Cloning, heterologous expression, and characterization of a metagenome-derived GH10 xylanase with salt and alkali tolerance from Xinjiang saline-alkali soil.

Heterologous expression in Pichia pastoris and characterization of a novel GH11 xylanase from saline-alkali soil with excellent tolerance to high pH, high salt concentrations and ethanol

Rhizosphere microorganisms can effectively promote the stress resistance of plants, and some beneficial rhizosphere microorganisms can significantly promote the growth of crops under salt stress, which has the potential to develop special microbial fertilizers for increasing the yield of saline-alkali land and provides a low-cost and environmentally friendly new strategy for improving the crop yield of saline-alkali cultivated land by using agricultural microbial technology. In May 2022, a field study in a completely randomized block design was conducted at the Heilongjiang Academy of Agricultural Sciences to explore the correlation between plant rhizosphere microorganisms and soybean growth in saline-alkali soil. Two soybean cultivars (Hening 531, a salt-tolerant variety, and 20_1846, a salt-sensitive variety) were planted at two experimental sites [Daqing (normal condition) and Harbin (saline-alkali conditions)], aiming to investigate the performance of soybean in saline-alkali environments. Soybeans grown in saline-alkali soil showed substantial reductions in key traits: plant height (25%), pod number (26.6%), seed yield (33%), and 100 seed weight (13%). This underscores the unsuitability of this soil type for soybean cultivation. Additionally, microbial analysis revealed 43 depleted and 56 enriched operational taxonomic units (OTUs) in the saline-alkali soil compared to normal soil. Furthermore, an analysis of ion-associated microbes identified 85 mOTUs with significant correlations with various ions. A co-occurrence network analysis revealed strong relationships between specific mOTUs and ions, such as Proteobacteria with multiple ions. In addition, the study investigated the differences in rhizosphere species between salt-tolerant and salt-sensitive soybean varieties under saline-alkali soil conditions. Redundancy analysis (RDA) indicated that mOTUs in saline-alkali soil were associated with pH and ions, while mOTUs in normal soil were correlated with Ca2+ and K+. Comparative analyses identified significant differences in mOTUs between salt-tolerant and salt-sensitive varieties under both saline-alkali and normal soil conditions. Planctomycetes, Proteobacteria, and Actinobacteria were dominant in the bacterial community of saline-alkali soil, with significant enrichment compared to normal soil. The study explored the functioning of the soybean rhizosphere key microbiome by comparing metagenomic data to four databases related to the carbon, nitrogen, phosphorus, and sulfur cycles. A total of 141 KOs (KEGG orthologues) were identified, with 66 KOs related to the carbon cycle, 16 KOs related to the nitrogen cycle, 48 KOs associated with the phosphorus cycle, and 11 KOs linked to the sulfur cycle. Significant correlations were found between specific mOTUs, functional genes, and phenotypic traits, including per mu yield (PMY), grain weight, and effective pod number per plant. Overall, this study provides comprehensive insights into the structure, function, and salt-related species of soil microorganisms in saline-alkali soil and their associations with salt tolerance and soybean phenotype. The identification of key microbial species and functional categories offers valuable information for understanding the mechanisms underlying plant-microbe interactions in challenging soil conditions.

Agaricus sinodeliciosus is a rare wild edible mushroom from northwest China, and grows naturally in mild saline-alkali soil, which is also unusual in mushrooms. A. sinodeliciosus represents a potential model organism for explaining saline-alkali tolerance mechanisms and revealing related physiological processes in mushrooms. Here, we provide a high-quality genome of A. sinodeliciosus. Comparative genomic analyses reveal A. sinodeliciosus has numerous changes to its genome organization after a solitary evolutionary history under saline-alkali environments, such as gene family contraction, retrotransposon expansion and rapid evolution of adaptative genes. Our saline and alkali tolerance tests show that mycelium growth and fruit body formation of this species are effected by mild alkalinity. Transcriptomic analyses reveal that genes involved in carbon and nitrogen utilization, cell stability and fruit body formation of A. sinodeliciosus could be activated under mildly alkaline conditions. In particular, the ‘starch and sucrose metabolism’, ‘biosynthesis of amino acids’ and ‘phenylpropanoid biosynthesis’ pathways are important for mildly alkaline tolerance of A. sinodeliciosus. Like plants and arbuscular mycorrhizal fungi, in the rot fungus A. sinodeliciosus, the biosynthesis of intracellular small molecules could be enhanced to counter osmotic and oxidative stresses caused by mild alkalinity, and the biosynthesis of monolignol could be suppressed to increase cell wall infiltrates under mildly alkaline conditions. This research provides an understanding of the genomic evolution and mechanisms of A. sinodeliciosus in tolerance to saline-alkali environments. The A. sinodeliciosus genome constitutes a valuable resource for evolutionary and ecological studies of Agaricus.

Superabsorbent polymer used for saline-alkali soil water retention

In this study, the first xylantic enzyme from the family Marinifilaceae, XynSPP2, was identified from Marinifilaceae bacterium strain SPP2. Amino acid sequence analysis revealed that XynSPP2 is a rare Fn3-fused xylanase, consisting of a signal peptide, a fibronectin type-III domain (Fn3), and a C-terminal catalytic domain belonging to glycoside hydrolase family 10 (GH10). The catalytic domain shared 17–46% identities to those of biochemically characterized GH10 xylanases. Structural analysis revealed that the conserved asparagine and glutamine at the glycone −2/−3 subsite of GH10 xylanases are substituted by a tryptophan and a serine, respectively, in XynSPP2. Full-length XynSPP2 and its Fn3-deleted variant (XynSPP2ΔFn3) were overexpressed in Escherichia coli and purified by Ni-affinity chromatography. The optimum temperature and pH for both recombinant enzymes were 50°C and 6, respectively. The enzymes were stable under alkaline condition and at temperature lower than 50°C. With beechwood xylan as the substrate, XynSPP2 showed 2.8 times the catalytic efficiency of XynSPP2ΔFn3, indicating that the Fn3 module promotes xylanase activity. XynSPP2 was active toward xylooligosaccharides (XOSs) longer than xylotriose. Such a substrate preference can be explained by the unique −2/−3 subsite composition in the enzyme which provides new insight into subsite interaction within the GH10 family. XynSPP2 hydrolyzed beechwood xylan into small XOSs (xylotriose and xylotetraose as major products). No monosaccharide was detected by thin-layer chromatography which may be ascribed to putative transxylosylation activity of XynSPP2. Preferring long XOS substrate and lack of monosaccharide production suggest its potential in probiotic XOS manufacture.

A bacterial strain (designated as Y-8), capable of efficiently degrading lignin under alkaline conditions, was isolated from saline-alkali soil and identified as Streptomyces sp. based on the morphological and physiological analyses as well as the gene sequences comparison of the 16 S rDNA. The alkali tolerance and lignin degradation capability of the isolated strain were determined. The results indicated that Streptomyces sp. Y-8 achieved the optimum lignin degradation at a temperature of 35 degrees C and a pH value of 9.5 with sucrose as the auxiliary carbon source and ammonium nitrate as the nitrogen source, reaching 32.6% in seven days. In addition, the activity of the three enzymes that were produced from the lignin degradation by Streptomyces sp. Y-8 under alkaline conditions (pH = 9.5), laccase (Lac), lignin peroxidase (Lip), and manganese peroxidase (MnP) was studied. The results indicated that the Lac and Lip activities reached their respective maximums on the fifth and fourth day of lignin degradation, respectively, being 4583.5 U/L, and 1163.2 U/L, respectively, and almost no MnP was detected throughout the process. This study is of great significance to the biological pretreatment of lignocellulose or the treatment of black liquid produced from papermaking under alkaline conditions.

Paper mulberry is a high-quality woody feed resource plant with high crude protein content. It is widely distributed in China and has excellent characteristics of salt and alkali tolerance. Paper mulberry has ecological and economic importance. Salt stress has become a critical factor with the increasing degree of soil salinity that restricts plant growth. In the saline-alkali environments, transcriptome expression is altered leading to phenotypic defects in most plants. However, the regulatory mechanism related to paper mulberry’s salt-stress (SS) response is not clearly understood. In the present study <i>de novo</i> transcriptomic assembly was performed, and gene expression levels were measured between different SS and natural conditions (25°C) as a control for paper mulberry plants. According to the results of our study, under NaCl stress conditions, the differential gene expression was observed in the leaves of paper mulberry compared with the control. A total of 2126 differentially expressed unigenes were observed. Among these unigenes the expression of 812 DEGs was up-regulated and the expression of 1,314 DEGs was down-regulated. Additionally, The GO and KEGG analyses regarding differentially expressed unigenes (DEUs) revealed that the observed critical transcriptomic alterations under salt stress (SS) conditions were associated with primary and secondary metabolism, photosynthesis, and plant hormone signaling pathways. Further investigations such as gene function studies regarding the unigenes depicting altered expression under salt stress conditions in paper mulberry will help understand the mechanism of salt tolerance, and this information can be utilized in paper mulberry breeding and improvement programs.

A neutral xylanase (XynII) from Volvariella volvacea was identified and characterized. Unlike other modular xylanases, it consists of only a single GH10 catalytic domain with a unique C-terminal sequence (W-R-W-F) and a phenylalanine and proline-rich motif (T-P-F-P-P-F) at N-terminus, indicating that it is a novel GH10 xylanase. XynII exhibited optimal activity at pH 7 and 60 °C and stability over a broad range of pH 4.0-10.0. XynII displayed extreme highly SDS resistance retaining 101.98, 92.99, and 69.84 % activity at the presence of 300 mM SDS on birchwood, soluble oat spelt, and beechwood xylan, respectively. It remained largely intact after 24 h of incubation with proteinase K at a protease to protein ratio of 1:50 at 37 °C. The kinetic constants K(m) value towards beechwood xylan was 0.548 mg ml⁻¹, and the k(cat)/K(m) ratio, reflecting the catalytic efficiency of the enzyme, was 126.42 ml mg⁻¹ s⁻¹ at 60 °C. XynII was a true endo-acting xylanase lacking cellulase activity. It has weak activity towards xylotriose but efficiently hydrolyzed xylans and xylooligosaccharides larger than xylotriose mainly to xylobiose. Synergistic action with acetyl xylan esterase (AXEI) from V. volvacea was observed for de-starched wheat bran. The highest degree of synergy (DS 1.42) was obtained in sequential reactions with AXEI digestion preceding XynII. The high SDS resistance and intrinsic stability suggested XynII may have potential applications in various industrial processes especially for the detergent and textile industries and animal feed industries.

Corynebacterium glutamicum is generally regarded as a moderately salt- and alkali-tolerant industrial organism. However, relatively little is known about the molecular mechanisms underlying these specific adaptations. Here, we found that the Mrp1 antiporter played crucial roles in conferring both environmental Na+ resistance and alkali tolerance whereas the Mrp2 antiporter was necessary in coping with high-KCl stress at alkaline pH. Furthermore, the Δmrp1 Δmrp2 double mutant showed the most-severe growth retardation and failed to grow under high-salt or alkaline conditions. Consistent with growth properties, the Na+/H+ antiporters of C. glutamicum were differentially expressed in response to specific salt or alkaline stress, and an alkaline stimulus particularly induced transcript levels of the Mrp-type antiporters. When the major Mrp1 antiporter was overwhelmed, C. glutamicum might employ alternative coordinate strategies to regulate antiport activities. Site-directed mutagenesis demonstrated that several conserved residues were required for optimal Na+ resistance, such as Mrp1A K299, Mrp1C I76, Mrp1A H230, and Mrp1D E136 Moreover, the chromosomal replacement of lysine 299 in the Mrp1A subunit resulted in a higher intracellular Na+ level and a more alkaline intracellular pH value, thereby causing a remarkable growth attenuation. Homology modeling of the Mrp1 subcomplex suggested two possible ion translocation pathways, and lysine 299 might exert its effect by affecting the stability and flexibility of the cytoplasm-facing channel in the Mrp1A subunit. Overall, these findings will provide new clues to the understanding of salt-alkali adaptation during C. glutamicum stress acclimatization.IMPORTANCE The capacity to adapt to harsh environments is crucial for bacterial survival and product yields, including industrially useful Corynebacterium glutamicum Although C. glutamicum exhibits a marked resistance to salt-alkaline stress, the possible mechanism for these adaptations is still unclear. Here, we present the physiological functions and expression patterns of C. glutamicum putative Na+/H+ antiporters and conserved residues of Mrp1 subunits, which respond to different salt and alkaline stresses. We found that the Mrp-type antiporters, particularly the Mrp1 antiporter, played a predominant role in maintaining intracellular nontoxic Na+ levels and alkaline pH homeostasis. Loss of the major Mrp1 antiporter had a profound effect on gene expression of other antiporters under salt or alkaline conditions. The lysine 299 residue may play its essential roles in conferring salt and alkaline tolerance by affecting the ion translocation channel of the Mrp1A subunit. These findings will contribute to a better understanding of Na+/H+ antiporters in sodium antiport and pH regulation.

The early-lineage, aerobic, zoosporic fungi from the Chytridiomycota constitute less than 1% of the described fungi and can use diverse sources of nutrition from plant or animal products. One of the ancestral sources of fungal nutrition could be products following enzymatic degradation of plant material. However, carbohydrate-active enzymes from these ancient fungi have been less studied. A GH11 xylanase (RrXyn11A) (EC 3.2.1.8) and a GH43 xylosidase (RrXyl43A) (EC 3.2.1.37) were identified from an early-lineage aerobic zoosporic fungus, Rhizophlyctis rosea NBRC 105426. Both genes were heterologously expressed in Pichia pastoris and the recombinant enzymes were purified and characterized. The optimal pH for recombinant RrXyn11A and RrXyl43A was pH 7. RrXyn11A had high stability over a wide range of pH (4–8) and temperature (25–70 °C). RrXyn11A also showed high substrate specificity on both azurine-cross-linked (AZCL) arabinoxylan and AZCL xylan. RrXyl43A had β-xylosidase and minor α-l-arabinofuranosidase activity. This enzyme showed low product inhibition and retained 51% activity in the presence of 100 mM xylose. A combination of RrXyn11A and RrXyl43A exhibited significantly higher hydrolytic and polymer degradation capability and xylose release on wheat bran and beechwood xylan compared to treatment with commercial enzymes. This study was the first to heterologously express and characterize the GH11 xylanase (RrXyn11A) and GH43 xylosidase (RrXyl43A) from the ancient fungus, R. rosea. Meanwhile, this study also demonstrated that the enzymes from the ancient fungus R. rosea can be easily handled and heterologously expressed in Pichia, which presents a promising path to a new source of enzymes for biomass degradation.

The rhizosphere microbiota plays a critical and crucial role in plant health and growth, assisting plants in resisting adverse stresses, including soil salinity. Plastic film mulching is an important method to adjust soil properties and improve crop yield, especially in saline-alkali soil. However, it remains unclear whether and to what extent the association between these improvements and rhizosphere microbiota exists. Here, from a field survey and a greenhouse mesocosm experiment, we found that mulching plastic films on saline-alkali soil can promote the growth of soybeans in the field. Results of the greenhouse experiment showed that soybeans grew better in unsterilized saline-alkali soil than in sterilized saline-alkali soil under plastic film mulching. By detecting the variations in soil properties and analyzing the high-throughput sequencing data, we found that with the effect of film mulching, soil moisture content was effectively maintained, soil salinity was obviously reduced, and rhizosphere bacterial and fungal communities were significantly changed. Ulteriorly, correlation analysis methods were applied. The optimization of soil properties ameliorated the survival conditions of soil microbes and promoted the increase in relative abundance of potential beneficial microorganisms, contributing to the growth of soybeans. Furthermore, the classification of potential key rhizosphere microbial OTUs were identified. In summary, our study suggests the important influence of soil properties as drivers on the alteration of rhizosphere microbial communities and indicates the important role of rhizosphere microbiota in promoting plant performance in saline-alkali soil under plastic film mulching.

Background: Most of the grasslands in China are experiencing varying degrees of degradation, desertification, and salinization (collectively referred to as the “three degradations”), posing a serious threat to the country’s ecological security. Agropyron desertorum, known for its wide distribution, strong adaptability, and resistance, is an excellent grass species for the ecological restoration of grasslands affected by the “three degradations”. This study focused on two currently popular varieties of A. desertorum, exploring their salt tolerance mechanisms and identifying candidate genes for salt and alkali tolerance. Methods: Transcriptome sequencing was performed on two varieties of A. desertorum during the seed germination and seedling stages under varying degrees of saline–alkali stress. At the seed stage, we measured the germination rate, relative germination rate, germination index, and salt injury rate under different NaCl concentrations. During the seedling stage, physiological indicators, including superoxide dismutase (SOD), peroxidase (POD), malondialdehyde (MDA), proline (PRO), soluble protein (SP), and catalase (CAT), were analyzed after exposure to 30, 60, 120, and 180 mM NaCl for 12 days. Analysis of differentially expressed genes (DEGs) at 6 and 24 h post-treatment with 120 mM NaCl revealed significant differences in the salt stress responses between the two cultivars. Results: Our study indicates that during the seed stage, A. desertorum (Schult.) exhibits a higher relative germination potential, relative germination rate, and relative germination index, along with a lower relative salt injury rate compared to A. desertorum cv. Nordan. Compared with A. desertorum cv. Nordan, A. desertorum (Schult.) has higher salt tolerance, which is related to its stronger antioxidant activity and different antioxidant-related pathways. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were used to identify the key biological processes and pathways involved in salt tolerance, including plant hormone signal transduction, antioxidant defense, and cell membrane stability. Conclusions: A. desertorum (Schult.) exhibits stronger salt tolerance than A. desertorum cv. Nordan. Salt stress at a concentration of 30–60 mM promotes the germination of the seeds of both Agropyron cultivars. The two Agropyron plants mainly overcome the damage caused by salt stress through the AsA-GSH pathway. This study provides valuable insights into the molecular mechanisms of salt tolerance in Agropyron species and lays the groundwork for future breeding programs aimed at improving salt tolerance in desert grasses.

A gene encoding glycoside hydrolase family 11 xylanase (HoXyn11B) from Hypocrea orientalis EU7-22 was expressed in Pichia pastoris with a high activity (413IU/ml). HoXyn11B was partly N-glycosylated and appeared two protein bands (19-29kDa) on SDS-PAGE. The recombinant enzyme exhibited optimal activity at pH 4.5 and 55°C, and retained more than 90% of the original activity after incubation at 50°C for 60min. The determined apparent K m and V max values using beechwood xylan were 10.43mg/ml and 3246.75IU/mg, respectively. The modes of action of recombinant HoXyn11B on xylo-oligosaccharides (XOSs) and beechwood xylan were investigated by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), which indicated that the modes of action of HoXyn11B are different from HoXyn11A since it is able to release a significant amount of xylose from various substrates. This study provides an opportunity to better understand the hydrolysis mechanisms of xylan by xylanases from Trichoderma.

A glycosyl hydrolase family 10 endoxylanase from Bacillus sp. HJ14 was grouped in a separated cluster with another six Bacillus endoxylanases which have not been characterized. These Bacillus endoxylanases showed less than 52% amino acid sequence identity with other endoxylanases and far distance with endoxylanases from most microorganisms. Signal peptide was not detected in the endoxylanase. The endoxylanase was expressed in Escherichia coli BL21 (DE3), and the purified recombinant enzyme (rXynAHJ14) was characterized. rXynAHJ14 was apparent optimal at 62.5 °C and pH 6.5 and retained more than 55% of the maximum activity when assayed at 40-75 °C, 23% at 20 °C, 16% at 85 °C, and even 8% at 0 °C. Half-lives of the enzyme were more than 60 min, approximately 25 and 4 min at 70, 75, and 80 °C, respectively. The enzyme exhibited more than 62% xylanase activity and stability at the concentration of 3-30% (w/v) NaCl. No xylanase activity was lost after incubation of the purified rXynAHJ14 with trypsin and proteinase K at 37 °C for 60 min. Different components of oligosaccharides were detected in the time-course hydrolysis of beechwood xylan by the enzyme. During the simulated intestinal digestion phase in vitro, 11.5-19.0, 15.3-19.0, 21.9-27.7, and 28.2-31.2 μmol/mL reducing sugar were released by the purified rXynAHJ14 from soybean meal, wheat bran, beechwood xylan, and rapeseed meal, respectively. The endoxylanase might be an alternative for potential applications in the processing of sea food and saline food and in aquaculture as agastric fish feed additive.

Salinity–alkalinity stress is one of the main abiotic factors limiting rice production worldwide. With the widespread use of rice direct seeding technology, it has become increasingly important to improve the tolerance to salinity–alkalinity of rice varieties at the germination stage. Although we have a more comprehensive understanding of salt tolerance in rice, the genetic basis of alkali tolerance in rice is still poorly understood. In this study, we measured seven germination-related traits under alkali stress and control conditions using 428 diverse rice accessions. The alkali tolerance levels of rice germplasms varied considerably during germination. Xian/indica accessions had generally higher tolerance to alkali stress than Geng/japonica accessions at the germination stage. Using genome-wide association analysis, 90 loci were identified as significantly associated with alkali tolerance. Eight genes (LOC_Os01g12000, LOC_Os03g60240, LOC_Os03g08960, LOC_Os04g41410, LOC_Os09g25060, LOC_Os11g35350, LOC_Os12g09350, and LOC_Os12g13300) were selected as important candidate genes for alkali tolerance based on the gene functional annotation and gene-CDS-haplotype analysis. According to the expression levels of LOC_Os09g25060 (OsWRKY76), it is likely to play a negative regulatory role in alkali tolerance during rice germination. An effective strategy for improving rice alkali tolerance may be to pyramid alkali-tolerant haplotypes of multiple candidate genes to obtain the optimal haplotype combination. Our findings may provide valuable genetic information and expand the use of alkali tolerance germplasm resources in rice molecular breeding to improve the alkali tolerance at the germination stage.