Strain M2 Research Articles

Antibiotic-Free High-Level l-Methionine Production in Engineered Escherichia coli.

l-Methionine, a valuable sulfur-containing amino acid, holds great significance as a feed additive, nutraceutical, pharmaceutical, or even in the cosmetic industry. However, achieving efficient microbial production of l-methionine remains challenging due to its complex biosynthetic pathway and plasmid loss during fermentation. Herein, l-methionine biosynthesis was improved by enhancing succinyl-CoA supply, introducing a direct-sulfurylation pathway, and weakening the l-threonine branched pathway. The engineered strain produced 21.55 g/L l-methionine with a yield of 0.14 g/g glucose in a 5 L bioreactor. To eliminate the need for antibiotics and minimize plasmid loss, the hok/sok system was incorporated into the plasmid. The resulting plasmid pAm10 enabled strain M2 thrBA1G to produce 20.39 g/L of l-methionine without antibiotics in 5 L of fed-batch cultivation, a 42.58% increase compared to the control. This study highlights the potential of plasmid-based antibiotic-free fermentation for efficient and cost-effective production of l-methionine, as well as other amino acids or chemicals.

Serine affects engulfment during the sporulation process in Clostridium perfringens strain SM101

ObjectivesAlthough Clostridium perfringens sporulation is a key event in the pathogenesis of food-borne illness, the molecules and underlying mechanisms responsible for regulating sporulation are incompletely understood. The present study sought to identify amino acids that affect sporulation in C. perfringens strain SM101. MethodsA C. perfringens strain was cultured in the chemically defined medium deficient in an amino acid. The bacterial growth was determined by spectrophotometrically measuring culture turbidity and by calculating colony-forming unit. Morphological characteristics were assessed by phase-contrast microscopy with fluorescent staining and by electron microscopy. ResultsThe amino acids Arg, Cys, Gly, His, Ile, Leu, Met, Phe, Thr, Trp, Tyr, and Val were important for sporulation, and furthermore, Ser reduced sporulation. The mechanism underlying Ser-induced prevention of sporulation was assessed morphologically. The numbers of bacterial cells in sporulation stage II were significantly higher in the presence than in the absence of Ser. In the presence of Ser, almost all cells were in stage II−III, characterized by polar septation−early engulfment, and did not proceed to late engulfment. ConclusionsThese results suggest that Ser accelerated the early stage of sporulation of C. perfringens strain SM101, but disturbed the engulfment process, resulting in reduction of sporulation. To the best of our knowledge, this is the first study reporting that an amino acid affects engulfment during the C. perfringens sporulation process.

Isolation of α-Amylase Producing Microorganisms from Soil of Kachchh, Gujarat

The purpose of this study is to explore the soil of the Gujarat, Kachchh region to identify amylase-producing bacteria and characterize them using molecular methods. The unique ecological characteristics of the Kachchh region may facilitate the isolation of these bacteria. Samples were collected from multiple locations within the Kachchh District, including Gandhidham, Rapar, Bhuj, Nakhatrana, Mandvi, and Mundra Talukas. These samples were then screened to isolate amylase-producing bacteria. A total of 27 different types of colonies were identified, out of which 16 exhibited amylase production (M1-M16). Out of 27 colonies identified, 16 showed amylase production. Strains M2, M7, and M13 exhibited high amylase activity, with M2 showing a consistent increase over 72 hours, making it a strong candidate for amylase production. Further identification of M2 stain identified M2 as a Gram-positive, spore-forming, capsulated, and motile bacillus, specifically Bacillus licheniformis. This was confirmed through DNA sequencing and analysis in the NCBI database, which showed a 99.15% similarity with Bacillus licheniformis. The study concludes that Soil in Kachchh is rich with microorganisms that produce amylase, an enzyme with diverse industrial applications. These organisms are valuable for sectors like food, textiles, paper, detergents, pharmaceuticals, and biofuel production.

Phosphorus-Solubilizing Bacteria Enhance Cadmium Immobilization and Gene Expression in Wheat Roots to Reduce Cadmium Uptake.

The application of phosphorus-solubilizing bacteria is an effective method for increasing the available phosphorus content and inhibiting wheat uptake of heavy metals. However, further research is needed on the mechanism by which phosphorus-solubilizing bacteria inhibit cadmium (Cd) uptake in wheat roots and its impact on the expression of root-related genes. Here, the effects of strain Klebsiella aerogenes M2 on Cd absorption in wheat and the expression of root-related Cd detoxification and immobilization genes were determined. Compared with the control, strain M2 reduced (64.1-64.6%) Cd uptake by wheat roots. Cd fluorescence staining revealed that strain M2 blocked the entry of exogenous Cd into the root interior and enhanced the immobilization of Cd by cell walls. Forty-seven genes related to Cd detoxification, including genes encoding peroxidase, chalcone synthase, and naringenin 3-dioxygenase, were upregulated in the Cd+M2 treatment. Strain M2 enhanced the Cd resistance and detoxification activity of wheat roots through the regulation of flavonoid biosynthesis and antioxidant enzyme activity. Moreover, strain M2 regulated the expression of genes related to phenylalanine metabolism and the MAPK signaling pathway to enhance Cd immobilization in roots. These results provide a theoretical basis for the use of phosphorus-solubilizing bacteria to remediate Cd-contaminated fields and reduce Cd uptake in wheat.

Immobilization of Heavy Metals by Phosphorus-solubilizing Bacteria and Inhibition of Cd and Pb Uptake by Wheat

Phosphorus-solubilizing microorganisms convert insoluble phosphorus in the soil into phosphorus that can be absorbed by plants. Soluble phosphate combines with heavy metals to form precipitation, reducing the content of available heavy metals, thereby reducing the absorption of heavy metals by crops, which plays an important role in the remediation of heavy metal-contaminated soil. The effects of the immobilization of Cd and Pb and the release of PO43- by the phosphorus-solubilizing bacterium Klebsiella sp. M2 were studied through solution culture experiments. In addition, the effects of strain M2 on wheat uptake of Cd and Pb and its microbiological mechanism were also explored through pot experiments. The results showed that strain M2 reduced the concentrations of Cd and Pb and increased the concentration of PO43- in the solution through cell wall adsorption and induced phosphate precipitation. Pot experiments showed that compared to those in the CK group and inactivated strain M2 group, inoculation with live strain M2 significantly increased （123%-293%） the contents of Ca2-P and Ca8-P in rhizosphere soil, decreased the content of DTPA-Cd （34.48%） and DTPA-Pb （36.72%） in wheat rhizosphere soil, and thus hindered the accumulation of Cd and Pb in wheat grains. Moreover, high-throughput sequencing results showed that strain M2 significantly increased the diversity of wheat rhizosphere bacterial communities； increased the relative abundance of Proteobacteria, Gemmatimonadetes, and Bacteroidota in wheat rhizosphere soil； and increased the proportion of heavy metal-immobilizing and phosphorus-promoting bacteria in wheat rhizosphere soil （mainly Sphingomonas, Nocardioides, Bacillus, Gemmatimonas, and Enterobacter）. These bacterial genera played an important role in immobilizing heavy metals and preventing wheat from absorbing heavy metals. These results provide bacterial resources and theoretical basis for the bioremediation of heavy metal-contaminated farmland.

Engineering carbon source division of labor for efficient α-carotene production in Corynebacterium glutamicum

Effective utilization of glucose, xylose, and acetate, common carbon sources in lignocellulose hydrolysate, can boost biomanufacturing economics. However, carbon leaks into biomass biosynthesis pathways instead of the intended target product remain to be optimized. This study aimed to enhance α-carotene production by optimizing glucose, xylose, and acetate utilization in a high-efficiency Corynebacterium glutamicum cell factory. Heterologous xylose pathway expression in C. glutamicum resulted in strain m4, exhibiting a two-fold increase in α-carotene production from xylose compared to glucose. Xylose utilization was found to boost the biosynthesis of pyruvate and acetyl-CoA, essential precursors for carotenoid biosynthesis. Additionally, metabolic engineering including pck, pyc, ppc, and aceE deletion, completely disrupted the metabolic connection between glycolysis and the TCA cycle, further enhancing α-carotene production. This strategic intervention directed glucose and xylose primarily towards target chemical production, while acetate supplied essential metabolites for cell growth recovery. The engineered strain C. glutamicum m8 achieved 30 mg/g α-carotene, 67% higher than strain m4. In fed-batch fermentation, strain m8 produced 1802 mg/L of α-carotene, marking the highest titer reported to date in microbial fermentation. Moreover, it exhibited excellent performance in authentic lignocellulosic hydrolysate, producing 216 mg/L α-carotene, 1.45 times higher than the initial strain (m4). These labor-division strategies significantly contribute to the development of clean processes for producing various valuable chemicals from lignocellulosic resources.

Biogenic Mn Oxide Generation and Mn(II) Removal by a Manganese Oxidizing Bacterium Bacillus sp. Strain M2.

Mn(II)-oxidizing bacteria (MOB) are widely distributed in natural environments and can convert soluble Mn(II) into insoluble Mn(III) and Mn(IV). The biogenic manganese oxides (BioMnOx) produced by MOB have been considered for remediating heavy metal pollution and degrading organic pollutants in an eco-friendly manner. In this study, a manganese-oxidizing bacterium was isolated from Mn-polluted rivulet sediment and identified as Bacillus sp. strain M2 by PCR, phylogenetic tree construction, transmission electron microscopy (TEM), and physiological and biochemical indices. Strain M2 grew well under Mn(II) stress. BioMnOx with nanosized irregular geometric shapes and loose structures generated by strain M2 were found on the surface of the bacterial cells. The content of Mn in the bacteria was as high as 5.36%. Approximately 71.24% and 47.52% of Mn(II) was oxidized to Mn(III/IV) in the cell and in the deposits, respectively, within 3 d of cultivation with Mn(II). Extracellular enzymes contributed to the Mn removal and oxidation. In conclusion, Bacillus sp. strain M2 has a high potential for use in the remediation of Mn-contaminated sites.

Ecological Characteristics of Culturable Myxobacteria in the Rhizosphere Soil of Saline-Tolerant Plants in the Ebinur Saline Wetlands

T his study investigated the ecological distribution of myxobacteria in the saline environment of the Ebinur Lake Wetland, in China, which provides a unique microecological environment for their survival. Culturable myxobacteria were isolated using traditional methods including Escherichia coli DH5α, rabbit feces, and filter paper flakes as food sources. Molecular identification along with physiological, biochemical assays and morphological characterization was carried out for the classification and identification of myxobacteria. Associations between soil environmental factors and myxobacterial diversity were analyzed using redundancy analysis. This study involved collecting rhizosphere soil from 15 species of salt-tolerant plants, ultimately yielding 15 strains of myxobacteria from eight of these species. Fifteen strains of myxobacteria were taxonomically placed by analyzing 16S rRNA homology, seven strains were closely related to Myxococcus virescens, six strains were closely related to Myxococcus fulvus and two strains were closely related to Myxococcus xanthus. Myxococcus virescens, the dominant cultivable species of myxobacteria in the Ebinur Lake Wetland, was isolated from various sampling sites using traditional methods. Analysis of antibiotic resistance revealed that myxobacterial strains M2, M6, M9, M13, and M15 are resistant to gentamicin. Furthermore, predation assays demonstrated that myxobacterial strain M14 exhibits the broadest spectrum of predation against both bacterial and fungal prey. The limited culturable myxobacterial diversity observed in the Ebinur Lake Wetland could potentially result from variations in soil physicochemical properties, vegetation type, and employed isolation methods. These findings enhance our comprehension of the myxobacterial resources within this specific salt desert ecosystem.

Proteomics and metabolic burden analysis to understand the impact of recombinant protein production in E. coli

The impact of recombinant protein production (RPP) on host cells and the metabolic burden associated with it undermine the efficiency of the production system. This study utilized proteomics to investigate the dynamics of parent and recombinant cells induced at different time points for RPP. The results revealed significant changes in both transcriptional and translational machinery that may have impacted the metabolic burden, growth rate of the culture and the RPP. The timing of protein synthesis induction also played a critical role in the fate of the recombinant protein within the host cell, affecting protein and product yield. The study identified significant differences in the expression of proteins involved in fatty acid and lipid biosynthesis pathways between two E. coli host strains (M15 and DH5⍺), with the E. coli M15 strain demonstrating superior expression characteristics for the recombinant protein. Overall, these findings contribute to the knowledge base for rational strain engineering for optimized recombinant protein production.

Overexpressing the cpr1953 Orphan Histidine Kinase Gene in the Absence of cpr1954 Orphan Histidine Kinase Gene Expression, or Vice Versa, Is Sufficient to Obtain Significant Sporulation and Strong Production of Clostridium perfringens Enterotoxin or Spo0A by Clostridium perfringens Type F Strain SM101.

The CPR1953 and CPR1954 orphan histidine kinases profoundly affect sporulation initiation and Clostridium perfringens enterotoxin (CPE) production by C. perfringens type F strain SM101, whether cultured in vitro (modified Duncan-Strong sporulation medium (MDS)) or ex vivo (mouse small intestinal contents (MIC)). To help distinguish whether CPR1953 and CPR1954 act independently or in a stepwise manner to initiate sporulation and CPE production, cpr1953 and cpr1954 null mutants of SM101 were transformed with plasmids carrying the cpr1954 or cpr1953 genes, respectively, causing overexpression of cpr1954 in the absence of cpr1953 expression and vice versa. RT-PCR confirmed that, compared to SM101, the cpr1953 mutant transformed with a plasmid encoding cpr1954 expressed cpr1954 at higher levels while the cpr1954 mutant transformed with a plasmid encoding cpr1953 expressed higher levels of cpr1953. Both overexpressing strains showed near wild-type levels of sporulation, CPE toxin production, and Spo0A production in MDS or MIC. These findings suggest that CPR1953 and CPR1954 do not function together in a step-wise manner, e.g., as a novel phosphorelay. Instead, it appears that, at natural expression levels, the independent kinase activities of both CPR1953 and CPR1954 are necessary for obtaining sufficient Spo0A production and phosphorylation to initiate sporulation and CPE production.

Comparative transcriptome analysis reveals the redirection of metabolic flux from cell growth to astaxanthin biosynthesis in Yarrowia lipolytica.

Engineering Yarrowia lipolytica to produce astaxanthin provides a promising route. Here, Y. lipolytica M2 producing a titer of 181 mg/L astaxanthin was isolated by iterative atmospheric and room-temperature plasma mutagenesis and diphenylamine-mediated screening. Interestingly, a negative correlation was observed between cell biomass and astaxanthin production. To reveal the underlying mechanism, RNA-seq analysis of transcriptional changes was performed in high producer M2 and reference strain M1, and a total of 1379 differentially expressed genes were obtained. Data analysis revealed that carbon flux was elevated through lipid metabolism, acetyl-CoA and mevalonate supply, but restrained through central carbon metabolism in strain M2. Moreover, upregulation of other pathways such as ATP-binding cassette transporter and thiamine pyrophosphate possibly provided more cofactors for carotenoid hydroxylase and relieved cell membrane stress caused by astaxanthin insertion. These results suggest that balancing cell growth and astaxanthin production may be important to promote efficient biosynthesis of astaxanthin in Y. lipolytica.

A novel Ruminiclostridium thermocellum cellulase system enhances cellulosic saccharification by elimination of cellobiose feedback inhibition

The Ruminiclostridium thermocellum cellulosome system is one of the most efficient cellulase systems in nature. The main hydrolysis product of cellulosic saccharification by R. thermocellum was cellobiose, which served as a strong feedback inhibitor to the cellulosome. In this study, R. thermocellum M3, which directly hydrolyzes cellulosic biomass into monosaccharides, was used to degrade cellobiose to investigate the kinetic characteristics of cellobiose degradation. Moreover, the transcriptomic characteristics of strain M3 under cellobiose and Avicel culture conditions were compared. The results indicated that strain M3 was able to grow and hydrolyze cellobiose under cellobiose concentrations higher than 20 g/L. The best growth performance and glucose production were obtained under 12 g/L cellobiose. Compared with that of Avicel, cellobiose had greater β-glucosidase activity under cellobiose culture, with a maximum glucose yield of 767.97±19.13 mg/g cellobiose. The transcription results indicated that R. thermocellum M3 resisted the feedback inhibition of cellobiose by regulating genes encoding β-glucosidase, cellobiose phosphorylase and ABC transporters. The high glucose yield of strain M3 was closely related to the expression of the bglX gene. In addition, the gene related to the chemotactic pathway of strain M3 also increased its perception and adaptation to cellobiose.

BCG-booster vaccination with HSP90-ESAT-6-HspX-RipA multivalent subunit vaccine confers durable protection against hypervirulent Mtb in mice

The quest for effective and enhanced multiantigenic tuberculosis (TB) subunit vaccine necessitates the induction of a protective pathogen-specific immune response while circumventing detrimental inflammation within the lung milieu. In line with this goal, we engineered a modified iteration of the quadrivalent vaccine, namely HSP90-ESAT-6-HspX-RipA (HEHR), which was coupled with the TLR4 adjuvant, CIA09A. The ensuing formulation was subjected to comprehensive assessment to gauge its protective efficacy against the hypervirulent Mycobacterium tuberculosis (Mtb) Haarlem clinical strain M2, following a BCG-prime boost regimen. Regardless of vaccination route, both intramuscular and subcutaneous administration with the HEHR vaccine exhibited remarkable protective efficacy in significantly reducing the Mtb bacterial burden and pulmonary inflammation. This underscores its notably superior protective potential compared to the BCG vaccine alone or a former prototype, the HSP90-E6 subunit vaccine. In addition, this superior protective efficacy was confirmed when testing a tag-free version of the HEHR vaccine. Furthermore, the protective immune determinant, represented by durable antigen-specific CD4+IFN-γ+IL-17A+ T-cells expressing a CXCR3+KLRG1- cell surface phenotype in the lung, was robustly induced in HEHR-boosted mice at 12 weeks post-challenge. Collectively, our data suggest that the BCG-prime HEHR boost vaccine regimen conferred improved and long-term protection against hypervirulent Mtb strain with robust antigen-specific Th1/Th17 responses.

New insights into bioaugmented removal of sulfamethoxazole in sediment microcosms: degradation efficiency, ecological risk and microbial mechanisms

BackgroundBioaugmentation has the potential to enhance the ability of ecological technology to treat sulfonamide-containing wastewater, but the low viability of the exogenous degraders limits their practical application. Understanding the mechanism is important to enhance and optimize performance of the bioaugmentation, which requires a multifaceted analysis of the microbial communities. Here, DNA-stable isotope probing (DNA-SIP) and metagenomic analysis were conducted to decipher the bioaugmentation mechanisms in stabilization pond sediment microcosms inoculated with sulfamethoxazole (SMX)-degrading bacteria (Pseudomonas sp. M2 or Paenarthrobacter sp. R1).ResultsThe bioaugmentation with both strains M2 and R1, especially strain R1, significantly improved the biodegradation rate of SMX, and its biodegradation capacity was sustainable within a certain cycle (subjected to three repeated SMX additions). The removal strategy using exogenous degrading bacteria also significantly abated the accumulation and transmission risk of antibiotic resistance genes (ARGs). Strain M2 inoculation significantly lowered bacterial diversity and altered the sediment bacterial community, while strain R1 inoculation had a slight effect on the bacterial community and was closely associated with indigenous microorganisms. Paenarthrobacter was identified as the primary SMX-assimilating bacteria in both bioaugmentation systems based on DNA-SIP analysis. Combining genomic information with pure culture evidence, strain R1 enhanced SMX removal by directly participating in SMX degradation, while strain M2 did it by both participating in SMX degradation and stimulating SMX-degrading activity of indigenous microorganisms (Paenarthrobacter) in the community.ConclusionsOur findings demonstrate that bioaugmentation using SMX-degrading bacteria was a feasible strategy for SMX clean-up in terms of the degradation efficiency of SMX, the risk of ARG transmission, as well as the impact on the bacterial community, and the advantage of bioaugmentation with Paenarthrobacter sp. R1 was also highlighted.8sNhd6qoUNMdHWMUGzd-tFVideo

Marker recycling in Lentinula edodes via 5-fluoroorotic acid counter-selection.

Shiitake (Lentinula edodes) contains various beneficial compounds and possesses several notable properties. However, there are few reports on its molecular breeding due to delay in development of its gene-modifying technology. Therefore, here, strain UV30 (pyrG -) was bred from the UV-irradiated protoplasts of strain M2. Strain UV30 was uracil-auxotrophic, and the phenylalanine residue in the active centre of orotidine-5-phosphate decarboxylase encoded by pyrG in the strain was substituted with a serine residue. Next, a recycling marker consisting of the upstream sequence of ku80, a repeat sequence (a portion of the downstream sequence of ku80), pyrG, and the downstream sequence of ku80 was introduced into the strain UV30. Consequently, the prototrophic strain ckp2-1, in which ku80 was replaced with the recycling marker, was obtained. After cultivation in complete medium, mycelia from the edges of ckp2-1 colonies were inoculated into a complete medium containing 5-Fluoroorotic acid (5-FOA). A 5-FOA-resistant strain KaM2, in which pyrG sequence was spliced from the recycling marker sequence via homologous recombination, was obtained. In this study, we developed the first marker recycling system for multigene targeting in L. edodes. Moreover, the resulting ∆ku80 strain may serve as a non-homologous end-joining deficient strain for further genetic manipulations.

Dynamic responses in Pseudomonas stutzeri M3 bioaugmentation of crude-oil-contaminated soil: Hydrocarbons, microbial community structures, and functional genes

Bioaugmentation is a cost-effective and environmentally friendly method for dealing with hazardous pollutants. However, bioaugmentation has some drawbacks, including poor bioactivities, inconsistent effects, and uncontrollability. Understanding the inter-relationships between actual microbial communities and various petroleum hydrocarbon components remains a key challenge for effective and efficient decontamination in oilfield soil. Here, dynamic changes of reconstructed PIONA (paraffins, iso-paraffins, olefins, naphthenes, and aromatics) fractions and microbial community structures were investigated in a 60-day laboratory-scale experiment with exogenous Pseudomonas stutzeri M3 supplementation. The results showed that effective biodegradation occurred between the 15th and 30th days of bioaugmentation, and the biodegradation efficiency of paraffin reached 96.5% after 60 days in 1% oil-contaminated soil. Moreover, supplementation with strain M3 significantly altered the microbial community structures, promoting the enrichment of Balneolaceae, Halolalama, and Woeserchaeia, while delaying the colonization of Alcanivorax. Weighted gene co-expression network analysis (WGCNA) showed that the positive interactions between hub bacteria were more significant than the negative interactions and revealed the synergistic degradation of petroleum constituents. The abundance of alcohol dehydrogenase, aldehyde dehydrogenase, catechol cleavage enzyme, cytochrome P450, gentisate dioxygenase, and benzoate/toluate dioxygenase increased abruptly in the initial 7 days with the inoculation of strain M3. This study provides insights into the inner mechanisms of petroleum hydrocarbon metabolism under the combined action of exogenous and indigenous microbes in oilfield soil.

A novel two-domain laccase with a middle redox potential: physicochemical and structural properties

The gene for a previously unexplored two-domain laccase was identified in the genome of the actinobacterium Streptomyces carpinensis VKM Ac-1300. A two-domain laccase, named ScaSL, was produced in a heterologous expression system (Escherichia coli strain M15 [pREP4]). The enzyme was purified to homogeneity using affinity chromatography. ScaSL laccase, like most two-domain laccases, exhibited activity in the homotrimer form. However, unlike most two-domain laccases, it was also active in multimeric forms. The enzyme exhibited maximum activity at 80°C and was thermally stable. The half-inactivation time of ScaSL at 80°C was 40 min. Laccase oxidized the non-phenolic organic compound ABTS at a maximum rate at pH 4.7; oxidized the phenolic compound 2,6-dimethoxyphenol at a maximum rate at pH 7.5. Laccase stability was observed in the pH range 9-11. At pH 7.5, laccase was weakly inhibited by sodium azide, sodium fluoride, and sodium chloride; at pH 4.5, laccase was completely inhibited by 100 mM sodium azide. Km and kcat of the enzyme for ABTS are 0.1 mM and 20 s-1, respectively. Km and kcat for 2,6-dimethoxyphenol are 0.84 mM and 0.36 s-1, respectively. ScaSL catalyzed the polymerization of humic acids and lignin. The redox potential of laccase was 0.472 ± 0.007 V. Thus, ScaSL laccase is the first characterized two-domain laccase with a middle redox potential. The crystal structure of ScaSL was determined with a resolution of 2.35 Å. A comparative analysis of the structures of ScaSL and other two-domain laccases suggested that the middle potential of ScaSL may be associated with conformational differences in the position of the side groups of amino acids at position 230 (in ScaSL numbering), which belong to the second coordination sphere of the copper atom of the T1 center.

A Novel Two-Domain Laccase with Middle Redox Potential: Physicochemical and Structural Properties.

The gene for a previously unexplored two-domain laccase was identified in the genome of actinobacterium Streptomyces carpinensis VKM Ac-1300. The two-domain laccase, named ScaSL, was produced in a heterologous expression system (Escherichia coli strain M15 [pREP4]). The enzyme was purified to homogeneity using affinity chromatography. ScaSL laccase, like most two-domain laccases, exhibited activity in the homotrimer form. However, unlike the most two-domain laccases, it was also active in multimeric forms. The enzyme exhibited maximum activity at 80°C and was thermally stable. Half-inactivation time of ScaSL at 80°C was 40 min. The laccase was able to oxidize a non-phenolic organic compound ABTS at a maximum rate at pH 4.7, and to oxidized a phenolic compound 2,6-dimethoxyphenol at a maximum rate at pH 7.5. The laccase stability was observed in the pH range 9-11. At pH 7.5, laccase was slightly inhibited by sodium azide, sodium fluoride, and sodium chloride; at pH 4.5, the laccase was completely inhibited by 100 mM sodium azide. The determined Km and kcat of the enzyme for ABTS were 0.1 mM and 20 s-1, respectively. The Km and kcat for 2,6-dimethoxyphenol were 0.84 mM and 0.36 s-1, respectively. ScaSL catalyzed polymerization of humic acids and lignin. Redox potential of the laccase was 0.472 ± 0.007 V. Thus, the ScaSL laccase is the first characterized two-domain laccase with a middle redox potential. Crystal structure of ScaSL was determined with 2.35 Å resolution. Comparative analysis of the structures of ScaSL and other two-domain laccases suggested that the middle potential of ScaSL may be associated with conformational differences in the position of the side groups of amino acids at position 230 (in ScaSL numbering), which belong to the second coordination sphere of the copper atom of the T1 center.

Simultaneous carbon catabolite repression governs sugar and aromatic co-utilization in Pseudomonas putida M2.

Pseudomonas putida have emerged as promising biocatalysts for the conversion of sugars and aromatic compounds obtained from lignocellulosic biomass. Understanding the role of carbon catabolite repression (CCR) in these strains is critical to optimize biomass conversion to fuels and chemicals. The CCR functioning in P. putida M2, a strain capable of consuming both hexose and pentose sugars as well as aromatic compounds, was investigated by cultivation experiments, proteomics, and CRISPRi-based gene repression. Strain M2 co-utilized sugars and aromatic compounds simultaneously; however, during cultivation with glucose and aromatic compounds (p-coumarate and ferulate) mixture, intermediates (4-hydroxybenzoate and vanillate) accumulated, and substrate consumption was incomplete. In contrast, xylose-aromatic consumption resulted in transient intermediate accumulation and complete aromatic consumption, while xylose was incompletely consumed. Proteomics analysis revealed that glucose exerted stronger repression than xylose on the aromatic catabolic proteins. Key glucose (Eda) and xylose (XylX) catabolic proteins were also identified at lower abundance during cultivation with aromatic compounds implying simultaneous catabolite repression by sugars and aromatic compounds. Reduction of crc expression via CRISPRi led to faster growth and glucose and p-coumarate uptake in the CRISPRi strains compared to the control, while no difference was observed on xylose+p-coumarate. The increased abundances of Eda and amino acid biosynthesis proteins in the CRISPRi strain further supported these observations. Lastly, small RNAs (sRNAs) sequencing results showed that CrcY and CrcZ homologues levels in M2, previously identified in P. putida strains, were lower under strong CCR (glucose+p-coumarate) condition compared to when repression was absent (p-coumarate or glucose only). IMPORTANCE A newly isolated Pseudomonas putida strain, P. putida M2, can utilize both hexose and pentose sugars as well as aromatic compounds making it a promising host for the valorization of lignocellulosic biomass. Pseudomonads have developed a regulatory strategy, carbon catabolite repression, to control the assimilation of carbon sources in the environment. Carbon catabolite repression may impede the simultaneous and complete metabolism of sugars and aromatic compounds present in lignocellulosic biomass and hinder the development of an efficient industrial biocatalyst. This study provides insight into the cellular physiology and proteome during mixed-substrate utilization in P. putida M2. The phenotypic and proteomics results demonstrated simultaneous catabolite repression in the sugar-aromatic mixtures, while the CRISPRi and sRNA sequencing demonstrated the potential role of the crc gene and small RNAs in carbon catabolite repression.

Identification of orphan histidine kinases that impact sporulation and enterotoxin production by Clostridium perfringens type F strain SM101 in a pathophysiologically-relevant ex vivo mouse intestinal contents model.

When causing food poisoning or antibiotic-associated diarrhea, Clostridium perfringens type F strains must sporulate to produce C. perfringens enterotoxin (CPE) in the intestines. C. perfringens is thought to use some of its seven annotated orphan histidine kinases to phosphorylate Spo0A and initiate sporulation and CPE production. We previously demonstrated the CPR0195 orphan kinase, but not the putative CPR1055 orphan kinase, is important when type F strain SM101 initiates sporulation and CPE production in modified Duncan-Strong (MDS) sporulation medium. Since there is no small animal model for C. perfringens sporulation, the current study used diluted mouse intestinal contents (MIC) to develop an ex vivo sporulation model and employed this model to test sporulation and CPE production by SM101 CPR0195 and CPR1055 null mutants in a pathophysiologically-relevant context. Surprisingly, both mutants still sporulated and produced CPE at wild-type levels in MIC. Therefore, five single null mutants were constructed that cannot produce one of the previously-unstudied putative orphan kinases of SM101. Those mutants implicated CPR1316, CPR1493, CPR1953 and CPR1954 in sporulation and CPE production by SM101 MDS cultures. Phosphorylation activity was necessary for CPR1316, CPR1493, CPR1953 and CPR1954 to affect sporulation in those MDS cultures, supporting their identity as kinases. Importantly, only the CPR1953 or CPR1954 null mutants exhibited significantly reduced levels of sporulation and CPE production in MIC cultures. These phenotypes were reversible by complementation. Characterization studies suggested that, in MDS or MIC, the CPR1953 and CPR1954 mutants produce less Spo0A than wild-type SM101. In addition, the CPR1954 mutant exhibited little or no Spo0A phosphorylation in MDS cultures. These studies, i) highlight the importance of using pathophysiologically-relevant models to investigate C. perfringens sporulation and CPE production in a disease context and ii) link the CPR1953 and CPR1954 kinases to C. perfringens sporulation and CPE production in disease-relevant conditions.