xylE Gene Research Articles

XylR regulates genes at xyl cluster, involved in D-xylose catabolism in Herbaspirillum seropedicae Z69.

D-xylose, one of the most abundant sugars in lignocellulosic biomass, is not widely used to produce bioproducts with added value, in part due to the absence of industrial microorganisms able to metabolize it efficiently. Herbaspirillum seropedicae Z69 is a β-proteobacterium able to accumulate poly-3-hydroxybutyrate, a biodegradable thermoplastic biopolymer, with contents higher than 50%. It metabolizes D-xylose by non-phosphorylative pathways. In the genome of Z69, we found the genes xylFGH (ABC D-xylose transporter), xylB, xylD, and xylC (superior non-phosphorylative pathway), and the transcriptional regulator xylR, forming the xyl cluster. We constructed the knock-out mutant Z69ΔxylR that has a reduced growth in D-xylose and in D-glucose, compared with Z69. In addition, we analyzed the expression of xyl genes by RT-qPCR and promoter fusion. These results suggest that XylR activates the expression of genes at the xyl cluster in the presence of D-xylose. On the other hand, XylR does not regulate the expression of xylA, mhpD (lower non-phosphorylative pathways) and araB (L-arabinose dehydrogenase) genes. The participation of D-glucose in the regulation mechanism of these genes must still be elucidated. These results contribute to the development of new strains adapted to consume lignocellulosic sugars for the production of value-added bioproducts.

XylR Overexpression in Escherichia coli Alleviated Transcriptional Repression by Arabinose and Enhanced Xylitol Bioproduction from Xylose Mother Liquor.

Xylitol is a polyol widely used in food, pharmaceuticals, and light industries. It is currently produced through the chemical catalytic hydrogenation of xylose and generates xylose mother liquor as a substantial byproduct in the procedure of xylose extraction. If xylose mother liquor could also be efficiently bioconverted to xylitol, the greenness and atom economy of xylitol production would be largely improved. However, xylose mother liquor contains a mixture of glucose, xylose, and arabinose, raising the issue of carbon catabolic repression in its utilization by microbial conversion. Targeting this challenge, the transcriptional activator XylR was overexpressed in a previously constructed xylitol-producing E. coli strain CPH. The resulting strain CPHR produced 16.61g/L of xylitol in shake-flask cultures from the mixture of corn cob hydrolysate and xylose mother liquor (1:1, v/v) with a xylose conversion rate of 90.1%, which were 2.23 and 2.15 times higher than the starting strain, respectively. Furthermore, XylR overexpression upregulated the expression levels of xylE, xylF, xylG, and xylH genes by 2.08-2.72 times in arabinose-containing medium, suggesting the alleviation of transcriptional repression of xylose transport genes by arabinose. This work lays the foundation for xylitol bioproduction from xylose mother liquor.

Oxygenation influences xylose fermentation and gene expression in the yeast genera Spathaspora and Scheffersomyces

BackgroundCost-effective production of biofuels from lignocellulose requires the fermentation of d-xylose. Many yeast species within and closely related to the genera Spathaspora and Scheffersomyces (both of the order Serinales) natively assimilate and ferment xylose. Other species consume xylose inefficiently, leading to extracellular accumulation of xylitol. Xylitol excretion is thought to be due to the different cofactor requirements of the first two steps of xylose metabolism. Xylose reductase (XR) generally uses NADPH to reduce xylose to xylitol, while xylitol dehydrogenase (XDH) generally uses NAD+ to oxidize xylitol to xylulose, creating an imbalanced redox pathway. This imbalance is thought to be particularly consequential in hypoxic or anoxic environments.ResultsWe screened the growth of xylose-fermenting yeast species in high and moderate aeration and identified both ethanol producers and xylitol producers. Selected species were further characterized for their XR and XDH cofactor preferences by enzyme assays and gene expression patterns by RNA-Seq. Our data revealed that xylose metabolism is more redox balanced in some species, but it is strongly affected by oxygen levels. Under high aeration, most species switched from ethanol production to xylitol accumulation, despite the availability of ample oxygen to accept electrons from NADH. This switch was followed by decreases in enzyme activity and the expression of genes related to xylose metabolism, suggesting that bottlenecks in xylose fermentation are not always due to cofactor preferences. Finally, we expressed XYL genes from multiple Scheffersomyces species in a strain of Saccharomyces cerevisiae. Recombinant S. cerevisiae expressing XYL1 from Scheffersomyces xylosifermentans, which encodes an XR without a cofactor preference, showed improved anaerobic growth on xylose as the primary carbon source compared to S. cerevisiae strain expressing XYL genes from Scheffersomyces stipitis.ConclusionCollectively, our data do not support the hypothesis that xylitol accumulation occurs primarily due to differences in cofactor preferences between xylose reductase and xylitol dehydrogenase; instead, gene expression plays a major role in response to oxygen levels. We have also identified the yeast Sc. xylosifermentans as a potential source for genes that can be engineered into S. cerevisiae to improve xylose fermentation and biofuel production.

Construction of an economical xylose-utilizing Saccharomyces cerevisiae and its ethanol fermentation.

Traditional industrial Saccharomyces cerevisiae could not metabolize xylose due to the lack of a specific enzyme system for the reaction from xylose to xylulose. This study aims to metabolically remould industrial S. cerevisiae for the purpose of utilizing both glucose and xylose with high efficiency. Heterologous gene xylA from Piromyces and homologous genes related to xylose utilization were selected to construct expression cassettes and integrated into genome. The engineered strain was domesticated with industrial material under optimizing conditions subsequently to further improve xylose utilization rates. The resulting S. cerevisiae strain ABX0928-0630 exhibits a rapid growth rate and possesses near 100% xylose utilization efficiency to produce ethanol with industrial material. Pilot-scale fermentation indicated the predominant feature of ABX0928-0630 for industrial application, with ethanol yield of 0.48g/g sugars after 48 hours and volumetric xylose consumption rate of 0.87g/l/h during the first 24 hours. Transcriptome analysis during the modification and domestication process revealed a significant increase in the expression level of pathways associated with sugar metabolism and sugar sensing. Meanwhile, genes related to glycerol lipid metabolism exhibited a pattern of initial increase followed by a subsequent decrease, providing a valuable reference for the construction of efficient xylose-fermenting strains.

Biodegradation of chloroethene compounds under microoxic conditions.

The biodegradation of chloroethene compounds under oxic and anoxic conditions is well established. However, the biological reactions that take place under microoxic conditions are unknown. Here, we report the biostimulated (BIOST: addition of lactate) and natural attenuated (NAT) degradation of chloroethene compounds under microoxic conditions by bacterial communities from chloroethene compounds-contaminated groundwater. The degradation of tetrachloroethene was significantly higher in NAT (15.14% on average) than in BIOST (10.13% on average) conditions at the end of the experiment (90 days). Sporomusa, Paracoccus, Sedimentibacter, Pseudomonas, and Desulfosporosinus were overrepresented in NAT and BIOST compared to the source groundwater. The NAT metagenome contains phenol hydrolase P1 oxygenase (dmpL), catechol-1,2-dioxygenase (catA), catechol-2,3-dioxygenases (dmpB, todE, and xylE) genes, which could be involved in the cometabolic degradation of chloroethene compounds; and chlorate reductase (clrA), that could be associated with partial reductive dechlorination of chloroethene compounds. Our data provide a better understanding of the bacterial communities, genes, and pathways potentially implicated in the reductive and cometabolic degradation of chloroethene compounds under microoxic conditions.

Codon Optimization Improves the Prediction of Xylose Metabolism from Gene Content in Budding Yeasts.

Xylose is the second most abundant monomeric sugar in plant biomass. Consequently, xylose catabolism is an ecologically important trait for saprotrophic organisms, as well as a fundamentally important trait for industries that hope to convert plant mass to renewable fuels and other bioproducts using microbial metabolism. Although common across fungi, xylose catabolism is rare within Saccharomycotina, the subphylum that contains most industrially relevant fermentative yeast species. The genomes of several yeasts unable to consume xylose have been previously reported to contain the full set of genes in the XYL pathway, suggesting the absence of a gene-trait correlation for xylose metabolism. Here, we measured growth on xylose and systematically identified XYL pathway orthologs across the genomes of 332 budding yeast species. Although the XYL pathway coevolved with xylose metabolism, we found that pathway presence only predicted xylose catabolism about half of the time, demonstrating that a complete XYL pathway is necessary, but not sufficient, for xylose catabolism. We also found that XYL1 copy number was positively correlated, after phylogenetic correction, with xylose utilization. We then quantified codon usage bias of XYL genes and found that XYL3 codon optimization was significantly higher, after phylogenetic correction, in species able to consume xylose. Finally, we showed that codon optimization of XYL2 was positively correlated, after phylogenetic correction, with growth rates in xylose medium. We conclude that gene content alone is a weak predictor of xylose metabolism and that using codon optimization enhances the prediction of xylose metabolism from yeast genome sequence data.

Silver and zinc oxide nanoparticles disrupt essential parasitism, neuropeptidergic, and expansion-like proteins genes in Meloidogyneincognita

Meloidogyne incognita leads to considerable losses in crop productivity. In this study, the impact of silver nanoparticles (AgNPs) and zinc oxide nanoparticles (ZnONPs) in 100, 200, or 300 ppm concentrations were investigated on some essential genes of M. incognita in vitro. For this purpose, AgNPs and ZnONPs were synthesized and characterized for their physicochemical properties. Thereafter, second-stage Juveniles (J2) of M. incognita were exposed to AgNPs and ZnONPs solution for 24 h. The LC50, LC90, and mortality rates were calculated for both nanoparticles. Finally, the expression of parasitism genes (Xyl-1; msp-20; 16D10), neuropeptidergic gene (Ace-2), expansion-like proteins (MAP-1), and oxidative stress gene (GSTS-1) was analyzed. The results showed a successful preparation of nanoparticles to obtain a pure, well-dispersed, and stable suspension, as revealed by physicochemical properties. ZnONPs showed LC50 and LC90 values of 63.56 and 208.5 ppm, respectively, while AgNPs recorded 11.78 and 28.59 ppm, respectively. AgNPs at concentrations 100, 200, and 300 ppm showed mortality rate 66%, 84%, and 100%, respectively, whereas ZnONPs at the same concentrations caused a 58%, 78%, and 91% mortality rate, respectively. Analysis of gene expression showed dose-dependent downregulation of each parasitism gene Xyl-1, 16D10, and msp-20 genes, neuropeptidergic gene (Ace-2), and expansion-like proteins MAP-1 after treatment with either AgNPs or ZnONPs. On the other hand, the oxidative stress response gene GSTS-1 showed upregulation with all concentrations of AgNPs and ZnONPs. The study concluded that the AgNPs and ZnONPs have efficient nematocidal activity and can be used in Meloidogyne incognita control.

Fermentation of D-xylose to Ethanol by Saccharomyces cerevisiae CAT-1 Recombinant Strains.

Ethanol production by the D-xylose fermentation of lignocellulosic biomass would augment environmental sustainability by increasing the yield of biofuel obtained per cultivated area. A set of recombinant strains derived from the industrial strain Saccharomyces cerevisiae CAT-1 was developed for this purpose. First, two recombinant strains were obtained by the chromosomal insertion of genes involved in the assimilation and transport of D-xylose (Gal2-N376F). Strain CAT-1-XRT was developed with heterologous genes for D-xylose metabolism from the oxo-reductive pathway of Scheffersomyces stipitis (XYL1-K270R, XYL2); and strain CAT-1-XIT, with D-xylose isomerase (xylA gene, XI) from Streptomyces coelicolor. Moreover, both recombinant strains contained extra copies of homologous genes for xylulose kinase (XK) and transaldolase (TAL1). Furthermore, plasmid (pRS42K::XI) was constructed with xylA from Piromyces sp. transferred to CAT-1, CAT-1-XRT, and CAT-1-XIT, followed by an evolution protocol. After 10 subcultures, CAT-1-XIT (pRS42K::XI) consumed 74% of D-xylose, producing 12.6 g/L ethanol (0.31 g ethanol/g D-xylose). The results of this study show that CAT-1-XIT (pRS42K::XI) is a promising recombinant strain for the efficient utilization of D-xylose to produce ethanol from lignocellulosic materials.

Fermentation performance of a Mexican native Clavispora lusitaniae strain for xylitol and ethanol production from xylose, glucose and cellobiose

Lignocellulose hydrolysates are rich in fermentable sugars such as xylose, cellobiose and glucose, with high potential in the biotechnology industry to obtain bioproducts of higher economic value. Thus, it is important to search for and study new yeast strains that co-consume these sugars to achieve better yields and productivity in the processes. The yeast Clavispora lusitaniae CDBB-L-2031, a native strain isolated from mezcal must, was studied under various culture conditions to potentially produce ethanol and xylitol due to its ability to assimilate xylose, cellobiose and glucose. This yeast produced ethanol under microaerobic conditions with yields of 0.451 gethanol/gglucose and 0.344 gethanol/gcellobiose, when grown on 1% glucose or cellobiose, respectively. In mixtures (0.5% each) of glucose:xylose and glucose:xylose:cellobiose the yields were 0.367 gethanol/gGX and 0. 380 gethanol/gGXC, respectively. Likewise, in identical conditions, C. lusitaniae produced xylitol from xylose with a yield of 0.421 gxylitol/gxylose. In 5% glucose or xylose, this yeast had better ethanol and xylitol titers and yields, respectively. However, glucose negatively affected xylitol production in the mixture of both sugars (3% each), producing only ethanol. Xylose reductase (XR) and xylitol dehydrogenase (XDH) activities were evaluated in cultures growing on xylose or glucose, obtaining the highest values in cultures on xylose at 8 h (25.9 and 6.22 mU/mg, respectively). While in glucose cultures, XR and XDH activities were detected once this substrate was consumed (4.06 and 3.32 mU/mg, respectively). Finally, the XYL1 and XYL2 genes encoding xylose reductase and xylitol dehydrogenase, respectively, were up-regulated by xylose, whereas glucose down-regulated their expression.

Phytochemical effects of Apium graveolens on the abundances of functional genes associated with PAH degradation in soil

The biodegradation of polycyclic aromatic hydrocarbons in soil is enhanced by chemical constituents of various plant species that support the growth and activity of PAH degrading bacteria in the rhizosphere. Here, we investigated the phytochemical effects of celeriac (Apium graveolens var. rapaceum) root tissues, a known stimulator of PAH degradation, on the copy numbers of nahAc, xylE, and 16S rRNA genes to determine the relative contributions of growth-linked degradation versus selective enrichment of PAH degraders. Real-time quantitative PCR analysis showed that the copy numbers of 16S rRNA genes, as a reporter of general bacterial population size, increased approximately 2- to 5-fold in soil amended with celeriac, but were not affected by exposure to naphthalene alone. In contrast, copy numbers of two functional genes (nahAc and xylE), commonly used as reporters for monitoring the population size of PAH degraders, were significantly increased following exposure to naphthalene but were differently affected by soil amendment with celeriac. As compared with the copy numbers of nahAc gene in unamended control soil, the nahAc copy numbers were increased by 460-fold in soil amended with naphthalene alone and by 610-fold in soil amended with both naphthalene and celeriac after 10 days of incubation. The copy numbers of xylE were increased by 20-fold in soil amended with naphthalene alone and increased by 200-fold in soil amended with both naphthalene and celeriac after 10 days of incubation. The result suggests that increased degradation rates of PAHs in the presence of celeriac involves both growth linked processes and selective enrichment of the degrader population.

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.

Biodegradation of p-xylene-a comparison of three psychrophilic Pseudomonas strains through the lens of gene expression.

p-Xylene is considered a recalcitrant compound despite showing a similar aromatic structure to other BTEXs (benzene, toluene, ethylbenzene, xylene isomers). This study evaluated the p-xylene biodegradation potential of three psychrophilic Pseudomonas strains (Pseudomonas putida S2TR-01, Pseudomonas synxantha S2TR-20, and Pseudomonas azotoformans S2TR-09). The p-xylene metabolism-related catabolic genes (xylM, xylA, and xylE) and the corresponding regulatory genes (xylR and xylS) of the selected strains were investigated. The biodegradation results showed that the P. azotoformans S2TR-09 strain was the only strain that was able to degrade 200mg/L p-xylene after 60h at 15°C. The gene expression study indicated that the xylE (encoding catechol 2,3-dioxygenase) gene represents the bottleneck in p-xylene biodegradation. A lack of xylE expression leads to the accumulation of intermediates and the inhibition of biomass production and complete carbon recovery. The activity of xylene monooxygenase and catechol 2,3-dioxygenase was significantly increased in P. azotoformans S2TR-09 (0.5 and 0.08 U/mg, respectively) in the presence of p-xylene. The expression of the ring cleavage enzyme and its encoding gene (xylE) and activator (xylS) explained the differences in the p-xylene metabolism of the isolated bacteria and can be used as a novel biomarker of efficient p-xylene biodegradation at contaminated sites.

Utilization of a Wheat Sidestream for 5-Aminovalerate Production in Corynebacterium glutamicum.

Production of plastics from petroleum-based raw materials extensively contributes to global pollution and CO2 emissions. Biotechnological production of functionalized monomers can reduce the environmental impact, in particular when using industrial sidestreams as feedstocks. Corynebacterium glutamicum, which is used in the million-ton-scale amino acid production, has been engineered for sustainable production of polyamide monomers. In this study, wheat sidestream concentrate (WSC) from industrial starch production was utilized for production of l-lysine–derived bifunctional monomers using metabolically engineered C. glutamicum strains. Growth of C. glutamicum on WSC was observed and could be improved by hydrolysis of WSC. By heterologous expression of the genes xylA Xc B Cg (xylA from Xanthomonas campestris) and araBAD Ec from E. coli, xylose, and arabinose in WSC hydrolysate (WSCH), in addition to glucose, could be consumed, and production of l-lysine could be increased. WSCH-based production of cadaverine and 5-aminovalerate (5AVA) was enabled. To this end, the lysine decarboxylase gene ldcC Ec from E. coli was expressed alone or for conversion to 5AVA cascaded either with putrescine transaminase and dehydrogenase genes patDA Ec from E. coli or with putrescine oxidase gene puo Rq from Rhodococcus qingshengii and patD Ec . Deletion of the l-glutamate dehydrogenase–encoding gene gdh reduced formation of l-glutamate as a side product for strains with either of the cascades. Since the former cascade (ldcC Ec -patDA Ec ) yields l-glutamate, 5AVA production is coupled to growth by flux enforcement resulting in the highest 5AVA titer obtained with WSCH-based media.

Comparison of Spathaspora passalidarum and recombinant Saccharomyces cerevisiae for integration of first- and second-generation ethanol production.

First-generation ethanol (E1G) is based on the fermentation of sugars released from saccharine or starch sources, while second-generation ethanol (E2G) is focused on the fermentation of sugars released from lignocellulosic feedstocks. During the fractionation process to release sugars from hemicelluloses (mainly xylose), some inhibitor compounds are released hindering fermentation. Thus, the biggest challenge of using hemicellulosic hydrolysate is selecting strains and processes able to efficiently ferment xylose and tolerate inhibitors. With the aim of diluting inhibitors, sugarcane molasses (80% of sucrose content) can be mixed to hemicellulosic hydrolysate in an integrated E1G-E2G process. Cofermentations of xylose and sucrose were evaluated for the native xylose consumer Spathaspora passalidarum and a recombinant Saccharomyces cerevisiae strain. The industrial S. cerevisiae strain CAT-1 was modified to overexpress the XYL1, XYL2 and XKS1 genes and a mutant ([4-59Δ]HXT1) version of the low-affinity HXT1 permease, generating strain MP-C5H1. Although S. passalidarum showed better results for xylose fermentation, this yeast showed intracellular sucrose hydrolysis and low sucrose consumption in microaerobic conditions. Recombinant S. cerevisiae showed the best performance for cofermentation, and a batch strategy at high cell density in bioreactor achieved unprecedented results of ethanol yield, titer and volumetric productivity in E1G-E2G production process.

Metabolic Engineering of E. coli for Xylose Production from Glucose as the Sole Carbon Source.

Xylose is the raw material for the synthesis of many important platform compounds. At present, xylose is commercially produced by chemical extraction. However, there are still some bottlenecks in the extraction of xylose, including complicated operation processes and the chemical substances introduced, leading to the high cost of xylose and of synthesizing the downstream compounds of xylose. The current market price of xylose is 8× that of glucose, so using low-cost glucose as the substrate to produce the downstream compounds of xylose can theoretically reduce the cost by 70%. Here, we designed a pathway for the biosynthesis of xylose from glucose in Escherichia coli. This biosynthetic pathway was achieved by overexpressing five genes, namely, zwf, pgl, gnd, rpe, and xylA, while replacing the native xylulose kinase gene xylB with araL from B. subtilis, which displays phosphatase activity toward d-xylulose 5-phosphate. The yield of xylose was increased to 3.3 g/L by optimizing the metabolic pathway. Furthermore, xylitol was successfully synthesized by introducing the xyl1 gene, which suggested that the biosynthetic pathway of xylose from glucose is universally applicable for the synthesis of xylose downstream compounds. This is the first study to synthesize xylose and its downstream compounds by using glucose as a substrate, which not only reduces the cost of raw materials, but also alleviates carbon catabolite repression (CCR), providing a new idea for the synthesis of downstream compounds of xylose.

Improved glucose and xylose co-utilization by overexpression of xylose isomerase and/or xylulokinase genes in oleaginous fungus Mucor circinelloides.

Most of the oleaginous microorganisms cannot assimilate xylose in the presence of glucose, which is the major bottleneck in the bioconversion of lignocellulose to biodiesel. Our present study revealed that overexpression of xylose isomerase (XI) gene xylA or xylulokinase (XK) gene xks1 increased the xylose consumption by 25 to 37% and enhanced the lipid content by 8 to 28% during co-fermentation of glucose and xylose. In xylA overexpressing strain Mc-XI, the activity of XI was 1.8-fold higher and the mRNA level of xylA at 24 h and 48 h was 11- and 13-fold higher than that of the control, respectively. In xks1 overexpressing strain Mc-XK, the mRNA level of xks1 was 4- to 11-fold of that of the control strain and the highest XK activity of 950 nmol min-1 mg-1 at 72 h which was 2-fold higher than that of the control. Additionally, expression of a translational fusion of xylA and xks1 further enhanced the xylose utilization rate by 45%. Our results indicated that overexpression of xylA and/or xks1 is a promising strategy to improve the xylose and glucose co-utilization, alleviate the glucose repression, and produce lipid from lignocellulosic biomass in the oleaginous fungus M. circinelloides. KEY POINTS: • Overexpressing xylA or xks1 increased the xylose consumption and the lipid content. • The xylose isomerase activity and the xylA mRNA level were enhanced in strain Mc-XI. • Co-expression of xylA and xks1 further enhanced the xylose utilization rate by 45%.

Whole-Genome Sequence Data Analysis of Anoxybacillus kamchatkensis NASTPD13 Isolated from Hot Spring of Myagdi, Nepal.

Anoxybacillus kamchatkensis NASTPD13 isolated from Paudwar hot spring of Myagdi, Nepal, upon morphological and biochemical analysis revealed to be Gram-positive, straight or slightly curved, rod-shaped, spore-forming, catalase, and oxidase-positive facultative anaerobes. It grows over a wide range of pH (5.0-11) and temperature (37-75°C), which showed growth in different reduced carbon sources such as starch raffinose, glucose, fructose, inositol, trehalose, sorbitol, mellobiose, and mannitol in aerobic conditions. Furthermore, the partial sequence obtained upon sequencing showed 99% sequence similarity in 16S rRNA gene sequence with A. kamchatkensis JW/VK-KG4 and was suggested to be Anoxybacillus kamchatkensis. Moreover, whole-genome analysis of NASTPD13 revealed 2,866,796 bp genome with a G+C content of 41.6%. Analysis of the genome revealed the presence of 102 RNA genes, which includes sequences coding for 19 rRNA and 79 tRNA genes. While the 16S rRNA gene sequence of strain NASTPD13 showed high similarity (>99%) to those of A. kamchatkensis JW/VK-KG4, RAST analysis of NASTPD13 genome suggested that A. kamchatkensis G10 is actually the closest neighbor in terms of sequence similarity. The genome annotation by RAST revealed various genes encoding glycoside hydrolases supporting that it can utilize several reduced carbon sources as observed and these genes could be important for carbohydrate-related industries. Xylanase pathway, particularly the genomic region encoding key enzymes for xylan depolymerization and xylose metabolism, further confirmed the presence of the complete gene in xylan metabolism. In addition, the complete xylose utilization gene locus analysis of NASTPD13 genome revealed all including D-xylose transport ATP-binding protein XylG and XylF, the xylose isomerase encoding gene XylA, and the gene XylB coding for a xylulokinase supported the fact that the isolate contains a complete set of genes related to xylan degradation, pentose transport, and metabolism. The results of the present study suggest that the isolated A. kamchatkensis NASTPD13 containing xylanase-producing genes could be useful in lignocellulosic biomass-utilizing industries where pentose polymers could also be utilized along with the hexose polymers.

Adaptation of phenol-degrading Pseudomonas putida KB3 to suboptimal growth condition: A focus on degradative rate, membrane properties and expression of xylE and cfaB genes

Detailed characterization of new Pseudomonas strains that degrade toxic pollutants is required and utterly necessary before their potential use in environmental microbiology and biotechnology applications. Therefore, phenol degradation by Pseudomonas putida KB3 under suboptimal temperatures, pH, and salinity was examined in this study. Parallelly, adaptive mechanisms of bacteria to stressful growth conditions concerning changes in cell membrane properties during phenol exposure as well as the expression level of genes encoding catechol 2,3-dioxygenase (xylE) and cyclopropane fatty acid synthase (cfaB) were determined. It was found that high salinity and the low temperature had the most significant effect on the growth of bacteria and the rate of phenol utilization. Degradation of phenol (300 mg L−1) proceeded 12-fold and seven-fold longer at 10 °C and 5% NaCl compared to the optimal conditions. The ability of bacteria to degrade phenol was coupled with a relatively high activity of catechol 2,3-dioxygenase. The only factor that inhibited enzyme activity by approximately 80% compared to the control sample was salinity. Fatty acid methyl ester (FAMEs) profiling, membrane permeability measurements, and hydrophobicity tests indicated severe alterations in bacteria membrane properties during phenol degradation in suboptimal growth conditions. The highest values of pH, salinity, and temperature led to a decrease in membrane permeability. FAME analysis showed fatty acid saturation indices and cyclopropane fatty acid participation at high temperature and salinity. Genetic data showed that suboptimal growth conditions primarily resulted in down-regulation of xylE and cfaB gene expression.

Sustainable Production of N-methylphenylalanine by Reductive Methylamination of Phenylpyruvate Using Engineered Corynebacterium glutamicum

N-alkylated amino acids occur widely in nature and can also be found in bioactive secondary metabolites such as the glycopeptide antibiotic vancomycin and the immunosuppressant cyclosporine A. To meet the demand for N-alkylated amino acids, they are currently produced chemically; however, these approaches often lack enantiopurity, show low product yields and require toxic reagents. Fermentative routes to N-alkylated amino acids like N-methyl-l-alanine or N-methylantranilate, a precursor of acridone alkaloids, have been established using engineered Corynebacterium glutamicum, which has been used for the industrial production of amino acids for decades. Here, we describe metabolic engineering of C. glutamicum for de novo production of N-methylphenylalanine based on reductive methylamination of phenylpyruvate. Pseudomonas putida Δ-1-piperideine-2-carboxylate reductase DpkA containing the amino acid exchanges P262A and M141L showed comparable catalytic efficiencies with phenylpyruvate and pyruvate, whereas the wild-type enzyme preferred the latter substrate over the former. Deletion of the anthranilate synthase genes trpEG and of the genes encoding branched-chain amino acid aminotransferase IlvE and phenylalanine aminotransferase AroT in a strain engineered to overproduce anthranilate abolished biosynthesis of l-tryptophan and l-phenylalanine to accumulate phenylpyruvate. Upon heterologous expression of DpkAP262A,M141L, N-methylphenylalanine production resulted upon addition of monomethylamine to the medium. In glucose-based minimal medium, an N-methylphenylalanine titer of 0.73 ± 0.05 g L−1, a volumetric productivity of 0.01 g L−1 h−1 and a yield of 0.052 g g−1 glucose were reached. When xylose isomerase gene xylA from Xanthomonas campestris and the endogenous xylulokinase gene xylB were expressed in addition, xylose as sole carbon source supported production of N-methylphenylalanine to a titer of 0.6 ± 0.04 g L−1 with a volumetric productivity of 0.008 g L−1 h−1 and a yield of 0.05 g g−1 xylose. Thus, a fermentative route to sustainable production of N-methylphenylalanine by recombinant C. glutamicum has been established.

Subcellular Architecture of the xyl Gene Expression Flow of the TOL Catabolic Plasmid of Pseudomonas putida mt-2.

ABSTRACTDespite intensive research on the biochemical and regulatory features of the archetypal catabolic TOL system borne by pWW0 of Pseudomonas putida strain mt-2, the physical arrangement and tridimensional logic of the xyl gene expression flow remains unknown. In this work, the spatial distribution of specific xyl mRNAs with respect to the host nucleoid, the TOL plasmid, and the ribosomal pool has been investigated. In situ hybridization of target transcripts with fluorescent oligonucleotide probes revealed that xyl mRNAs cluster in discrete foci, adjacent but clearly separated from the TOL plasmid and the cell nucleoid. Also, they colocalize with ribosome-rich domains of the intracellular milieu. This arrangement was maintained even when the xyl genes were artificially relocated to different chromosomal locations. The same held true when genes were expressed through a heterologous T7 polymerase-based system, which likewise led to mRNA foci outside the DNA. In contrast, rifampin treatment, known to ease crowding, blurred the confinement of xyl transcripts. This suggested that xyl mRNAs exit from their initiation sites to move to ribosome-rich points for translation—rather than being translated coupled to transcription. Moreover, the results suggest the distinct subcellular motion of xyl mRNAs results from both innate properties of the sequences and the physical forces that keep the ribosomal pool away from the nucleoid in P. putida. This scenario is discussed within the background of current knowledge on the three-dimensional organization of the gene expression flow in other bacteria and the environmental lifestyle of this soil microorganism.