Acid Synthesis Research Articles

Characterization of Plant Growth Promoting Rhizobacteria and Their Benefits on Soybean Growth

Background: Soybean is the most important legume in the world and its seeds contains 40% protein and 20% oil. In recent years, more attention has been paid to the use of plant growth-promoting (PGP) rhizobacteria as a biofertilizer alternative to chemical fertilizers, which may pose risks to the environment. The rhizobia inoculation of soybean is a sustainable practice to induce atmospheric nitrogen fixation and subsequently improve crop productivity and soil fertility. Methods: In this study, 19 indigenous rhizobia isolated from soybean were tested in vitro for their plant growth-promoting properties (PGPR), while genotypic characterization included sequencing of the 16S rRNA gene. Result: Morphological characterisation has shown that all of the isolates were rod-shaped gram negative bacteria. The sequencing has shown that 12 out of 19 isolates belong to the genus Pseudomonas, four of which belong to P. fluorescens species. Isolates which belong to the Pseudomonas genus have shown the highest ability of indole-3-acetic acid synthesis, phosphorous solubilization and along with isolates SGN6, SGN7 and SGS4 (Sphingomonas sanguinis) potassium solubilisation too. All the isolates from Pseudomonas genus as well as those belonging to B. japonicum species have shown the protease synthesis abilities while amylase synthesis abilities was observed only in the isolate SAK2 (P. chlororaphis). The most efficient strains in in vitro biological nitrogen fixation assay belong to Pseudomonas and Rhizobium genera. Most of the isolates were positive to the organic acid production while all of them have shown the ability of the exopolysaccharide production.

Physiological Basis of Plant Growth Promotion in Rice by Rhizosphere and Endosphere Associated Streptomyces Isolates from India

This study demonstrated the plant growth-promoting capabilities of native actinobacterial strains obtained from different regions of the rice plant, including the rhizosphere (FT1, FTSA2, FB2, and FH7) and endosphere (EB6). We delved into the molecular mechanisms underlying the beneficial effects of these plant-microbe interactions by conducting a transcriptional analysis of a select group of key genes involved in phytohormone pathways. Through in vitro screening for various plant growth-promoting (PGP) traits, all tested isolates exhibited positive traits for indole-3-acetic acid synthesis and siderophore production, with FT1 being the sole producer of hydrogen cyanide (HCN). All isolates were identified as members of the Streptomyces genus through 16S rRNA amplification. In pot culture experiments, rice seeds inoculated with strains FB2 and FTSA2 exhibited significant increases in shoot dry mass by 7% and 34%, respectively, and total biomass by 8% and 30%, respectively. All strains led to increased leaf nitrogen levels, with FTSA2 demonstrating the highest increase (4.3%). On the contrary, strains FB2 and FT1 increased root length, root weight ratio, root volume, and root surface area, leading to higher root nitrogen content. All isolates, except for FB2, enhanced total chlorophyll and carotenoid levels. Additionally, qRT-PCR analysis supported these findings, revealing differential gene expression in auxin (OsAUX1, OsIAA1, OsYUCCA1, OsYUCCA3), gibberellin (OsGID1, OsGA20ox-1), and cytokinin (OsIPT3, OsIPT5) pathways in response to specific actinobacterial treatments. These actinobacterial strains, which enhance both aboveground and belowground crop characteristics, warrant further evaluation in field trials, either as individual strains or in consortia. This could lead to the development of commercial bioinoculants for use in integrated nutrient management practices.

Aspects of the joint cultivation of <i>Bacillus amyloliquefaciens</i> and <i>Bacillus aryabhattai</i> for the intensification of growth-stimulating substances synthesis

Growth-stimulating properties of Bacillus amyloliquefaciens B-11265 and Bacillus aryabhattai B-13192 strains were studied, and the possibility of their co-cultivation for activity intensification was evaluated. The strains under study were found to be able to synthesize indolyl-3-acetic and gibberellic acids, siderophores: B. amyloliquefaciens B-11265 in the amount of 5.23 µg/ml, 1.05 mg/ml, 46.15%; B. aryabhattai B-13192 – 3.07 µg/ml, 0.84 mg/ml, 35.50%, respectively. The research results showed that the strains were biocompatible and did not inhibit each other's development, allowing them to be included in the same consortium. Joint cultivation of the strains showed that the ratio of microorganisms in the nutrient substrate has a significant effect on the yield of the target substances. At the ratio of B. amyloliquefacien and B. aryabhattai 1 : 1, inhibition of indolyl-3-acetic acid synthesis was observed; ratios of 2 : 3 and 3 : 2 led to a decrease in gibberellic acid production. It was found that three (B. amyloliquefacien) to one (B. aryabhattai) was the optimal ratio to increase the yield of the target substances compared to the average values characteristic of individual strains. In this variant, the synthesis of growth-stimulating substances by the consortium was: indolyl-3-acetic acid – 5.31 μg/ml, gibberellic acid – 1.23 mg/ml, siderophores – 51.27%. The results obtained indicate the high potential of the engineered consortium for agricultural applications. However, in order to develop a growth-stimulating preparation based on it, it is necessary to conduct a number of additional studies aimed at investigating the effect of the strain on agricultural crops in laboratory and field experiments.

Soil microbial communities and degradation of pesticides in greenhouse effluent through a woodchip bioreactor

Pesticides, including insecticides and fungicides, are major contaminants in the effluent from intensive agricultural systems, such as greenhouses. Because of their constant use and persistence, some pesticides can accumulate in soil and/or run off into adjacent waterways. Microbial communities in soil can degrade some pesticides, and bioreactors with enhanced microbial communities have the potential to facilitate decontamination before the effluent is released into the environment. In this study, we sampled the soil along a gradient from immediately below greenhouses, into, through and below a bioreactor. Multi-analyte pesticide screening was undertaken along with shotgun metagenomic sequencing, to assess microbial community taxonomic profiles and metabolic pathway responses for functional analysis. Two insecticides (imidacloprid and fipronil) and nine fungicides were identified in the soil samples, with a general decrease in most pesticides with increasing distance from the greenhouses. Diversity indexes of taxonomic profiles show changes in the microbial community along the gradient. In particular, microbial communities were significantly different in the bioreactor, with lower Shannon diversity compared to immediately below the greenhouses, in the channels leading into the bioreactor and further downstream. Metabolic pathway analysis revealed significant changes in a wide range of core housekeeping genes such as protein/amino acid synthesis and lipid/fatty acid biosynthesis among the sampling sites. The result demonstrates that the composition and potential functional pathways of the microbial community shifted towards an increased tendency for phytol and contaminant degradation in the bioreactor, facilitated by high organic matter content. This highlights the potential to use enhanced microbial communities within bioreactors to reduce contamination by some pesticides in sediment receiving run-off from greenhouses.

Elimination of ineffective inorganic salt component in medium for indole‐3‐acetic acid synthesis by Serratia plymuthica UBCF_13 and its effect on the growth of chili seedlings

Indole‐3‐acetic acid (IAA) is an essential phytohormone that controls a variety of plant growth mechanisms. Bacteria can produce IAA to stimulate plant growth, with its production influenced by the culture conditions. Serratia plymuthica UBCF_13 is recognized as an IAA‐producing bacterium, exhibiting maximum IAA production in a yeast medium comprising yeast extract, sucrose, K2HPO4, MgSO4, NaCl, and CaCO3. However, prior studies optimizing individual inorganic salt components indicated minimal impact on IAA synthesis within this medium. This study aimed to eliminate the unnecessary inorganic salt components and the medium was then applied to investigate the IAA biosynthesis pathway and the plant growth‐promoting assay. The elimination assay consisted of yeast sucrose medium devoid of K2HPO4, MgSO4, NaCl, or CaCO3, and yeast sucrose medium containing only MgSO4 and CaCO3. Various indole compounds were then added to the revised medium composition to investigate the IAA biosynthesis pathway of UBCF_13 using high‐performance liquid chromatography (HPLC). Furthermore, the effect of UBCF_13 culture supernatant, cultivated in the new medium, on chili plant growth was evaluated. The highest IAA production (138.8 µg/mL) was observed in the yeast sucrose with CaCO3 and MgSO4 (elimination of K2HPO4 and NaCl). The presence of indole‐3‐acetamide (IAM) compound from the medium extracts, supplemented with multiple indole compounds, revealed that UBCF_13 may use the IAM pathway. The application of UBCF_13 supernatant enhanced the shoot, root length, fresh weight, and germination time of chili seeds by 37.7%, 49.3%, 204.3%, and 38.6%, respectively. This study demonstrated that eliminating K2HPO4 and NaCl provided a new culture medium composition conducive to IAA production by UBCF_13. Moreover, the UBCF_13 extract has the potential to promote plant growth.

Бактерии-деструкторы с ростстимулирующими свойствами для использования в экологической

A topical research trend in ecological biotechnology is the search and study of microorganisms-destructor resistant to additional pollutants and growth-stimulating to remediant-plants. Four strains of hydrocarbon-oxidizing microorganisms isolated from soil samples were identified on the basis of cultural-morphological, physiological-biochemical properties and a comparative analysis of the gene encoding 16S rRNA. The bacteria Acinetobacter calcoaceticus UOM 22 and UOM 29, A. courvalinii UOM 35 showed a significant degree of oil biodegradation in a liquid medium at 24 and 8 °C (93.0–95.6% and 78.4–81.1%, respectively). In the same conditions these indicators were 44.8 and 33.7%, respectively for strain Rhodococcus erythropolis UOM 33. All microorganisms grew in the presence of aromatic hydrocarbons (including polycyclic hydrocarbons), were resistant to NaCl (5–7%) and lead ions (1.00–1.25 g/L). They were capable of nitrogen fixation and inorganic phosphate dissolution, indole-3-acetic acid synthesis (150–1416 ng/ml of culture liquid) and hydrolytic enzyme lipase production. We found that A. calcoaceticus UOM 22 and A. courvalinii UOM 35 strains increased the barley seeds germination by 8.6 and 10.5%, as well as A. calcoaceticus UOM 29 and A. courvalinii UOM 35 stimulated the barley root growth by 16.3 and 18.1% respectively. The results of the experiments indicate the possibility of using A. calcoaceticus UOM 22 and UOM 29, and A. courvalinii UOM 35 strains for cleaning oil-contaminated soils, including in combination with remediant-plants.

Prohexadione-calcium improves grape quality by regulating endogenous hormones, sugar and acid metabolism and related enzyme activities in grape berries.

Prohexadione-Calcium (Pro-Ca) plays key roles in improving fruit quality and yield by regulating various aspects of plant growth. However, the effects of how Pro-Ca regulates the regulation of sugar and acid balance and its impact on the production of volatile aroma substances during fruit growth and development are poorly understood. In this study, the Pro-Ca solutions developed at concentrations of 200, 400, 600 and 800 mg·L-1 were sprayed on the entire "Chardonnay" grape tree 22, 42, 62 and 82 days after initial flowering. The values of endogenous hormones, sugar and acid content, enzyme activities and flavor content were then measured in grapes 45, 65, 85 and 105 days (ripeness stage) after the initial flowering. The results showed that Pro-Ca had significant effects on fruits during development, including reducing ABA content, increasing ZT, GA3 and IAA levels, promoting fruit ripening and enhancing enzymes, which are involved in sugar and acid synthesis. Consequently, these effects led to an increase in sugar and acid content in the berries. Particularly during the ripening phase, the application of 600 mg L-1 Pro-Ca resulted in an increase in soluble sugar content of 11.28% and a significant increase in citric acid and malic acid content of 97.80% and 68.86%, respectively. Additionally, Pro-Ca treatment enhanced both the variety and quantity of aroma compounds present in the berries, with the 600 mg·L-1 Pro-Ca treatment showcasing the most favorable impact on volatile aroma compounds in 'Chardonnay' grapes. The levels of aldehydes, esters, alcohols, phenols, acids, ketones, and terpenes were significantly higher under the 600 mg·L-1 Pro-Ca treatment compared to those of control with 51.46 - 423.85% increase. In conclusion, Pro-Ca can regulate the content of endogenous hormones and the activities of enzymes related to sugar and acid metabolism in fruit, thereby increasing the content of soluble sugar and organic acid in fruit and the diversity and concentration of fruit aroma substances. Among them, foliar spraying 600mg · L-1 Pro-Ca has the best effect. In the future, we need to further understand the molecular mechanism of Pro-Ca in grape fruit to lay a solid foundation for quality improvement breeding.

Novel recombinant aminoacylase from Paraburkholderia monticola capable of N-acyl-amino acid synthesis

N-Acyl-amino acids can act as mild biobased surfactants, which are used, e.g., in baby shampoos. However, their chemical synthesis needs acyl chlorides and does not meet sustainability criteria. Thus, the identification of biocatalysts to develop greener synthesis routes is desirable. We describe a novel aminoacylase from Paraburkholderia monticola DSM 100849 (PmAcy) which was identified, cloned, and evaluated for its N-acyl-amino acid synthesis potential. Soluble protein was obtained by expression in lactose autoinduction medium and co-expression of molecular chaperones GroEL/S. Strep-tag affinity purification enriched the enzyme 16-fold and yielded 15 mg pure enzyme from 100 mL of culture. Biochemical characterization revealed that PmAcy possesses beneficial traits for industrial application like high temperature and pH-stability. A heat activation of PmAcy was observed upon incubation at temperatures up to 80 °C. Hydrolytic activity of PmAcy was detected with several N-acyl-amino acids as substrates and exhibited the highest conversion rate of 773 U/mg with N-lauroyl-L-alanine at 75 °C. The enzyme preferred long-chain acyl-amino-acids and displayed hardly any activity with acetyl-amino acids. PmAcy was also capable of N-acyl-amino acid synthesis with good conversion rates. The best synthesis results were obtained with the cationic L-amino acids L-arginine and L-lysine as well as with L-leucine and L-phenylalanine. Exemplarily, L-phenylalanine was acylated with fatty acids of chain lengths from C8 to C18 with conversion rates of up to 75%. N-lauroyl-L-phenylalanine was purified by precipitation, and the structure of the reaction product was verified by LC–MS and NMR.Key points• A novel aminoacylase from Paraburkholderia monticola was cloned, expressed in E. coli and purified.• The enzyme PmAcy exhibits exceptional temperature and pH stability and a broad substrate spectrum.• Synthesis of acyl amino acids was achieved in good yields.

Enhanced poly-γ-glutamic acid synthesis in Corynebacterium glutamicum by reconstituting PgsBCA complex and fermentation optimization

Previously, a novel Corynebacterium glutamicum strain for the de novo biosynthesis of tailored poly-γ-glutamic acid (γ-PGA) has been constructed by our group. The strain was based on the γ-PGA synthetase complex, PgsBCA, which is the only polyprotein complex responsible for γ-PGA synthesis in Bacillus spp. In the present study, PgsBCA was reconstituted and overexpressed in C. glutamicum to further enhance γ-PGA synthesis. First, we confirmed that all the components (PgsB, PgsC, and PgsA) of γ-PGA synthetase derived from B. licheniformis are necessary for γ-PGA synthesis, and γ-PGA was detected only when PgsB, PgsC, and PgsA were expressed in combination in C. glutamicum. Next, the expression level of each pgsB, pgsC, and pgsA was tuned in order to explore the effect of expression of each of the γ-PGA synthetase subunits on γ-PGA production. Results showed that increasing the transcription levels of pgsB or pgsC and maintaining a medium-level transcription level of pgsA led to 35.44% and 76.53% increase in γ-PGA yield (γ-PGA yield-to-biomass), respectively. Notably, the expression level of pgsC had the greatest influence (accounting for 68.24%) on γ-PGA synthesis, followed by pgsB. Next, genes encoding for PgsC from four different sources (Bacillus subtilis, Bacillus anthracis, Bacillus methylotrophicus, and Bacillus amyloliquefaciens) were tested in order to identify the influence of PgsC-encoding orthologues on γ-PGA production, but results showed that in all cases the synthesis of γ-PGA was significantly inhibited. Similarly, we also explored the influence of gene orthologues encoding for PgsB on γ-PGA production, and found that the titer increased to 17.14 ± 0.62 g/L from 8.24 ± 0.10 g/L when PgsB derived from B. methylotrophicus replaced PgsB alone in PgsBCA from B. licheniformis. The resulting strain was chosen for further optimization, and we achieved a γ-PGA titer of 38.26 g/L in a 5 L fermentor by optimizing dissolved oxygen level. Subsequently, by supplementing glucose, γ-PGA titer increased to 50.2 g/L at 48 h. To the best of our knowledge, this study achieved the highest titer for de novo production of γ-PGA from glucose, without addition of L-glutamic acid, resulting in a novel strategy for enhancing γ-PGA production.

Maize Dek407 Encodes the Nitrate Transporter 1.5 and Is Required for Kernel Development.

The kernel serves as the storage organ and harvestable component of maize, and it plays a crucial role in determining crop yield and quality. Understanding the molecular and genetic mechanisms of kernel development is of considerable importance for maize production. In this study, we obtained a mutant, which we designated defective kernel 407 (dek407), through ethyl methanesulfonate mutagenesis. The dek407 mutant exhibited reduced kernel size and kernel weight, as well as delayed grain filling compared with those of the wild type. Positional cloning and an allelism test revealed that Dek407 encodes a nitrate transporter 1/peptide transporter family (NPF) protein and is the allele of miniature 2 (mn2) that was responsible for a poorly filled defective kernel phenotype. A transcriptome analysis of the developing kernels showed that the mutation of Dek407 altered the expression of phytohormone-related genes, especially those genes associated with indole-3-acetic acid synthesis and signaling. Phytohormone measurements and analysis indicated that the endogenous indole-3-acetic acid content was significantly reduced by 66% in the dek407 kernels, which may be the primary cause of the defective phenotype. We further demonstrated that natural variation in Dek407 is associated with kernel weight and kernel size. Therefore, Dek407 is a potential target gene for improvement of maize yield.

Melatonin: A biomolecule for mitigating postharvest chilling injury in fruits and vegetables

AbstractThe best strategy to extend the shelf life of fresh horticultural produce is to keep it at a low temperature. However, storing fruits and vegetables at a temperature lower than their recommended storage temperature and above the freezing point for a prolonged time leads to the development of chilling injury (CI). Melatonin (MT) being a versatile biological signal has been reported to possess antioxidant properties that aid in mediating stress response and extending the postharvest storage life of horticultural crops. Among CI‐induced mechanisms, MT substantially reduces oxidative stress by elevating the reactive oxygen species scavenging and triggering the phenol accumulation in association with 2, 2‐diphenyl‐1 picryl hydrazyl scavenging activity. Besides, MT increases intracellular energy by maintaining a proper ratio of unsaturated to saturated fatty acid and improving cellular fluidity. Additionally, MT regulates arginine metabolism by maintaining the γ‐aminobutyric acid synthesis. Furthermore, MT encodes the mitogen‐activated protein kinases, and calcium‐dependent protein kinases cascade and acclimate to cold stress by activating C‐repeat binding factors and helps in maintaining postharvest quality. This review reports the recent progress of MT‐induced physiological, biochemical, and molecular mechanisms for enhancing chilling tolerance, which can elevate the marketability and earnings from postharvest horticultural crops.

Sustainably Scaled Electrochemical Synthesis of 3‐Propyladipic Acid in Line with Fluctuating Grid Supply

AbstractChemical production is a significant contributor to global climate change, which expedites the growing demand for transitioning to more sustainable and climate‐friendly methodologies. Ideally this should include high compatibility with the fluctuating electricity supply which results from renewable energy sources in the electrical grid. Here we show an electrochemical path for the 3‐propyladipic acid synthesis from 4‐propylcyclohexanol implementing a semi‐technical electrochemical continuously stirred tank reactor. Following a Design of Experiments approach, we found a strong influence of the biphasic electrolyte mixing and the continuous feeding in of the substrate. By switching to an electrolyte recirculation mode and efficient mixing, the isolated product yield could be increased up to 31 % for a 10 L total reaction volume, indicating the potential for further scale‐up into the technical range. This reaction proceeds while forming several by‐products, which have not been fully described yet. A proposal for the formation mechanism is included.

Comparative Proteome-wide Characterization of Three Different Tissues of High-Protein Mutant and Wild Type Unravels Protein Accumulation Mechanisms in Rice Seeds.

Improving the proteins and amino acid contents of rice seeds is one of the prime objectives of plant breeders. We recently developed an EMS mutant/high-protein mutant (HPM) of rice that exhibits 14.8% of the total protein content as compared to its parent Dharial (wild-type), which shows only 9.3% protein content in their mature seeds. However, the mechanisms underlying the higher protein accumulation in these HPM seeds remain largely elusive. Here, we utilized high-throughput proteomics to examine the differences in the proteome profiles of the embryo, endosperm, and bran tissues of Dharial and HPM seeds. Utilizing a label-free quantitative proteomic and subsequent functional analyses of the identified proteins revealed that nitrogen compound biosynthesis, intracellular transport, protein/amino acid synthesis, and photosynthesis-related proteins were specifically enriched in the endosperm and bran of the high-protein mutant seed. Our data have uncovered proteome-wide changes highlighting various functions of metabolic pathways associated with protein accumulation in rice seeds.

Increased yield of 2-O-α-d-glucopyranosyl-l-ascorbic acid synthesis by α-glucosidase using rational design that regulating the ground state of enzyme and substrate complex.

α-Glucosidase (AG) is a bifunctional enzyme, it has a capacity to synthesize 2-O-α-d-glucopyranosyl-l-ascorbic acid (AA-2G) from l-ascorbic acid (L-AA) and low-cost maltose under mild conditions, but it can also hydrolyze AA-2G, which leads to low synthesis efficiency of AA-2G. This study introduces a rational molecular design strategy to regulate enzymatic reactions based on inhibiting the formation of ground state of enzyme-substrate complex. Y215 was analyzed as the key amino acid site affecting the affinity of AG to AA-2G and L-AA. For the purpose of reducing the hydrolysis efficiency of AA-2G, the mutant Y215W was obtained by analyzing the molecular docking binding energy and hydrogen bond formation between AG and the substrates. Compared with the wild-type, isothermal titration calorimetry (ITC) results showed that the equilibrium dissociation constant (KD ) of the mutant for AA-2G was doubled; the Michaelis constant (Km ) for AA-2G was reduced by 1.15 times; and the yield of synthetic AA-2G was increased by 39%. Our work also provides a new reference strategy for the molecular modification of multifunctional enzymes and other enzymes in cascade reactions system.

α,β-Dehydroamino Acid Synthesis through Proline-Catalyzed Aldol Condensation with a Glycine Schiff Base.

A general protocol for the synthesis of α,β-dehydroamino acids and their peptides was developed. Proline efficiently catalyzed an aldol condensation reaction of a glycine Schiff base with a variety of aldehydes. The hydroxy group on the benzophenone imine was crucial for high Z/E selectivity and further transimination for protecting group-free α,β-dehydroamino esters. Peptide elongation of both the C- and N-terminals highlighted the usefulness of our present protocol.

Transition-Metal-Free Upscaling of 2,5-Furandicarboxylic Acid Synthesis and Investigation of the Reaction Mechanism.

An environmentally friendly oxidation system has proposed for the practical and scalable production of value-added 2,5-furandicarboxylic acid from 1 kg of 5-hydroxymethylfurfural. The system is composed of a simple base, oxygen, and a green solvent, thereby providing a sustainable and economical approach to organic synthesis. To gain insight into the mechanism of this oxidation process, NMR spectroscopic analysis and kinetic study are used for the mechanistic investigation of this environmentally friendly oxidation process.

Characterization and genome analysis of Acinetobacter oleivorans S4 as an efficient hydrocarbon-degrading and plant-growth-promoting rhizobacterium

Plant-growth-promoting rhizobacteria (PGPR) have received increasing attention for assisting phytoremediation. However, the effect of PGPR on total petroleum hydrocarbon (TPH) degradation and plant growth promotion and its underlying mechanism is not well understood. In this study, phenotypic analysis and whole genome sequencing were conducted to comprehensively characterize a newly isolated rhizobacterium strain S4, which was identified as Acinetobacter oleivorans, from a TPH-contaminated soil. The strain degraded 62.5% of initially spiked diesel (1%) in minimal media within six days and utilized n-alkanes with a wide range of chain length (i.e., C12 to C40). In addition, the strain showed phenotypic traits beneficial to plant growth, including siderophore production, indole-3-acetic acid synthesis and phosphate solubilization. Potential metabolic pathways and genes encoding proteins responsible for the phenotypic traits were identified. In a real TPH-contaminated soil, inoculation of Acinetobacter oleivorans S4 significantly enhanced the growth of tall fescue relative to the soil without inoculation. In contrast, inoculation of Bacillus sp. Z7, a hydrocarbon-degrading strain, showed a negligible effect on the growth of tall fescue. The removal efficiency of TPH with inoculation of Acinetobacter oleivorans S4 was significantly higher than those without inoculation or inoculation of Bacillus sp. Z7. These results suggested that traits of PGPR beneficial to plant growth are critical to assist phytoremediation. Furthermore, heavy metal resistance genes and benzoate and phenol degradation genes were found in the genome of Acinetobacter oleivorans S4, suggesting its application potential in broad scenarios.

Enhancing 2-O-α-D-glucopyranosyl-L-ascorbic acid synthesis by weakening the acceptor specificity of CGTase toward glucose and maltose.

2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) is a stable derivative of L-ascorbic acid (L-AA), which has been widely used in food and cosmetics industries. Sugar molecules, such as glucose and maltose produced by cyclodextrin glycosyltransferase (CGTase) during AA-2G synthesis may compete with L-AA as the acceptors, resulting in low AA-2G yield. Multiple sequence alignment combined with structural simulation analysis indicated that residues at positions 191 and 255 of CGTase may be responsible for the difference in substrate specificity. To investigate the effect of these two residues on the acceptor preference and the AA-2G yield, five single mutants Bs F191Y, Bs F255Y, Bc Y195F, Pm Y195F and Pm Y260F of three CGTases from Bacillus stearothermophilus NO2 (Bs), Bacillus circulans 251 (Bc) and Paenibacillus macerans (Pm) were designed for AA-2G synthesis. Under optimal conditions, the AA-2G yields of the mutants Bs F191Y and Bs F255Y AA-2G were 34.3% and 7.9% lower than that of Bs CGTase, respectively. The AA-2G yields of mutant Bc Y195F, Pm Y195F and Pm Y260F were 45.8%, 36.9% and 12.6% higher than those of wild-type CGTases, respectively. Kinetic studies revealed that the residues at positions 191 and 255 of the three CGTases were F, which decreased glucose and maltose specificity and increased L-AA specificity. This study not only proposes for the first time that the AA-2G yield can be improved by weakening the acceptor specificity of CGTase toward sugar byproducts, but also provides new insight on the modification of CGTase that catalyze the double-substrate transglycosylation reaction.

Chaperone assisted recombinant expression of a mycobacterial aminoacylase in Vibrio natriegens and Escherichia coli capable of N-lauroyl-L-amino acid synthesis

BackgroundAminoacylases are highly promising enzymes for the green synthesis of acyl-amino acids, potentially replacing the environmentally harmful Schotten-Baumann reaction. Long-chain acyl-amino acids can serve as strong surfactants and emulsifiers, with application in cosmetic industries. Heterologous expression of these enzymes, however, is often hampered, limiting their use in industrial processes.ResultsWe identified a novel mycobacterial aminoacylase gene from Mycolicibacterium smegmatis MKD 8, cloned and expressed it in Escherichia coli and Vibrio natriegens using the T7 overexpression system. The recombinant enzyme was prone to aggregate as inclusion bodies, and while V. natriegens Vmax™ could produce soluble aminoacylase upon induction with isopropyl β-d-1-thiogalactopyranoside (IPTG), E. coli BL21 (DE3) needed autoinduction with lactose to produce soluble recombinant protein. We successfully conducted a chaperone co-expression study in both organisms to further enhance aminoacylase production and found that overexpression of chaperones GroEL/S enhanced aminoacylase activity in the cell-free extract 1.8-fold in V. natriegens and E. coli. Eventually, E. coli ArcticExpress™ (DE3), which co-expresses cold-adapted chaperonins Cpn60/10 from Oleispira antarctica, cultivated at 12 °C, rendered the most suitable expression system for this aminoacylase and exhibited twice the aminoacylase activity in the cell-free extract compared to E. coli BL21 (DE3) with GroEL/S co-expression at 20 °C. The purified aminoacylase was characterized based on hydrolytic activities, being most stable and active at pH 7.0, with a maximum activity at 70 °C, and stability at 40 °C and pH 7.0 for 5 days. The aminoacylase strongly prefers short-chain acyl-amino acids with smaller, hydrophobic amino acid residues. Several long-chain amino acids were fairly accepted in hydrolysis as well, especially N-lauroyl-L-methionine. To initially evaluate the relevance of this aminoacylase for the synthesis of N-acyl-amino acids, we demonstrated that lauroyl-methionine can be synthesized from lauric acid and methionine in an aqueous system.ConclusionOur results suggest that the recombinant enzyme is well suited for synthesis reactions and will thus be further investigated.

β,γ,δ,ε-Amino Acid Synthesis by Photocatalytic Carboxylation with CO2

Key words β,γ,δ,ε-amino acids - photocatalysis - carboxylation - carbon dioxide