applications, the demand to enhance poly-γ-glutamic acid (γ-PGA) production while decreasing the cost has increased in the past decade. Here, xylose/glucose mixture and corncob hydrolysate (CCH) was evaluated as alternatives for γ-PGA production by Bacillus amyloliquefaciens C1. Although both have been validated to support cell growth, glucose and xylose were not simutaneously consumed and exhibited a diauxic growth pattern due to carbon catabolite repression (CCR) in B. amyloliquefaciens C1, while the enhanced transcription of araE alleviated the xylose transport bottleneck across a cellular membrane. Additionally, the xyl operon (xylA and xylB), which was responsible for xylose metabolism, was strongly induced by xylose at the transcriptional level. When cultured in a mixed medium, xylR was sharply induced to 3.39-folds during the first 8-h while reduced to the base level similar to that in xylose medium. Finally, pre-treated CCH mainly contained a mixture of glucose and xylose was employed for γ-PGA fermentation, which obtained a final concentration of 6.56 ± 0.27g/L. Although the glucose utilization rate (84.91 ± 1.81%) was lower than that with chemical substrates, the xylose utilization rate (43.41 ± 2.14%) and the sodium glutamate conversion rate (77.22%) of CCH were acceptable. Our study provided a promising approach for the green production of γ-PGA from lignocellulosic biomass and circumvent excessive non-food usage of glucose.

Production of polyhydroxyalkanoates (PHA) by a thermophilic strain of Schlegelella thermodepolymerans from xylose rich substrates

The aim of this work was to investigate the thermophilic bacterium Schelegelella thermodepolymerans DSM 15344 in terms of its polyhydroxyalkanoates (PHA) biosynthesis capacity. The bacterium is capable of converting various sugars into PHA with the optimal growth temperature of 55 °C; therefore, the process of PHA biosynthesis could be robust against contamination. Surprisingly, the highest yield was gained on xylose. Results suggested that S. thermodepolymerans possess unique xylose metabolism since xylose is utilized preferentially with the highest consumption rate as compared to other sugars. In the genome of S. thermodepolymerans DSM 15344, a unique putative xyl operon consisting of genes responsible for xylose utilization and also for its transport was identified, which is a unique feature among PHA producers. The bacterium is capable of biosynthesis of copolymers containing 3-hydroxybutyrate and also 3-hydroxyvalerate subunits. Hence, S.thermodepolymerans seems to be promising candidate for PHA production from xylose rich substrates.

Development of a strictly regulated xylose-induced expression system in Streptomyces

BackgroundGenetic tools including constitutive and inducible promoters have been developed over the last few decades for strain engineering in Streptomyces. Inducible promoters are useful for controlling gene expression, however only a limited number are applicable to Streptomyces. The aim of this study is to develop a controllable protein expression system based on an inducible promoter using sugar inducer, which has not yet been widely applied in Streptomyces.ResultsTo determine a candidate promoter, inducible protein expression was first examined in Streptomyces avermitilis MA-4680 using various carbon sources. Xylose isomerase (xylA) promoter derived from xylose (xyl) operon was selected due to strong expression of xylose isomerase (XylA) in the presence of d-xylose. Next, a xylose-inducible protein expression system was constructed by investigating heterologous protein expression (chitobiase as a model protein) driven by the xylA promoter in Streptomyces lividans. Chitobiase activity was detected at high levels in S. lividans strain harboring an expression vector with xylA promoter (pXC), under both xylose-induced and non-induced conditions. Thus, S. avermitilis xylR gene, which encodes a putative repressor of xyl operon, was introduced into constructed vectors in order to control protein expression by d-xylose. Among strains constructed in the study, XCPR strain harboring pXCPR vector exhibited strict regulation of protein expression. Chitobiase activity in the XCPR strain was observed to be 24 times higher under xylose-induced conditions than that under non-induced conditions.ConclusionIn this study, a strictly regulated protein expression system was developed based on a xylose-induced system. As far as we could ascertain, this is the first report of engineered inducible protein expression in Streptomyces by means of a xylose-induced system. This system might be applicable for controllable expression of toxic products or in the field of synthetic biology using Streptomyces strains.

Engineering genome-reduced Bacillus subtilis for acetoin production from xylose.

To investigate the capacity of a genome-reduced Bacillus subtilis strain as chassis cell for acetoin production from xylose. To endow the genome-reduced Bacillus subtilis strain BSK814 with the ability to utilize xylose, we inserted a native xyl operon into its genome and deleted the araR gene. The resulting strain BSK814A2 produced 2.94g acetoin/l from 10g xylose/l, which was 39% higher than control strain BSK19A2. The deletion of the bdhA and acoA genes further improved xylose utilization efficiency and increased acetoin production to 3.71g/l in BSK814A4. Finally, BSK814A4 produced up to 23.3g acetoin/l from 50g xylose/l, with a yield of 0.46g/g xylose. Both the titer and yield were 39% higher than those of control strain BSK19A4. As a chassis cell, genome-reduced B. subtilis showed significantly improved capacity for the production of the overflow product acetoin from xylose compared with wild-type strain.

Complete genome sequence of probiotic Bacillus coagulans HM-08: A potential lactic acid producer

Bacillus coagulans HM-08 is a commercialized probiotic strain in China. Its genome contains a 3.62Mb circular chromosome with an average GC content of 46.3%. In silico analysis revealed the presence of one xyl operon as well as several other genes that are correlated to xylose utilization. The genetic information provided here may help to expand its future biotechnology potential in lactic acid production.

Catabolite responsive element deficiency ofxyloperon resulting in carbon catabolite derepression inLactobacillus fermentum1001

To explore the molecular mechanism of the carbon catabolite derepression in Lactobacillus fermentum 1001 when this strain consumed xylose and glucose simultaneously. The transcriptional level of ccpAf was measured by real-time qPCR, revealing that ccpAf transcribed mRNA normally in Lact.fermentum 1001. The ccpAf gene could complement the ccpA-deficiency of a Lactococcus lactis mutant. Moreover, when the phosphofructokinase from Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 was expressed in Lact.fermentum 1001, the recombinant preferred glucose to fructose rather than to xylose. All data suggested that CcpAf was functional in Lact.fermentum 1001. In addition, the promoter (Plx) activity of the xyl operon from Lact.fermentum 1001 was further test in Lactobacillus casei BL23, and it could drive the expression of green fluorescent protein in the presence of glucose. The ability of Lact.fermentum 1001 to co-utilize xylose and glucose resulted from the deficiency of catabolite responsive element in P1x rather than the null mutation of the ccpAf gene. Lactobacillus fermentum 1001 is a potential candidate as a CCR-absent cell factory to transform biomass to high-value-added products. P1x was provided for engineering LAB to enhance fermentation efficiency by avoiding CCR.

High-resolution analysis of the m-xylene/toluene biodegradation subtranscriptome of Pseudomonas putida mt-2.

Pseudomonas putida mt-2 metabolizes m-xylene and other aromatic compounds through the enzymes encoded by the xyl operons of the TOL plasmid pWW0 along with other chromosomally encoded activities. Tiling arrays of densely overlapping oligonucleotides were designed to cover every gene involved in this process, allowing dissection of operon structures and exposing the interplay of plasmid and chromosomal functions. All xyl sequences were transcribed in response to aromatic substrates and the 3'-termini of both upper and lower mRNA operons extended beyond their coding regions, i.e. the 3'-end of the lower operon mRNA penetrated into the convergent xylS regulatory gene. Furthermore, xylR mRNA for the master m-xylene responsive regulator of the system was decreased by aromatic substrates, while the cognate upper operon mRNA was evenly stable throughout its full length. RNA sequencing confirmed these data at a single nucleotide level and refined the formerly misannotated xylL sequence. The chromosomal ortho route for degradation of benzoate (the ben, cat clusters and some pca genes) was activated by this aromatic, but not by the TOL substrates, toluene or m-xylene. We advocate this scenario as a testbed of natural retroactivity between a pre-existing metabolic network and a new biochemical pathway implanted through gene transfer.

TheTOLnetwork ofPseudomonas putidamt‐2 processes multiple environmental inputs into a narrowresponse space

The TOL system encoded by plasmid pWW0 of Pseudomonas putida mt-2 is able to sense a large number of both exogenous and endogenous signals as inputs for the genetic and metabolic circuit that determines the biodegradation of m-xylene. However, whether the enormous combinatorial space of inputs is translated into an equally variable response landscape or is processed into very few outcomes remains unclear. To address this question, we set out to define the number of states that can be obtained by a network of a given set of genes under the control of a specified regulatory circuit that is exposed to all possible combinations of inputs. To this end, the TOL network and its regulatory wiring were formalized as a synchronous logic Boolean circuit that had 10 signals (i.e. pathway substrates, temperature, sugars, amino acids, metabolic regimes and global regulators) as possible inputs. The analysis of the attractors of the circuit using a satisfiability (SAT) algorithm revealed that only eight transcriptional states are reached in response to the 1024 possible combinations of stimuli. The experimental validation resulted in a refinement of the model through the addition of a previously unknown interaction that controls the meta catabolic pathway. The full induction of the two xyl operons occurred with only 1.6% of the input combinations, which suggests that the architecture of the network allows the expression of the xyl genes only under a very narrow range of conditions. These data not only explain much of the unusual layout of the TOL circuit but also strengthen the view of the regulatory circuits of environmental bacteria as digital decision-making devices.

Deepening TOL and TOU catabolic pathways of Pseudomonas sp. OX1: Cloning, sequencing and characterization of the lower pathways

Pseudomonas sp. OX1 is able to metabolize toluene and o-xylene through the TOU catabolic pathway, whereas its mutant M1 strain was found to be able to use m- and p-xylene as carbon and energy source, using the TOL catabolic pathway. Here we report the complete nucleotide sequence of the phe lower operon of the TOU catabolic pathway, and the sequence of the last four genes of the xyl-like lower operon of the TOL catabolic pathway. DNA sequence analysis shows the gene order within the operons to be pheCDEFGHI (phe operon) and xyl-likeQKIH (xyl-like operon), identical to the order found for the isofunctional genes of meta operons in the toluene/xylene pathway of TOL plasmid pWW0 from Pseudomonas putida mt-2 and the phenol/methylphenol pathway of pVIl50 from Pseudomonas sp. CF600. The nucleotide and the deduced amino acid sequences are homologous to the equivalent gene and enzyme sequences from other Pseudomonas meta pathways. Recombinant 2-hydroxymuconic semialdehyde dehydrogenase (HMSD) and 2-hydroxymuconic semialdehyde hydrolase (HMSH), coded by pheCD genes, respectively, and ADA and HOA enzymes from both phe and xyl operons were expressed in E.coli and steady-state kinetic analysis was carried out. The analysis of the kinetic parameters of HMSD and HMSH showed that the enzymes from Pseudomonas sp. OX1 are more specialized to channel metabolites into the two branches of the lower pathway than homologous enzymes from other pseudomonads. The kinetics parameters of recombinant ADA from phe and xyl-like operon were found to be similar to those of homologous enzymes from other Pseudomonas strains. In addition, the enzyme from xyl-like operon showed a substrate affinity three times higher than the enzyme from phe operon.

Design and Characterization of an Aequorin‐based Bacterial Biosensor for Detection of Toluene and Related Compounds

An aequorin-based Escherichia coli strain JM109 biosensor was constructed and characterized for its potential to detect toluene and related compounds in aqueous solutions. The biosensor was constructed based on a PGL2 plasmid carrying the lower pathway promoter (Pu) of the xyl operon of Pseudomonas putida mt-2, which was incorporated with transcriptional activator xylR and fused to aequorin cDNA named pGL2-aequorin. Binding of xylR protein to a subset of toluene-like compounds activates transcription at the Pu promoter, thus expression of aequorin is controlled by xylR and Pu. In this work we have compared the effect of Shine-Dalgarno (SD) and T2 rrnβ terminator sequence in the expression of aequorin. According to the sensitivity of aequorin and increase in the signal-to-noise ratio, this reporter enzyme has reasonable sensitivity compared with other reporter systems. The results indicate higher expression of aequorin in the presence of SD and T2 rrnβ. The activity of aequorin in recombinant whole-cell biosensor was linear from 1 to 500 μm of toluene. The bioluminescence response was specific for toluene-like molecules, so this biosensor cells would be able to detect toluene derivative contamination in environmental samples, accurately.

The regulatory logic of m‐xylene biodegradation by Pseudomonas putida mt‐2 exposed by dynamic modelling of the principal node Ps/Pr of the TOL plasmid

The structure of the extant transcriptional control network of the TOL plasmid pWW0 born by Pseudomonas putida mt-2 for biodegradation of m-xylene is far more complex than one would consider necessary from a mere engineering point of view. In order to penetrate the underlying logic of such a network, which controls a major environmental cleanup bioprocess, we have developed a dynamic model of the key regulatory node formed by the Ps/Pr promoters of pWW0, where the clustering of control elements is maximal. The model layout was validated with batch cultures estimating parameter values and its predictive capability was confirmed with independent sets of experimental data. The model revealed how regulatory outputs originated in the divergent and overlapping Ps/Pr segment, which expresses the transcription factors XylS and XylR respectively, are computed into distinct instructions to the upper and lower catabolic xyl operons for either simultaneous or stepwise consumption of m-xylene and/or succinate. In this respect, the model reveals that the architecture of the Ps/Pr is poised to discriminate the abundance of alternative and competing C sources, in particular m-xylene versus succinate. The proposed framework provides a first systemic understanding of the causality and connectivity of the regulatory elements that shape this exemplary regulatory network, facilitating the use of model analysis towards genetic circuit optimization.

Cloning and overexpression of the xylose isomerase gene from Burkholderia sacchari and production of polyhydroxybutyrate from xylose

A different organization for the xyl operon was found in different genomes of Burkholderia and Pseudomomas species. Degenerated primers were designed based on Burkholderia genomes and used to amplify the xylose isomerase gene (xylA) from Burkholderia sacchari IPT101. The gene encoded a protein of 329 amino acids, which showed the highest similarity (90%) to the homologous gene of Burkholderia dolosa. It was cloned in the broad host range plasmid pBBR1MCS-2, which partially restored growth and polyhydroxybutyrate production capability in xylose to a B. sacchari xyl- mutant. When xylA was overexpressed in the wild-type strain, it was not able to increase growth and polyhydroxybutyrate production, suggesting that XylA activity is not limiting for xylose utilization in B. sacchari.

Relaxed control of sugar utilization in Lactobacillus brevis

Prioritization of sugar consumption is a common theme in bacterial growth and a problem for complete utilization of five and six carbon sugars derived from lignocellulose. Growth studies show that Lactobacillus brevis simultaneously consumes numerous carbon sources and appears to lack normal hierarchical control of carbohydrate utilization. Analysis of several independent L. brevis isolates indicated that co-utilization of xylose and glucose is a common trait for this species. Moreover, carbohydrates that can be used as a single carbon source are simultaneously utilized with glucose. Analysis of the proteome of L. brevis cells grown on glucose, xylose or a glucose/xylose mixture revealed the constitutive expression of the enzymes of the heterofermentative pathway. In addition, fermentative mass balances between mixed sugar inputs and end-products indicated that both glucose and xylose are simultaneously metabolized through the heterofermentative pathway. Proteomic and mRNA analyses revealed that genes in the xyl operon were expressed in the cells grown on xylose or on glucose/xylose mixtures but not in those grown on glucose alone. However, the expression level of XylA and XylB proteins in cells grown on a glucose/xylose mixture was reduced 2.7-fold from that observed in cells grown solely on xylose. These results suggest that regulation of xylose utilization in L. brevis is not stringently controlled as seen in other lactic acid bacteria, where carbon catabolite repression operates to prioritize carbohydrate utilization more rigorously.

Evidence that Bacillus catabolite control protein CcpA interacts with RNA polymerase to inhibit transcription

Summary Bacilluscatabolite control protein (CcpA) mediates carbon catabolite repression (CCR) by controlling expression of catabolite responsive (CR) genes or operons through interaction with catabolite responsive elements (cres) located within or outside of CR promoters. Here, we investigated how CcpA inhibits the transcription of CR promoters in vitro. CcpA has different affinities for different cres, but this does not correlate with its ability to inhibit transcription. In the amyE promoter, which overlaps a CcpA binding site (amyE cre centred at +4.5), CcpA does not prevent RNA polymerase (RNAP) binding to the promoter; it may even interact with RNAP. Inserting non-integral turns of helix (1.5 and 2.5) between the amyE promoter (-10 hexamer) and the amyE cre relieved CCR of amyE expression. In the xyl operon, despite the downstream location of its cre (a major cre centred at +130.5), CcpA blocked transcription initiation, not elongation (roadblock) at the site of the cre. Taken together, our results strongly suggest that CcpA requires interactions with RNAP to inhibit transcription.

Monitoring aromatic hydrocarbons by whole cell electrochemical biosensors

In this article, we describe a bacterial whole cell electrochemical biosensors system that can be used for monitoring aromatic hydrocarbons. These bacterial biosensors are based on fusions of a promoter that is sensitive to aromatic compounds (the promoter region of the xylS gene and the xylR gene coding for the transcriptional regulator of the xyl operon) to reporter genes that can be monitored electrochemically at real-time and on-line. The xylS promoter was fused upstream of two promoterless genes coding the lacZ gene and phoA. These constructs reacted specifically to aromatic compounds but not to nonaromatic compounds, and we could detect, within minutes, micromolar concentrations of different aromatic hydrocarbons such as xylene and toluene. The use of two different reporter genes allows the future construction of a multianalyte detection system for simultaneous monitoring of several pollutants. These whole cell biosensors are potentially useful for on-line and in situ detection of aromatic compounds and as early warning systems of environmental hazards.

Quantification of the Influence of HPrSer46P on CcpA– cre Interaction

Carbon catabolite repression (CCR) of the Bacillus megaterium xyl operon is dependent on the catabolite responsive element cre, the catabolite control protein (CcpA) and the histidine-containing phosphocarrier protein phosphorylated at the serine 46 residue (HPrSer46P). The latter is formed in the presence of glucose and mediates CCR via CcpA. We present evidence for the presence of HPrSer46P in a ternary complex with CcpA and cre. We also demonstrate increased stability of this complex compared to the CcpA– cre complex by electrophoretic mobility shift analysis (EMSA). This stabilization by HPrSer46P is the same for the xyl cre and an improved cre. Thus, HPrSer46P is a co-repressor for CcpA. In addition, surface plasmon resonance (SPR) experiments yielded binding constants of CcpA and the CcpA–HPrSer46P complex with cre. HPrSer46P stimulated CcpA binding to cre 50-fold. The binding constant is 4.9(±0.5)×10 6 M −1. Non-phosphorylated HPr did not affect the complex formation between CcpA and cre. Previously proposed effects by glucose-6-phosphate, fructose-1,6-diphosphate and NADP on CcpA– cre or CcpA–HPrSer46P– cre formation were not found in EMSA and SPR experiments.

Cooperative and non-cooperative DNA binding modes of catabolite control protein CcpA from Bacillus megaterium result from sensing two different signals

Carbon catabolite repression (CCR) of several operons in Bacillus subtilis and Bacillus megaterium is mediated by the cis-acting cre sequence and trans-acting catabolite control protein (CcpA). We describe purification of CcpA from B. megaterium and its interaction with regulatory sequences from the xyl operon. Specific interaction of CcpA with cre as scored by DNase I footprints at concentrations similar to the in vivo situation requires the presence of effectors. We have found two molecular effectors for CcpA activity, which lead to different recognition modes of DNA. The heat-stable phosphotransfer protein HPr from the PTS sugar uptake system triggers non-cooperative binding of CcpA to cre when phosphorylated at Ser46 (HPr-Ser46-P). Glucose 6-phosphate (Glc-6-P) triggers cooperative binding of CcpA to cre and two auxiliary cre∗ sites, one of which overlaps the −35 box of the xyl promoter. Binding to cre∗ depends on the presence of the functional cre sequence. A mutation in cre abolishes carbon catabolite repression in vivo and binding of CcpA to cre and cre∗ in vitro, indicating looping of the intervening DNA. The two triggers are not simultaneously active. The acidity of the buffer determines which of them activates CcpA when both are present in vitro. Glc-6-P is preferred at pH values below 5.4, and HPr-Ser46-P is preferred at neutral pH. The CcpA dimers present at neutral pH form tetramers and higher oligomers at pH 4.6, explaining cooperativity of binding to DNA. CcpA is the first member of the LacI/GalR family of regulators, for which oligomerization without the leucine zipper at the C terminus is demonstrated.

Regulation of expression, genetic organization and substrate specificity of xylose uptake in Bacillus megaterium

Xylose uptake in Bacillus megaterium depends on expression of a putative H+/xylose symporter encoded by xylT, the last gene in the xyl operon. Insertional inactivation of xylT leads to an apparent uptake deficiency determined with whole cells and severely slower growth on xylose as sole carbon source. Expression of xylT is xylose inducible and subject to carbon catabolite repression mediated by CcpA and cre. Northern analysis of the xyl mRNA reveals that a potential stem-loop structure located in the non-translated region between xylA and xylB presumably acts as a transcriptional terminator, as it leads to different amounts of the respective mRNA sections: the 5'-xylA portion is very abundant, while the 3'-xylBT portion constitutes only a fraction of it. XylT has an apparent Michaelis constant (KM) of approx. 100 microM and is competitively inhibited by glucose with an inhibitor constant KI of 16 mM.