Isolation and characterization of polyester polyurethane-degrading bacterium Bacillus sp. YXP1.

Microorganisms have the potential to be applied for the degradation or depolymerization of polyurethane (PU) and other plastic waste, which have attracted global attention. The appropriate strain or enzyme that can effectively degrade PU is the key to treat PU plastic wastes by biological methods. Here, a polyester PU-degrading bacterium Bacillus sp. YXP1 was isolated and identified from a plastic landfill. Three PU substrates with increasing structure complexities, including Impranil DLN, poly (1,4-butylene adipate)-based PU (PBA-PU), and polyester PU foam, were used to evaluate the degradation capacity of Bacillus sp. YXP1. Under optimal conditions, strain YXP1 could completely degrade 0.5% Impranil DLN within 7 days. After 30 days, the weight loss of polyester PU foam by strain YXP1 was as high as 42.1%. In addition, PBA-PU was applied for degradation pathway analysis due to its clear composition and chemical structure. Five degradation intermediates of PBA-PU were identified, including 4,4'-methylenedianiline (MDA), 1,4-butanediol, adipic acid, and two MDA derivates, indicating that strain YXP1 could depolymerize PBA-PU by the hydrolysis of ester and urethane bonds. Furthermore, the extracellular enzymes produced by strain YXP1 could hydrolyze PBA-PU to generate MDA. Together, this study provides a potential bacterium for the biological treatment of PU plastic wastes and for the mining of functional enzymes.

[Isolation and identification of a polyester-polyurethane degrading bacterium Bacillus altitudinis YX8-1].

Although polyurethane (PUR) plastics play important roles in daily life, its wastes bring serious environmental pollutions. Biological (enzymatic) degradation is considered as an environmentally friendly and low-cost method for PUR waste recycling, in which the efficient PUR-degrading strains or enzymes are crucial. In this work, a polyester PUR-degrading strain YX8-1 was isolated from the surface of PUR waste collected from a landfill. Based on colony morphology and micromorphology observation, phylogenetic analysis of 16S rDNA and gyrA gene, as well as genome sequence comparison, strain YX8-1 was identified as Bacillus altitudinis. The results of high performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) showed that strain YX8-1 was able to depolymerize self-synthesized polyester PUR oligomer (PBA-PU) to produce a monomeric compound 4, 4'-methylene diphenylamine. Furthermore, strain YX8-1 was able to degrade 32% of the commercialized polyester PUR sponges within 30 days. This study thus provides a strain capable of biodegradation of PUR waste, which may facilitate the mining of related degrading enzymes.

Improved Whole-Cell Biocatalyst for the Synthesis of Vitamin E Precursor 2,3,5-Trimethylhydroquinone.

2,3,5-Trimethylhydroquinone (2,3,5-TMHQ) is the key precursor in the synthesis of vitamin E. It is still a major challenge to produce 2,3,5-TMHQ under mild reaction conditions by chemical methods. The monooxygenase system MpdAB can specifically catalyze the conversion of 2,3,6-trimethylphenol (2,3,6-TMP) to 2,3,5-TMHQ. However, the weak catalytic capacity of wild-type MpdA and the cytotoxicity of the substrate limited the production efficiency of 2,3,5-TMHQ. Here, homologous modeling and saturation mutation were performed to increase the catalytic activity of MpdA. Two variants, L128A and L128K, with higher activity toward 2,3,6-TMP (1.86-1.87-fold) were obtained. On the other hand, an evolved strain B5-4M-evolved with enhanced resistance to 2,3,6-TMP (8.15-fold higher for 1000 µM 2,3,6-TMP) was obtained through adaptive laboratory evolution. Subsequently, a 5.29-fold (or 4.87-fold) improvement in 2,3,5-TMHQ production was achieved by a strain B5-4M-evolved harboring L128K (or L128A) and MpdB, in comparison with that of the wild type (strain B5-4M expressing MpdAB). This study provides better genetic resources for producing 2,3,5-TMHQ and proves that the synthesis efficiency of 2,3,5-TMHQ can be improved through enzyme modification and adaptive laboratory evolution.

Characterization of the 2,6-Dimethylphenol Monooxygenase MpdAB and Evaluation of Its Potential in Vitamin E Precursor Synthesis.

2,6-Dimethylphenol (2,6-DMP) is a widely used chemical intermediate whose residue has been frequently detected in the environment, posing a threat to some aquatic organisms. Microbial degradation is an effective method to eliminate 2,6-DMP in nature. However, the genetic and biochemical mechanisms of 2,6-DMP metabolism remain unknown. Mycobacterium neoaurum B5-4 is a 2,6-DMP-degrading bacterium isolated in our previous study. Here, a 2,6-DMP degradation-deficient mutant of strain B5-4 was screened. Comparative genomic, transcriptomic, gene disruption, and genetic complementation data indicated that mpdA and mpdB are responsible for the initial step of 2,6-DMP degradation in M. neoaurum B5-4. MpdAB was predicted to be a two-component flavin-dependent monooxygenase system, which shows 32% and 36% identities with HsaAB from Mycobacterium tuberculosis CDC1551. The transcription of mpdA and mpdB was substantially increased upon exposure to 2,6-DMP. Nuclear magnetic resonance analysis showed that purified 6×His-MpdA and 6×His-MpdB hydroxylated 2,6-DMP and 2,3,6-trimethylphenol (2,3,6-TMP) at the para-position using NADH and flavin adenine dinucleotide (FAD) as cofactors. The apparent Km values of MpdAB for 2,6-DMP and 2,3,6-TMP were 0.12 ± 0.01 and 0.17 ± 0.01 mM, respectively, and the corresponding kcat/Km values were 4.02 and 2.84 s-1 mM-1, respectively. Since para-hydroxylated 2,3,6-TMP is a major precursor for vitamin E synthesis, the potential of MpdAB in vitamin E synthesis was preliminarily evaluated using whole-cell catalysis. Low expression levels of MpdA and 2,3,6-TMP cytotoxicity limited the efficiency of whole-cell catalysis. Together, this study reveals the genetic and biochemical basis for the initial step of 2,6-DMP biodegradation and provides candidate enzymes for vitamin E synthesis. IMPORTANCE Although the microbial degradation of the six isomers of dimethylphenol has been extensively studied, the genetic and biochemical mechanisms of 2,6-DMP degradation remain unclear. This study identified the genes responsible for the initial step in the 2,6-DMP catabolic pathway in M. neoaurum B5-4. Moreover, MpdAB also catalyzed the transformation of 2,3,6-TMP to 2,3,5-trimethylhydroquinone (2,3,5-TMHQ), a crucial step in vitamin E synthesis. Overall, this study provides candidate enzymes for both the bioremediation of 2,6-DMP contamination and the development of a green method to synthesize vitamin E.

Biodegradation of plastic monomer 2,6-dimethylphenol by Mycobacterium neoaurum B5-4.

2,6-Dimethylphenol (2,6-DMP), an important chemical intermediate and the monomer of plastic polyphenylene oxide, is widely used in chemical and plastics industry. However, the pollution problem of 2,6-DMP residues is becoming increasingly serious, which is harmful to some aquatic animals. Microbial degradation provided an effective approach to eliminate DMPs in nature, which is considered as a prospective way to remediate DMPs-contaminated environments. But the 2,6-DMP-degrading bacteria is not available and the molecular mechanism of 2,6-DMP degradation is unclear as well. Here, a 2,6-DMP-degrading bacterium named B5-4 was isolated and identified as Mycobacterium neoaurum. M. neoaurum B5-4 could utilize 2,6-DMP as the sole carbon source for growth. Furthermore, M. neoaurum B5-4 could degrade 2,6-DMP with concentrations ranging from 1 to 500 mg L-1. Six intermediate metabolites of 2,6-DMP were identified and a metabolic pathway of 2,6-DMP in M. neoaurum B5-4 was proposed, in which 2,6-DMP was initially converted to 2,6-dimethyl-hydroquinone and 2,6-dimethyl-3-hydroxy-hydroquinone by two consecutive hydroxylations at C-4 and γ position; 2,6-dimethyl-3-hydroxy-hydroquinone was then subjected to aromatic ring ortho-cleavage to produce 2,4-dimethyl-3-hydroxymuconic acid, which was further transformed to citraconate, and subsequently into TCA cycle. In addition, toxicity bioassay of 2,6-DMP in water using zebrafish indicates that 2,6-DMP is toxic to zebrafish and M. neoaurum B5-4 could effectively eliminate 2,6-DMP in water to protect zebrafish from 2,6-DMP-induced death. This work provides a potential strain for bioremediation of 2,6-DMP-contaminated environments and lays a foundation for elucidating the molecular mechanism and genetic determinants of 2,6-DMP degradation.

Expansion of Thaumarchaeota habitat range is correlated with horizontal transfer of ATPase operons.

Thaumarchaeota are responsible for a significant fraction of ammonia oxidation in the oceans and in soils that range from alkaline to acidic. However, the adaptive mechanisms underpinning their habitat expansion remain poorly understood. Here we show that expansion into acidic soils and the high pressures of the hadopelagic zone of the oceans is tightly linked to the acquisition of a variant of the energy-yielding ATPases via horizontal transfer. Whereas the ATPase genealogy of neutrophilic Thaumarchaeota is congruent with their organismal genealogy inferred from concatenated conserved proteins, a common clade of V-type ATPases unites phylogenetically distinct clades of acidophilic/acid-tolerant and piezophilic/piezotolerant species. A presumptive function of pumping cytoplasmic protons at low pH is consistent with the experimentally observed increased expression of the V-ATPase in an acid-tolerant thaumarchaeote at low pH. Consistently, heterologous expression of the thaumarchaeotal V-ATPase significantly increased the growth rate of E. coli at low pH. Its adaptive significance to growth in ocean trenches may relate to pressure-related changes in membrane structure in which this complex molecular machine must function. Together, our findings reveal that the habitat expansion of Thaumarchaeota is tightly correlated with extensive horizontal transfer of atp operons.

The substrate specificity of aniline dioxygenase is mainly determined by two of its components: glutamine synthetase-like enzyme and oxygenase.

The residues of aniline and its derivatives are serious environment pollutants. Aniline dioxygenase (AD) derived from aerobic bacteria catalyzes the conversion of aniline to catechol, which has potential use in the bioremediation of aromatic amines and biorefining process. AD contains four components: a glutamine synthetase (GS)-like enzyme, a glutamine amidotransferase (GAT)-like enzyme, oxygenase, and reductase. ADs from diverse hosts exhibit different substrate specificities against aniline derivatives. However, what component of AD determines AD's substrate specificity is still unknown which limits the effects of extending AD's substrate spectrum through mutagenesis. Here, each component of two ADs (AtdA1A2A3A4A5 and AdoQTA1A2B) which have different substrate ranges was heterologously expressed and purified. The activity of both ADs was successfully constructed in vitro using the purified components. To identify the component that affects the substrate specificity of the ADs, the substrate specificity of each component was studied. The inability of AtdA1A2A3A4A5 to catalyze 4-methylaniline was determined with GS-like enzyme AtdA1; its inability to convert 2-isopropylaniline was caused by the oxygenase component, and its inability to convert 4-isopropylaniline was caused by both GS-like enzyme AtdA1 and oxygenase components. The inability of AdoQTA1A2B to catalyze 2-methylaniline was determined by GS-like enzyme AdoQ; its inability to convert 2-isopropylaniline was caused by both GS-like enzyme AdoQ and oxygenase components. Together, these results show that GS-like enzyme and oxygenase but not GAT-like enzyme or reductase play dominant roles in the substrate specificity of AD, and this finding will facilitate the engineering of AD to expand its substrate range.

Hydrolase CehA and Monooxygenase CfdC Are Responsible for Carbofuran Degradation in Sphingomonas sp. Strain CDS-1.

Carbofuran, a broad-spectrum systemic insecticide, has been extensively used for approximately 50 years. Diverse carbofuran-degrading bacteria have been described, among which sphingomonads have exhibited an extraordinary ability to catabolize carbofuran; other bacteria can only convert carbofuran to carbofuran phenol, while all carbofuran-degrading sphingomonads can degrade both carbofuran and carbofuran phenol. However, the genetic basis of carbofuran catabolism in sphingomonads has not been well elucidated. In this work, we sequenced the draft genome of Sphingomonas sp. strain CDS-1 that can transform both carbofuran and carbofuran phenol but fails to grow on them. On the basis of the hypothesis that the genes involved in carbofuran catabolism are highly conserved among carbofuran-degrading sphingomonads, two such genes, cehACDS-1 and cfdCCDS-1, were predicted from the 84 open reading frames (ORFs) that share ≥95% nucleic acid similarities between strain CDS-1 and another sphingomonad Novosphingobium sp. strain KN65.2 that is able to mineralize the benzene ring of carbofuran. The results of the gene knockout, genetic complementation, heterologous expression, and enzymatic experiments reveal that cehACDS-1 and cfdCCDS-1 are responsible for the conversion of carbofuran and carbofuran phenol, respectively, in strain CDS-1. CehACDS-1 hydrolyzes carbofuran to carbofuran phenol. CfdCCDS-1, a reduced flavin mononucleotide (FMNH2)- or reduced flavin adenine dinucleotide (FADH2)-dependent monooxygenase, hydroxylates carbofuran phenol at the benzene ring in the presence of NADH, FMN/FAD, and the reductase CfdX. It is worth noting that we found that carbaryl hydrolase CehAAC100, which was previously demonstrated to have no activity toward carbofuran, can actually convert carbofuran to carbofuran phenol, albeit with very low activity.IMPORTANCE Due to the extensive use of carbofuran over the past 50 years, bacteria have evolved catabolic pathways to mineralize this insecticide, which plays an important role in eliminating carbofuran residue in the environment. This study revealed the genetic determinants of carbofuran degradation in Sphingomonas sp. strain CDS-1. We speculate that the close homologues cehA and cfdC are highly conserved among other carbofuran-degrading sphingomonads and play the same roles as those described here. These findings deepen our understanding of the microbial degradation mechanism of carbofuran and lay a foundation for the better use of microbes to remediate carbofuran contamination.

Complete Genome Sequence of Alcaligenes Faecalis Strain JQ135, a Bacterium Capable of Efficiently Degrading Nicotinic Acid.

Nicotinic acid (NA), known as vitamin B3, is ubiquitous in nature and plays an important role in living organisms. The microbial catabolism of NA is highly diverse. However, the NA degradation by Alcaligenes faecalis strains has been poorly investigated. In this study, we report the complete genome sequence of A. faecalis JQ135 (4.08 Mbp) and several essential genes for NA degradation. This genome sequence will facilitate to elucidate the molecular metabolism of NA and advance the potential biotechnological applications of A. faecalis strains.

Molecular Mechanism and Genetic Determinants of Buprofezin Degradation.

Buprofezin is a widely used insect growth regulator whose residue has been frequently detected in the environment, posing a threat to aquatic organisms and nontarget insects. Microorganisms play an important role in the degradation of buprofezin in the natural environment. However, the relevant catabolic pathway has not been fully characterized, and the molecular mechanism of catabolism is still completely unknown. Rhodococcus qingshengii YL-1 can utilize buprofezin as a sole source of carbon and energy for growth. In this study, the upstream catabolic pathway in strain YL-1 was identified using tandem mass spectrometry. Buprofezin is composed of a benzene ring and a heterocyclic ring. The degradation is initiated by the dihydroxylation of the benzene ring and continues via dehydrogenation, aromatic ring cleavage, breaking of an amide bond, and the release of the heterocyclic ring 2-tert-butylimino-3-isopropyl-1,3,5-thiadiazinan-4-one (2-BI). A buprofezin degradation-deficient mutant strain YL-0 was isolated. A comparative genomic analysis combined with gene deletion and complementation experiments revealed that the gene cluster bfzBA3A4A1A2C is responsible for the upstream catabolic pathway of buprofezin. The bfzA3A4A1A2 cluster encodes a novel Rieske nonheme iron oxygenase (RHO) system that is responsible for the dihydroxylation of buprofezin at the benzene ring; bfzB is involved in dehydrogenation, and bfzC is in charge of benzene ring cleavage. Furthermore, the products of bfzBA3A4A1A2C can also catalyze dihydroxylation, dehydrogenation, and aromatic ring cleavage of biphenyl, flavanone, flavone, and bifenthrin. In addition, a transcriptional study revealed that bfzBA3A4A1A2C is organized in one transcriptional unit that is constitutively expressed in strain YL-1.IMPORTANCE There is an increasing concern about the residue and environmental fate of buprofezin. Microbial metabolism is an important mechanism responsible for the buprofezin degradation in the natural environment. However, the molecular mechanism and genetic determinants of microbial degradation of buprofezin have not been well identified. This work revealed that gene cluster bfzBA3A4A1A2C is responsible for the upstream catabolic pathway of buprofezin in Rhodococcus qingshengii YL-1. The products of bfzBA3A4A1A2C could also degrade bifenthrin, a widely used pyrethroid insecticide. These findings enhance our understanding of the microbial degradation mechanism of buprofezin and benefit the application of strain YL-1 and bfzBA3A4A1A2C in the bioremediation of buprofezin contamination.

Artificial induction of genetic competence in Bacillus amyloliquefaciens isolates.

OBJECTIVES: To induce natural genetic competence in Bacillus amyloliquefaciens isolates through overexpression of the master regulator, ComK, from B. subtilis (ComK Bsu ). RESULTS: Plasmid pUBXC carrying the xylose-inducible comK expression cassette was constructed using plasmid pUB110 as a backbone. Plasmid pUBXC could be transferred from B. subtilis to B. amyloliquefaciens through plasmid pLS20-mediated biparental conjugation. After being induced by xylose, four B. amyloliquefaciens strains harbouring plasmid pUBXC developed genetic competence. Under optimal conditions, the transformation efficiencies of plasmid DNA ranged from 129 ± 20.6 to 1.7 ± 0.1 × 105 cfu (colony-forming units) per µg DNA, and the transformation efficiencies of PCR-assembled deletion constructs ranged from 3.2 ± 0.76 to 3.5 ± 0.42 × 104 cfu per µg DNA in the four tested strains. CONCLUSION: Artificial induction of genetic competence through overexpressing ComK Bsu in B. amyloliquefaciens completed the tasks of replicative plasmid delivery and gene knockout via direct transformation of PCR-generated deletion cassettes.