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Characterizing microorganisms and enzymes involved in lignin biodegradation in thermal ecosystems can identify thermostable biocatalysts. We integrated stable isotope probing (SIP), genome-resolved metagenomics, and enzyme characterization to investigate the degradation of high-molecular weight, 13C-ring-labeled synthetic lignin by microbial communities from moderately thermophilic hot spring sediment (52 °C) and a woody “hog fuel” pile (53 and 62 °C zones). 13C-Lignin degradation was monitored using IR-GCMS of 13CO2, and isotopic enrichment of DNA was measured with UHLPC-MS/MS. Assembly of 42 metagenomic libraries (72 Gb) yielded 344 contig bins, from which 125 draft genomes were produced. Fourteen genomes were significantly enriched with 13C from lignin, including genomes of Actinomycetes (Thermoleophilaceae, Solirubrobacteraceae, Rubrobacter sp.), Firmicutes (Kyrpidia sp., Alicyclobacillus sp.) and Gammaproteobacteria (Steroidobacteraceae). We employed multiple approaches to screen genomes for genes encoding putative ligninases and pathways for aromatic compound degradation. Our analysis identified several novel laccase-like multi-copper oxidase (LMCO) genes in 13C-enriched genomes. One of these LMCOs was heterologously expressed and shown to oxidize lignin model compounds and minimally transformed lignin. This study elucidated bacterial lignin depolymerization and mineralization in thermal ecosystems, establishing new possibilities for the efficient valorization of lignin at elevated temperature.

The valorization of lignin is critical to establishing sustainable biorefineries as we transition away from petroleum-derived feedstocks. Advances in lignin fractionation and depolymerization are yielding new opportunities for the biocatalytic upgrading of lignin-derived aromatic compounds (LDACs) using microbial cell factories. Given their roles in lignin metabolism and their catalytic versatility, cytochromes P450 are attractive enzymes in engineering such biocatalysts. Here we highlight P450s that catalyze aromatic O-demethylation, a rate-limiting step in the conversion of LDACs to valuable chemicals, including efforts to engineer the specificity of these enzymes and to use them in developing biocatalysts. We also discuss broader opportunities at the intersection of biochemistry, structure-guided enzyme engineering, and metabolic engineering for application of P450s in the emerging area of microbial lignin valorization.

Despite the deployment of combination tuberculosis (TB) chemotherapy, efforts to identify shorter, nonrelapsing treatments have resulted in limited success. Recent evidence indicates that GSK2556286 (GSK286), which acts via Rv1625c, a membrane-bound adenylyl cyclase in Mycobacterium tuberculosis , shortens treatment in rodents relative to standard of care drugs.

Despite the deployment of combination tuberculosis (TB) chemotherapy, efforts to identify shorter, nonrelapsing treatments have resulted in limited success. Recent evidence indicates that GSK2556286 (GSK286), which acts via Rv1625c, a membrane-bound adenylyl cyclase in Mycobacterium tuberculosis, shortens treatment in rodents relative to standard of care drugs. Moreover, GSK286 can replace linezolid in the three-drug, Nix-TB regimen. Given its therapeutic potential, we sought to better understand the mechanism of action of GSK286. The compound blocked growth of M. tuberculosis in cholesterol media and increased intracellular cAMP levels ~50-fold. GSK286 did not inhibit growth of an rv1625c transposon mutant in cholesterol media and did not induce cyclic AMP (cAMP) production in this mutant, suggesting that the compound acts on this adenylyl cyclase. GSK286 also induced cAMP production in Rhodococcus jostii RHA1, a cholesterol-catabolizing actinobacterium, when Rv1625c was heterologously expressed. However, these elevated levels of cAMP did not inhibit growth of R. jostii RHA1 in cholesterol medium. Mutations in rv1625c conferred cross-resistance to GSK286 and the known Rv1625c agonist, mCLB073. Metabolic profiling of M. tuberculosis cells revealed that elevated cAMP levels, induced using either an agonist or a genetic tool, did not significantly affect pools of steroid metabolites in cholesterol-incubated cells. Finally, the inhibitory effect of agonists was not dependent on the N-acetyltransferase MtPat. Together, these data establish that GSK286 is an Rv1625c agonist and sheds light on how cAMP signaling can be manipulated as a novel antibiotic strategy to shorten TB treatments. Nevertheless, the detailed mechanism of action of these compounds remains to be elucidated.

Bacterial catabolic pathways have considerable potential as industrial biocatalysts for the valorization of lignin, a major component of plant-derived biomass. Here, we describe a pathway responsible for the catabolism of acetovanillone, a major component of several industrial lignin streams. Rhodococcus rhodochrous GD02 was previously isolated for growth on acetovanillone. A high-quality genome sequence of GD02 was generated. Transcriptomic analyses revealed a cluster of eight genes up-regulated during growth on acetovanillone and 4-hydroxyacetophenone, as well as a two-gene cluster up-regulated during growth on acetophenone. Bioinformatic analyses predicted that the hydroxyphenylethanone (Hpe) pathway proceeds via phosphorylation and carboxylation, before β-elimination yields vanillate from acetovanillone or 4-hydroxybenzoate from 4-hydroxyacetophenone. Consistent with this prediction, the kinase, HpeHI, phosphorylated acetovanillone and 4-hydroxyacetophenone. Furthermore, HpeCBA, a biotin-dependent enzyme, catalyzed the ATP-dependent carboxylation of 4-phospho-acetovanillone but not acetovanillone. The carboxylase’s specificity for 4-phospho-acetophenone ( k cat / K M = 34 ± 2 mM −1 s −1 ) was approximately an order of magnitude higher than for 4-phospho-acetovanillone. HpeD catalyzed the efficient dephosphorylation of the carboxylated products. GD02 grew on a preparation of pine lignin produced by oxidative catalytic fractionation, depleting all of the acetovanillone, vanillin, and vanillate. Genomic and metagenomic searches indicated that the Hpe pathway occurs in a relatively small number of bacteria. This study facilitates the design of bacterial strains for biocatalytic applications by identifying a pathway for the degradation of acetovanillone.

Mycobacterium abscessus , an opportunistic pathogen responsible for pulmonary infections, contains genes predicted to encode two steroid catabolic pathways: a cholesterol catabolic pathway similar to that of Mycobacterium tuberculosis and a 4-androstenedione (4-AD) catabolic pathway. Consistent with this prediction, M. abscessus grew on both steroids. In contrast to M. tuberculosis , Rhodococcus jostii RHA1, and other Actinobacteria, the cholesterol and 4-AD catabolic gene clusters of the M. abscessus complex lack genes encoding HsaD, the meta -cleavage product (MCP) hydrolase. However, M. abscessus ATCC 19977 harbors two hsaD homologs elsewhere in its genome. Only one of the encoded enzymes detectably transformed steroid metabolites. Among tested substrates, HsaD Mab and HsaD Mtb of M. tuberculosis had highest substrate specificities for MCPs with partially degraded side chains thioesterified with coenzyme A ( k cat / K M = 1.9 × 10 4 and 5.7 × 10 3 mM −1 s −1 , respectively). Consistent with a dual role in cholesterol and 4-AD catabolism, HsaD Mab also transformed nonthioesterified substrates efficiently, and a Δ hsaD mutant of M. abscessus grew on neither steroid. Interestingly, both steroids prevented growth of the mutant on acetate. The Δ hsaD mutant of M. abscessus excreted cholesterol metabolites with a fully degraded side chain, while the corresponding RHA1 mutant excreted metabolites with partially degraded side chains. Finally, the Δ hsaD mutant was not viable in macrophages. Overall, our data establish that the cholesterol and 4-AD catabolic pathways of M. abscessus are unique in that they converge upstream of where this occurs in characterized steroid-catabolizing bacteria. The data further indicate that cholesterol is a substrate for intracellular bacteria and that cholesterol-dependent toxicity is not strictly dependent on coenzyme A sequestration.

The development of microbial cell factories requires robust synthetic biology tools to reduce design uncertainty and accelerate the design-build-test-learn process. Herein, we developed a suite of integrative genetic tools to facilitate the engineering of Rhodococcus, a genus of bacteria with considerable biocatalytic potential. We first created pRIME, a modular, copy-controlled integrative-vector, to provide a robust platform for strain engineering and characterizing genetic parts. This vector was then employed to benchmark a series of strong promoters. We found PM6 to be the strongest constitutive rhodococcal promoter, 2.5- to 3-fold stronger than the next in our study, while overall promoter activities ranged 23-fold between the weakest and strongest promoters during exponential growth. Next, we used an optimized variant of PM6 to develop hybrid-promoters and integrative vectors to allow for tetracycline-inducible gene expression in Rhodococcus. The best of the resulting hybrid-promoters maintained a maximal activity of ∼50% of PM6 and displayed an induction factor of ∼40-fold. Finally, we developed and implemented a uLoop-derived Golden Gate assembly strategy for high-throughput DNA assembly in Rhodococcus. To demonstrate the utility of our approaches, pRIME was used to engineer Rhodococcus jostii RHA1 to grow on vanillin at concentrations 10-fold higher than what the wild-type strain tolerated. Overall, this study provides a suite of tools that will accelerate the engineering of Rhodococcus for various biocatalytic applications, including the sustainable production of chemicals from lignin-derived aromatics.

Dye-decolorizing peroxidases (DyPs) comprise a recently discovered family of heme peroxidases with considerable biotechnological potential. However, the roles of the distal aspartate and arginine i...

Cytochrome P450 enzymes have tremendous potential as industrial biocatalysts, including in biological lignin valorization. Here, we describe P450s that catalyze the O-demethylation of lignin-derived guaiacols with different ring substitution patterns. Bacterial strains Rhodococcus rhodochrous EP4 and Rhodococcus jostii RHA1 both utilized alkylguaiacols as sole growth substrates. Transcriptomics of EP4 grown on 4-propylguaiacol (4PG) revealed the up-regulation of agcA, encoding a CYP255A1 family P450, and the aph genes, previously shown to encode a meta-cleavage pathway responsible for 4-alkylphenol catabolism. The function of the homologous pathway in RHA1 was confirmed: Deletion mutants of agcA and aphC, encoding the meta-cleavage alkylcatechol dioxygenase, grew on guaiacol but not 4PG. By contrast, deletion mutants of gcoA and pcaL, encoding a CYP255A2 family P450 and an ortho-cleavage pathway enzyme, respectively, grew on 4-propylguaiacol but not guaiacol. CYP255A1 from EP4 catalyzed the O-demethylation of 4-alkylguaiacols to 4-alkylcatechols with the following apparent specificities (k(cat)/K(M)): propyl > ethyl > methyl > guaiacol. This order largely reflected AgcA’s binding affinities for the different guaiacols and was the inverse of GcoA(EP4)’s specificities. The biocatalytic potential of AgcA was demonstrated by the ability of EP4 to grow on lignin-derived products obtained from the reductive catalytic fractionation of corn stover, depleting alkylguaiacols and alkylphenols. By identifying related P450s with complementary specificities for lignin-relevant guaiacols, this study facilitates the design of these enzymes for biocatalytic applications. We further demonstrated that the metabolic fate of the guaiacol depends on its substitution pattern, a finding that has significant implications for engineering biocatalysts to valorize lignin.

Steroid-degrading bacteria, including Mycobacterium tuberculosis (Mtb), utilize an architecturally distinct subfamily of acyl coenzyme A dehydrogenases (ACADs) for steroid catabolism. These ACADs are α2β2 heterotetramers that are usually encoded by adjacent fadE-like genes. In mycobacteria, ipdE1 and ipdE2 (formerly fadE30 and fadE33) occur in divergently transcribed operons associated with the catabolism of 3aα-H-4α(3′-propanoate)-7aβ-methylhexahydro-1,5-indanedione (HIP), a steroid metabolite. In Mycobacterium smegmatis, ΔipdE1 and ΔipdE2 mutants had similar phenotypes, showing impaired growth on cholesterol and accumulating 5-OH HIP in the culture supernatant. Bioinformatic analyses revealed that IpdE1 and IpdE2 share many of the features of the α- and β-subunits, respectively, of heterotetrameric ACADs that are encoded by adjacent genes in many steroid-degrading proteobacteria. When coproduced in a rhodococcal strain, IpdE1 and IpdE2 of Mtb formed a complex that catalyzed the dehydrogenation of 5OH-HIP coenzyme A (5OH-HIP-CoA) to 5OH-3aα-H-4α(3′-prop-1-enoate)-7aβ-methylhexa-hydro-1,5-indanedione coenzyme A ((E)-5OH-HIPE-CoA). This corresponds to the initial step in the pathway that leads to degradation of steroid C and D rings via β-oxidation. Small-angle X-ray scattering revealed that the IpdE1-IpdE2 complex was an α2β2 heterotetramer typical of other ACADs involved in steroid catabolism. These results provide insight into an important class of steroid catabolic enzymes and a potential virulence determinant in Mtb.

The actinobacterium Rhodococcus jostii RHA1 grows on a remarkable variety of aromatic compounds and has been studied for applications ranging from the degradation of polychlorinated biphenyls to the valorization of lignin, an underutilized component of biomass. In RHA1, the catabolism of two classes of lignin-derived compounds, alkylphenols and alkylguaiacols, involves a phylogenetically distinct extradiol dioxygenase, AphC, previously misannotated as BphC, an enzyme involved in biphenyl catabolism. To better understand the role of AphC in RHA1 catabolism, we first showed that purified AphC had highest apparent specificity for 4-propylcatechol (k

Characterizing microorganisms and enzymes involved in lignin biodegradation in thermal ecosystems can identify thermostable biocatalysts. We integrated stable isotope probing (SIP), genome-resolved metagenomics, and enzyme characterization to investigate the degradation of high-molecular weight

Encapsulin nanocompartments are an emerging class of prokaryotic protein-based organelle consisting of an encapsulin protein shell that encloses a protein cargo. Genes encoding nanocompartments are widespread in bacteria and archaea, and recent works have characterized the biochemical function of several cargo enzymes. However, the importance of these organelles to host physiology is poorly understood. Here, we report that the human pathogen Mycobacterium tuberculosis (Mtb) produces a nanocompartment that contains the dye-decolorizing peroxidase DyP. We show that this nanocompartment is important for the ability of Mtb to resist oxidative stress in low pH environments, including during infection of host cells and upon treatment with a clinically relevant antibiotic. Our findings are the first to implicate a nanocompartment in bacterial pathogenesis and reveal a new mechanism that Mtb uses to combat oxidative stress.

Phthalate, a plasticizer, endocrine disruptor, and potential carcinogen, is degraded by a variety of bacteria. This degradation is initiated by phthalate dioxygenase (PDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of phthalate to a dihydrodiol. PDO has long served as a model for understanding ROs despite a lack of structural data. Here we purified PDOKF1 from Comamonas testosteroni KF1 and found that it had an apparent kcat/Km for phthalate of 0.58 ± 0.09 μM−1s−1, over 25-fold greater than for terephthalate. The crystal structure of the enzyme at 2.1 A resolution revealed that it is a hexamer comprising two stacked α3 trimers, a configuration not previously observed in RO crystal structures. We show that within each trimer, the protomers adopt a head-to-tail configuration typical of ROs. The stacking of the trimers is stabilized by two extended helices, which make the catalytic domain of PDOKF1 larger than that of other characterized ROs. Complexes of PDOKF1 with phthalate and terephthalate revealed that Arg207 and Arg244, two residues on one face of the active site, position these substrates for regiospecific hydroxylation. Consistent with their roles as determinants of substrate specificity, substitution of either residue with alanine yielded variants that did not detectably turnover phthalate. Together, these results provide critical insights into a pollutant-degrading enzyme that has served as a paradigm for ROs and facilitate the engineering of this enzyme for bioremediation and biocatalytic applications.

Encapsulin nanocompartments are an emerging class of prokaryotic protein-based organelle consisting of an encapsulin protein shell that encloses a protein cargo. Genes encoding nanocompartments are widespread in bacteria and archaea, and recent works have characterized the biochemical function of several cargo enzymes. However, the importance of these organelles to host physiology is poorly understood. Here, we report that the human pathogen Mycobacterium tuberculosis (Mtb) produces a nanocompartment that contains the dye-decolorizing peroxidase DyP. We show that this nanocompartment is important for the ability of Mtb to resist oxidative stress in low pH environments, including during infection of host cells and upon treatment with a clinically relevant antibiotic. Our findings are the first to implicate a nanocompartment in bacterial pathogenesis and reveal a new mechanism that Mtb uses to combat oxidative stress.

Significance Pyridoxal phosphate (PLP)-dependent enzymes rarely react with oxygen, but an emerging group of oxygen-, PLP-dependent enzymes oxidize l -arginine. Two types of oxidases are known: hydroxylases and desaturases. We demonstrate that arginine desaturases have a minor hydroxylase activity and then show through X-ray crystallographic, mutagenesis, spectroscopic, and computational studies that their mechanism involves two rounds of single-electron transfer to oxygen and superoxide rebound, ultimately giving a conjugated hydroperoxyl intermediate. Water can attack to give a hydroxylated product and release H 2 O 2 , but with water absent, the intermediate can be deprotonated, instead giving a desaturated product and H 2 O 2 . Our work outlines the unique mechanism and evolutionary history of these enzymes and sets the stage toward engineering these enzymes to catalyze oxidative reactions.

The valorization of lignin, a major component of plant-derived biomass, is essential to sustainable biorefining. We identified the major monoaromatic compounds present in black liquor, a lignin-rich stream generated in the kraft pulping process, and investigated their bacterial transformation. Among tested solvents, acetone extracted the greatest amount of monoaromatic compounds from softwood black liquor, with guaiacol, vanillin, and acetovanillone, in an approximately 4:3:2 ratio, constituting ~90% of the total extracted monoaromatic content. 4-Ethanol guaiacol, vanillate, and 4-propanol guaiacol were also present. Bacterial strains that grew on minimal media supplemented with the BL extracts at 1mM total aromatic compounds included Pseudomonas putida KT2442, Sphingobium sp. SYK-6, and Rhodococcus rhodochrous EP4. By contrast, the extracts inhibited the growth of Rhodococcus jostii RHA1 and Rhodococcus opacus PD630, strains extensively studied for lignin valorization. Of the strains that grew on the extracts, only R. rhodochrous GD01 and GD02, isolated for their ability to grow on acetovanillone, depleted the major extracted monoaromatics. Genomic analyses revealed that EP4, GD01, and GD02 share an average nucleotide identity (ANI) of 98% and that GD01 and GD02 harbor a predicted three-component carboxylase not present in EP4. A representative carboxylase gene was upregulated ~100-fold during growth of GD02 on a mixture of the BL monoaromatics, consistent with the involvement of the enzyme in acetovanillone catabolism. More generally, quantitative RT-PCR indicated that GD02 catabolizes the BL compounds in a convergent manner via the β-ketoadipate pathway. Overall, these studies help define the catabolic capabilities of potential biocatalytic strains, describe new isolates able to catabolize the major monoaromatic components of BL, including acetovanillone, and facilitate the design of biocatalysts to valorize under-utilized components of industrial lignin streams.

The development of microbial cell factories requires robust synthetic biology tools to reduce design uncertainty and accelerate the design-build-test-learn process. Herein, we developed a suite of integrative genetic tools to facilitate the engineering of

Funding Information: This work was supported by the Government of Ontario for the project “Forest FAB: Applied Genomics for Functionalized Fibre and Biochemicals” (grant number ORF-RE-05-005), the Natural Sciences and Engineering Research Council (NSERC) of Canada for the Strategic Network Grant “Industrial Biocatalysis Network,” and Genome Canada for the LSARP project “SYNBIOMICS – Functional genomics and techno-economic models for advanced biopolymer synthesis” (grant number 10405) to ERM as well as NSERC Discovery Grant 171359 to LDE. LDE is the recipient of a Tier 1 Canada Research Chair in Microbial Catabolism and Biocatalysis. Publisher Copyright: © Copyright © 2021 Vuong, Singh, Eltis and Master. The relative ability of the small laccase (sLac) and dye-decoloring peroxidase (DyP2) from Amycolatopsis sp. 75iv2 to transform a variety of lignins was investigated using time-of-flight secondary ion mass spectrometry (ToF-SIMS). The enzymes modified organosolv hardwood lignin to different extents even in the absence of an added mediator. More particularly, sLac decreased the lignin modification metric S (S-lignin)/Ar (total aromatics) by 58% over 16h, while DyP2 lowered this ratio by 31% in the absence of exogenous H2O2. When used on their own, both sLac and DyP2 also modified native lignin present in aspen wood powder, albeit to lesser extents than in the organosolv lignin. The addition of ABTS for sLac and Mn2+ as well as H2O2 for DyP2 led to increased lignin modification in aspen wood powder as reflected by a decrease in the G/Ar metric by up to a further 13%. This highlights the importance of exogenous mediators for transforming lignin within its native matrix. Furthermore, the addition of ABTS reduced the selectivity of sLac for S-lignin over G-lignin, indicating that the mediator also altered the product profiles. Finally, when sLac was included in reactions containing DyP2, in part to generate H2O2in situ, the relative abundance of lignin products differed from individual enzymatic treatments. Overall, these results identify possible routes to tuning lignin modification or delignification through choice of enzyme and mediator. Moreover, the current study expands the application of ToF-SIMS to evaluating enzyme action on technical lignins, which can accelerate the discovery and engineering of industrially relevant enzymes for lignin valorization.

Chelator-mediated Fenton chemistry is capable of reducing non-stochiometric amounts of iron via hydroquinone oxidation. These types of reactions have previously been demonstrated to be promoted by some lignocellulose degrading fungi in generating hydroxyl radicals to permit lignified plant cell wall deconstruction. Here we demonstrate that lignocellulose surfaces, when exposed by chemical treatment or fragmentation, can promote a similar multi-oxidative mechanism in the presence of iron. Iron reduction by lignin surfaces permits the generation of hydroxyl radicals in the cell wall to help explain fungal non-enzymatic cell wall deconstruction, and it also provides an explanation for certain phenomenon such as the anthropogenic generation of formaldehyde by wood. The mechanism also provides a basis for the generation of electrons by lignin that are required by certain fungal redox enzymes active in plant cell wall degrading systems. Overall, the data demonstrate that iron found naturally in lignocellulose materials will promote the oxidation of phenolic lignin compounds in the naturally low pH environments occurring within lignified plant cell walls, and that this activity is promoted by cell wall fragmentation.

Oleaginous microorganisms, such as those found in the Rhodococcus genus, have considerable potential for the sustainable production of lipid-based chemicals. Herein, we rewired lipid accumulation in Rhodococcus jostii RHA1 to create an industrially viable biocatalyst for the production of high-value wax esters (WEs). To efficiently manipulate these non-model bacteria, we first expanded the genetic tools available in rhodococci, creating pSYN, an integrative, modular expression vector. We employed this vector to screen predicted promoters, creating a library of strong constitutive promoters. RHA1 strains with a chromosomal insertion of fcrA, encoding a fatty acyl-CoA reductase, under the control of constitutive promoters accumulated WEs. We next screened wax synthases, identifying WS2 of Marinobacter hydrocarbonoclasticus DSM 8798 as the most effective at increasing WE levels in RHA1. Cassettes for the co-expression of chromosomally integrated fcrA and ws2 were created and transformed into RHA1, yielding a biocatalyst that, when grown in flasks, accumulated WEs to greater than 15% CDW, at yields of 0.05 g per g glucose, while maintaining 80% of the specific growth rate of WT. The accumulated WEs were 29 to 38 carbon atoms in length, of which 75% were unsaturated, with a ∼2 : 1 mix of mono- and diunsaturated species. In fed-batch fermentations, the biocatalyst produced WEs with a titer, rate, and yield of approximately 5 g L−1, 1 g L−1 day−1, and 0.025 g per g glucose, respectively. Overall, these results highlight the potential of Rhodococcus for the sustainable production of high-value unsaturated WEs, and facilitate the development of this genus for biocatalytic applications.

Lignostilbene-α,β-dioxygenases (LSDs) are iron-dependent oxygenases involved in the catabolism of lignin-derived stilbenes. Sphingobium sp. SYK-6 contains eight LSD homologs with undetermined physiological roles. To investigate which homologs are involved in the catabolism of dehydrodiconiferyl alcohol (DCA), derived from β-5 linked lignin subunits, we heterologously produced the enzymes and screened their activities in lysates. The seven soluble enzymes all cleaved lignostilbene, but only LSD2, LSD3, and LSD4 exhibited high specific activity for 3-(4-hydroxy-3-(4-hydroxy-3-methoxystyryl)-5-methoxyphenyl) acrylate (DCA-S) relative to lignostilbene. LSD4 catalyzed the cleavage of DCA-S to 5-formylferulate and vanillin and cleaved lignostilbene and DCA-S (∼106 M−1 s−1) with tenfold greater specificity than pterostilbene and resveratrol. X-ray crystal structures of native LSD4 and the catalytically inactive cobalt-substituted Co-LSD4 at 1.45 A resolution revealed the same fold, metal ion coordination, and edge-to-edge dimeric structure as observed in related enzymes. Key catalytic residues, Phe-59, Tyr-101, and Lys-134, were also conserved. Structures of Co-LSD4·vanillin, Co-LSD4·lignostilbene, and Co-LSD4·DCA-S complexes revealed that Ser-283 forms a hydrogen bond with the hydroxyl group of the ferulyl portion of DCA-S. This residue is conserved in LSD2 and LSD4 but is alanine in LSD3. Substitution of Ser-283 with Ala minimally affected the specificity of LSD4 for either lignostilbene or DCA-S. By contrast, substitution with phenylalanine, as occurs in LSD5 and LSD6, reduced the specificity of the enzyme for both substrates by an order of magnitude. This study expands our understanding of an LSD critical to DCA catabolism as well as the physiological roles of other LSDs and their determinants of substrate specificity.

Valorization of lignin, an abundant component of plant cell walls, is critical to enabling the lignocellulosic bioeconomy. Biological funneling using microbial biocatalysts has emerged as an attractive approach to convert complex mixtures of lignin depolymerization products to value-added compounds. Ideally, biocatalysts would convert aromatic compounds derived from the three canonical types of lignin: syringyl (S), guaiacyl (G), and p-hydroxyphenyl (H). Pseudomonas putida KT2440 (hereafter KT2440) has been developed as a biocatalyst owing in part to its native catabolic capabilities but is not known to catabolize S-type lignin-derived compounds. Here, we demonstrate that syringate, a common S-type lignin-derived compound, is utilized by KT2440 only in the presence of another energy source or when vanAB was overexpressed, as syringate was found to be O-demethylated to gallate by VanAB, a two-component monooxygenase, and further catabolized via extradiol cleavage. Unexpectedly, the specificity (kcat/KM) of VanAB for syringate was within 25% that for vanillate and O-demethylation of both substrates was well-coupled to O2 consumption. However, the native KT2440 gallate-cleaving dioxygenase, GalA, was potently inactivated by 3-O-methylgallate. To engineer a biocatalyst to simultaneously convert S-, G-, and H-type monomers, we therefore employed VanAB from Pseudomonas sp. HR199, which has lower activity for 3MGA, and LigAB, an extradiol dioxygenase able to cleave protocatechuate and 3-O-methylgallate. This strain converted 93% of a mixture of lignin monomers to 2-pyrone-4,6-dicarboxylate, a promising bio-based chemical. Overall, this study elucidates a native pathway in KT2440 for catabolizing S-type lignin-derived compounds and demonstrates the potential of this robust chassis for lignin valorization.

Encapsulin nanocompartments are an emerging class of prokaryotic protein-based organelles consisting of an encapsulin protein shell that encloses a protein cargo1. Genes encoding nanocompartments are widespread in bacteria and archaea, and recent works have characterized the biochemical function of several cargo enzymes2. However, the importance of these organelles to host physiology is poorly understood. Here, we report that the human pathogenMycobacterium tuberculosis(Mtb) produces a nanocompartment that contains the dye-decolorizing peroxidase DypB. We show that this nanocompartment is important for the ability of Mtb to resist oxidative stress in low pH environments, including during infection of host cells and upon treatment with a clinically relevant antibiotic. Our findings are the first to implicate a nanocompartment in bacterial pathogenesis and reveal a new mechanism that Mtb uses to combat oxidative stress.

Thermal swamps are unique ecosystems where geothermally warmed waters mix with decomposing woody biomass, hosting novel biogeochemical-cycling and lignin-degrading microbial consortia. Assembly of shotgun metagenome libraries resolved 351 distinct genomes from hot-spring (30–45 °C) and mesophilic (17 °C) sediments. Annotation of 39 refined draft genomes revealed metabolism consistent with oligotrophy, including pathways for degradation of aromatic compounds, such as syringate, vanillate, p-hydroxybenzoate, and phenol. Thermotolerant Burkholderiales, including Rubrivivax ssp., were implicated in diverse biogeochemical and aromatic transformations, highlighting their broad metabolic capacity. Lignin catabolism was further investigated using metatranscriptomics of sediment incubated with milled or Kraft lignin at 45 °C. Aromatic compounds were depleted from lignin-amended sediment over 148 h. The metatranscriptomic data revealed upregulation of des/lig genes predicted to specify the catabolism of syringate, vanillate, and phenolic oligomers in the sphingomonads Altererythrobacter ssp. and Novosphingobium ssp., as well as in the Burkholderiales genus, Rubrivivax. This study demonstrates how temperature structures biogeochemical cycling populations in a unique ecosystem, and combines community-level metagenomics with targeted metatranscriptomics to identify pathways with potential for bio-refinement of lignin-derived aromatic compounds. In addition, the diverse aromatic catabolic pathways of Altererythrobacter ssp. may serve as a source of thermotolerant enzymes for lignin valorization.

Steryl esters (SEs) are important storage compounds in many eukaryotes and are often prominent components of intracellular lipid droplets. Here we demonstrate that selected Actino- and Proteobacteria growing on sterols are also able to synthesize SEs and to sequester them in cytoplasmic lipid droplets. We found cholesteryl ester (CE) formation in members of the actinobacterial genera Rhodococcus, Mycobacterium, and Amycolatopsis as well as several members of the proteobacterial Cellvibrionales order. CEs maximally accumulated under nitrogen-limiting conditions, suggesting that steryl ester formation plays a crucial role for storing excess energy and carbon under adverse conditions. Rhodococcus jostii RHA1 was able to synthesize phytosteryl- and cholesteryl esters, the latter reaching up to 7% of its cellular dry weight and 69% of its lipid droplets. Purified lipid droplets from RHA1 contained CEs, free cholesterol and triacylglycerols. In addition, we found formation of CEs in Mycobacterium tuberculosis when grown with cholesterol plus an additional fatty acid substrate. This study provides a basis for the application of bacterial whole cell systems in the biotechnological production of SEs for use in functional foods and cosmetics.IMPORTANCEOleaginous bacteria exhibit great potential for the production of high-value neutral lipids, such as triacylglycerols and wax esters. This study describes the formation of steryl esters (SEs) as neutral lipid storage compounds in sterol-degrading oleaginous bacteria, providing a basis for biotechnological production of SEs using bacterial systems with potential applications in the functional food, nutraceutical, and cosmetic industries. We found cholesteryl ester (CE) formation in several sterol-degrading Actino- and Proteobacteria under nitrogen limiting conditions, suggesting an important role of this process in storing energy and carbon under adverse conditions. In addition, Mycobacterium tuberculosis grown on cholesterol accumulated CEs in the presence of an additional fatty acid substrate.

meta-Cleavage product (MCP) hydrolases catalyze the hydrolysis of a carbon carbon (C C) bond in the aerobic catabolism of aromatic compounds by bacteria. This activity has evolved in at least two superfamilies, the α/β-hydrolases and the amidohydrolases, providing a fascinating example of convergent evolution. Despite their different catalytic machinery, MCP hydrolases from the two superfamilies use similar catalytic strategies in which the enzyme first catalyzes an enol-to-keto tautomerization to generate a requisite electron sink with concomitant activation of the nucleophile: serine and water in the case of α/β-hydrolases and amidohydrolases, respectively. The two reactions proceed via similar, orange-colored intermediates. This article reviews what is known about the catalytic mechanism of the MCP hydrolases, highlighting how the catalytic machinery of serine- and metal-dependent hydrolases have been adapted for C C bond hydrolysis. Avenues for future work, including the identification of a possible third family of MCP hydrolases, are discussed.

Enzymes that catalyze hydroxylation of unactivated carbons normally contain heme and nonheme iron cofactors. By contrast, how a pyridoxal phosphate (PLP)-dependent enzyme could catalyze such a hydroxylation was unknown. Here, we investigate RohP, a PLP-dependent enzyme that converts l-arginine to (S)-4-hydroxy-2-ketoarginine. We determine that the RohP reaction consumes oxygen with stoichiometric release of H2O2. To understand this unusual chemistry, we obtain ∼1.5 A resolution structures that capture intermediates along the catalytic cycle. Our data suggest that RohP carries out a four-electron oxidation and a stereospecific alkene hydration to give the (S)-configured product. Together with our earlier studies on an O2, PLP-dependent l-arginine oxidase, our work suggests that there is a shared pathway leading to both oxidized and hydroxylated products from l-arginine.

IsdB is a receptor on the surface of the bacterial pathogen Staphylococcus aureus that extracts heme from hemoglobin (Hb) to enable growth on Hb as a sole iron source. IsdB is critically important both for in vitro growth on Hb and in infection models and is also highly up-regulated in blood, serum, and tissue infection models, indicating a key role of this receptor in bacterial virulence. However, structural information for IsdB is limited. We present here a crystal structure of a complex between human Hb and IsdB. In this complex, the α subunits of Hb are refolded with the heme displaced to the interface with IsdB. We also observe that atypical residues of Hb, His58 and His89 of αHb, coordinate to the heme iron, which is poised for transfer into the heme-binding pocket of IsdB. Moreover, the porphyrin ring interacts with IsdB residues Tyr440 and Tyr444 Previously, Tyr440 was observed to coordinate heme iron in an IsdB·heme complex structure. A Y440F/Y444F IsdB variant we produced was defective in heme transfer yet formed a stable complex with Hb (Kd = 6 ± 2 μm) in solution with spectroscopic features of the bis-His species observed in the crystal structure. Haptoglobin binds to a distinct site on Hb to inhibit heme transfer to IsdB and growth of S. aureus, and a ternary complex of IsdB·Hb·Hp was observed. We propose a model for IsdB heme transfer from Hb that involves unfolding of Hb and heme iron ligand exchange.

The bacterial catabolism of aromatic compounds has considerable promise to convert lignin depolymerization products to commercial chemicals. Alkylphenols are a key class of depolymerization products whose catabolism is not well elucidated. We isolatedRhodococcus rhodochrousEP4 on 4-ethylphenol and applied genomic and transcriptomic approaches to elucidate alkylphenol catabolism in EP4 andRhodococcus jostiiRHA1. RNA-Seq and RT-qPCR revealed a pathway encoded by theaphABCDEFGHIQRSgenes that degrades 4-ethylphenol via themeta-cleavage of 4-ethylcatechol. This process was initiated by a two-component alkylphenol hydroxylase, encoded by theaphABgenes, which were up-regulated ~3,000-fold. Purified AphAB from EP4 had highest specific activity for 4-ethylphenol and 4-propylphenol (~2000 U/mg) but did not detectably transform phenol. Nevertheless, a ΔaphAmutant in RHA1 grew on 4-ethylphenol by compensatory up-regulation of phenol hydroxylase genes (pheA1-3). Deletion ofaphC, encoding an extradiol dioxygenase, prevented growth on 4-alkylphenols but not phenol. Disruption ofpcaLin the β-ketoadipate pathway prevented growth on phenol but not 4-alkylphenols. Thus, 4-ethylphenol and 4-propylphenol are catabolized exclusively viameta-cleavage in rhodococci while phenol is subject toortho-cleavage. Putative genomic islands encodingaphgeneswere identified in EP4 and several other rhodococci. Overall, this study identifies a 4-alkylphenol pathway in rhodococci, demonstrates key enzymes involved, and presents evidence that the pathway is encoded in a genomic island. These advances are of particular importance for wide-ranging industrial applications of rhodococci, including upgrading of lignocellulose biomass.ImportanceElucidation of bacterial alkylphenol catabolism is important for the development of biotechnologies to upgrade the lignin component of plant biomass. We isolated a new strain,Rhodococcus rhodochrousEP4, on 4-ethylphenol, an alkylphenol that occurs in lignin-derived streams, including reductive catalytic fractionation products of corn stover. We further demonstrated its degradation via ameta-cleavage pathway (Aph) with transcriptomics. A new class of Actinobacterial hydroxylase, AphAB, acts specifically on alkylphenols. Phylogenomic analysis indicated that theaphgenes occur on putative genomic islands in several rhodococcal strains. These genes were identified in the genetically-tractable strainRhodococcus jostiiRHA1. Strains missing this element cannot metabolise 4-ethylphenol and 4-propylphenol. Overall, we advanced the understanding of how aromatic compounds are degraded by environmental bacteria and identified enzymes that can be employed in the transition away from petro-chemicals towards renewable alternatives.

Lignostilbene-α,β-dioxygenase A (LsdA) from the bacterium Sphingomonas paucimobilis TMY1009 is a nonheme iron oxygenase that catalyzes the cleavage of lignostilbene, a compound arising in lignin transformation, to two vanillin molecules. To examine LsdA's substrate specificity, we heterologously produced the dimeric enzyme with the help of chaperones. When tested on several substituted stilbenes, LsdA exhibited the greatest specificity for lignostilbene (k(cat)(app) = 1.00 ± 0.04 × 10(6) m(−1) s(−1)). These experiments further indicated that the substrate's 4-hydroxy moiety is required for catalysis and that this moiety cannot be replaced with a methoxy group. Phenylazophenol inhibited the LsdA-catalyzed cleavage of lignostilbene in a reversible, mixed fashion (K(ic) = 6 ± 1 μm, K(iu) = 24 ± 4 μm). An X-ray crystal structure of LsdA at 2.3 Å resolution revealed a seven-bladed β-propeller fold with an iron cofactor coordinated by four histidines, in agreement with previous observations on related carotenoid cleavage oxygenases. We noted that residues at the dimer interface are also present in LsdB, another lignostilbene dioxygenase in S. paucimobilis TMY1009, rationalizing LsdA and LsdB homo- and heterodimerization in vivo. A structure of an LsdA·phenylazophenol complex identified Phe(59), Tyr(101), and Lys(134) as contacting the 4-hydroxyphenyl moiety of the inhibitor. Phe(59) and Tyr(101) substitutions with His and Phe, respectively, reduced LsdA activity (k(cat)(app)) ∼15- and 10-fold. The K134M variant did not detectably cleave lignostilbene, indicating that Lys(134) plays a key catalytic role. This study expands our mechanistic understanding of LsdA and related stilbene-cleaving dioxygenases.

Laccases are multicopper oxidases that are being studied for their potential application in pretreatment strategies of lignocellulosic feedstocks for bioethanol production. Here, we report the expression and characterization of a predicted laccase (LAC_2.9) from the thermophilic bacterial strain Thermus sp. 2.9 and investigate its capacity to delignify lignocellulosic biomass. The purifed enzyme displayed a blue color typical of laccases, showed strict copper dependence and retained 80% of its activity after 16 h at 70 °C. At 60 °C, the enzyme oxidized 2,2′-azino-di-(3-ethylbenzthiazoline sulfonate) (ABTS) and 2,6-dimethoxyphenol (DMP) at optimal pH of 5 and 6, respectively. LAC_2.9 had higher substrate specifcity (kcat/KM) for DMP with a calculated value that accounts for one of the highest reported for laccases. Further, the enzyme oxidized a phenolic lignin model dimer. The incubation of steam-exploded eucalyptus biomass with LAC_2.9 and 1-hydroxybenzotriazole (HBT) as mediator changed the structural properties of the lignocellulose as evidenced by Fourier transform infrared (FTIR) spectroscopy and thermo-gravimetric analysis (TGA). However, this did not increase the yield of sugars released by enzymatic saccharifcation. In conclusion, LAC_2.9 is a thermostable laccase with potential application in the delignifcation of lignocellulosic biomass. Fil: Navas, Laura Emilce. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; Argentina Fil: Martínez, Fernando D.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; Argentina Fil: Taverna, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina. Universidad Tecnológica Nacional. Facultad Regional San Francisco; Argentina Fil: Fetherolf, Morgan M.. University of British Columbia; Canadá Fil: Eltis, Lindsay D.. University of British Columbia; Canadá Fil: Nicolau, Verónica. Universidad Tecnológica Nacional. Facultad Regional San Francisco; Argentina Fil: Estenoz, Diana Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Campos, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina Fil: Benintende, Graciela Beatriz. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Berretta, Marcelo Facundo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; Argentina

Cholesterol can be a major carbon source for Mycobacterium tuberculosis during infection, both at an early stage in the macrophage phagosome and later within the necrotic granuloma. KstR is a highly conserved TetR family transcriptional repressor that regulates a large set of genes responsible for cholesterol catabolism. Many genes in this regulon, including kstR, are either induced during infection or are essential for survival of M. tuberculosis in vivo. In this study, we identified two ligands for KstR, both of which are CoA thioester cholesterol metabolites with four intact steroid rings. A metabolite in which one of the rings was cleaved was not a ligand. We confirmed the ligand-protein interactions using intrinsic tryptophan fluorescence and showed that ligand binding strongly inhibited KstR-DNA binding using surface plasmon resonance (IC50 for ligand = 25 nm). Crystal structures of the ligand-free form of KstR show variability in the position of the DNA-binding domain. In contrast, structures of KstR·ligand complexes are highly similar to each other and demonstrate a position of the DNA-binding domain that is unfavorable for DNA binding. Comparison of ligand-bound and ligand-free structures identifies residues involved in ligand specificity and reveals a distinctive mechanism by which the ligand-induced conformational change mediates DNA release.

Methicillin-resistant Staphylococcus aureus (MRSA) causes serious community-acquired and nosocomial infections worldwide. MRSA strains are resistant to a variety of antibiotics, including the classic penicillin and cephalosporin classes of β-lactams, making them intractable to treatment. Although β-lactam resistance in MRSA has been ascribed to the acquisition and activity of penicillin-binding protein 2a (PBP2a, encoded by mecA), it has recently been observed that resistance can also be mediated by penicillin-binding protein 4 (PBP4). Previously, we have shown that broad-spectrum β-lactam resistance can arise following serial passaging of a mecA-negative COL strain of S. aureus, creating the CRB strain. This strain has two missense mutations in pbp4 and a mutation in the pbp4 promoter, both of which play an instrumental role in β-lactam resistance. To better understand PBP4's role in resistance, here we have characterized its kinetics and structure with clinically relevant β-lactam antibiotics. We present the first crystallographic PBP4 structures of apo and acyl-enzyme intermediate forms complexed with three late-generation β-lactam antibiotics: ceftobiprole, ceftaroline, and nafcillin. In parallel, we characterized the structural and kinetic effects of the PBP4 mutations present in the CRB strain. Localized within the transpeptidase active-site cleft, the two substitutions appear to have different effects depending on the drug. With ceftobiprole, the missense mutations impaired the K(m) value 150-fold, decreasing the proportion of inhibited PBP4. However, ceftaroline resistance appeared to be mediated by other factors, possibly including mutation of the pbp4 promoter. Our findings provide evidence that S. aureus CRB has at least two PBP4-mediated resistance mechanisms.

A series of alkylated 2,3-dihydroxybiphenyls has been prepared on the gram scale by using an effective Directed ortho Metalation-Suzuki-Miyaura cross-coupling strategy. These compounds have been used to investigate the substrate specificity of the meta-cleavage dioxygenase BphC, a key enzyme in the microbial catabolism of biphenyl. Isolation and characterization of the meta-cleavage products will allow further study of related processes, including the catabolism of lignin-derived biphenyls.

All steroid-degrading bacteria utilize IpdAB, a predicted CoA transferase (CoT) that has been implicated in the hydrolysis of a carbon–carbon bond, an unprecedented reaction in CoTs. In Mycobacterium tuberculosis, IpdAB is required for degrading host cholesterol and virulence. We used a combination of X-ray crystallographic and biochemical studies to elucidate the mechanism of IpdAB. Superposition of the IpdABMtb active site with those of CoTs reveals distinct architectural features which, in conjunction with the biochemical data, indicate that IpdAB catalyzes a retro-Claisen-like ring-opening reaction. This reaction is unique for a member of the CoT superfamily. This study provides insights into bacterial steroid catabolism and facilitates the development of potential antituberculosis therapeutics targeting IpdAB.

Mycobacterium tuberculosis (Mtb) grows on host-derived cholesterol during infection. IpdAB, found in all steroid-degrading bacteria and a determinant of pathogenicity, has been implicated in the hydrolysis of the last steroid ring. Phylogenetic analyses revealed that IpdAB orthologs form a clade of CoA transferases (CoTs). In a coupled assay with a thiolase, IpdAB transformed the cholesterol catabolite (R)-2-(2-carboxyethyl)-3-methyl-6-oxocyclohex-1-ene-1-carboxyl-CoA (COCHEA-CoA) and CoASH to 4-methyl-5-oxo-octanedioyl-CoA (MOODA-CoA) and acetyl-CoA with high specificity (kcat/Km = 5.8 ± 0.8 × 104 M-1⋅s-1). The structure of MOODA-CoA was consistent with IpdAB hydrolyzing COCHEA-CoA to a β-keto-thioester, a thiolase substrate. Contrary to characterized CoTs, IpdAB exhibited no activity toward small CoA thioesters. Further, IpdAB lacks the catalytic glutamate residue that is conserved in the β-subunit of characterized CoTs and a glutamyl-CoA intermediate was not trapped during turnover. By contrast, Glu105A, conserved in the α-subunit of IpdAB, was essential for catalysis. A crystal structure of the IpdAB·COCHEA-CoA complex, solved to 1.4 A, revealed that Glu105A is positioned to act as a catalytic base. Upon titration with COCHEA-CoA, the E105AA variant accumulated a yellow-colored species (λmax = 310 nm; Kd = 0.4 ± 0.2 μM) typical of β-keto enolates. In the presence of D2O, IpdAB catalyzed the deuteration of COCHEA-CoA adjacent to the hydroxylation site at rates consistent with kcat Based on these data and additional IpdAB variants, we propose a retro-Claisen condensation-like mechanism for the IpdAB-mediated hydrolysis of COCHEA-CoA. This study expands the range of known reactions catalyzed by the CoT superfamily and provides mechanistic insight into an important determinant of Mtb pathogenesis.

Enzymes that catalyze hydroxylation of unactivated carbons normally contain heme and nonheme iron cofactors. By contrast, how a pyridoxal phosphate (PLP)-dependent enzyme could catalyze such a hydroxylation was unknown. Here, we investigate RohP, a PLP-dependent enzyme that converts l-arginine to ( S)-4-hydroxy-2-ketoarginine. We determine that the RohP reaction consumes oxygen with stoichiometric release of H

Bacteria have tremendous potential as biocatalysts to valorize lignin streams. This biocatalytic potential derives from the recent characterization of their lignolytic enzymes and, more importantly, their inherent ability to catabolize a wide variety of aromatic compounds, transforming them into central metabolites through a limited suite of shared intermediates. This convergent catabolism facilitates the engineering of “biological funneling” to transform lignin depolymerization products to commodity chemicals. Indeed, biological funneling has the potential to overcome a major challenge of lignin valorization by converting complex mixtures of compounds into single chemical species in high atom yield. In this chapter, we first highlight newly developed deconstruction technologies and recent advances in our understanding of bacterial lignin catabolism. We then summarize features of this catabolism that are particularly advantageous to biotechnological applications and discuss characteristics of specific strains that make them good chasses for lignin-transforming biocatalysts. Examples of tailoring bacterial strains to transform specific lignin streams into target end-products are provided. Finally, we discuss advances in genome-editing tools, bioprospecting, and metabolic modeling that are essential to developing next generation bacterial biocatalysts for economically viable lignin valorization technologies.

CouR, a MarR-type transcriptional repressor, regulates the cou genes, encoding p-hydroxycinnamate catabolism in the soil bacterium Rhodococcus jostii RHA1. The CouR dimer bound two molecules of the catabolite p-coumaroyl–CoA (Kd = 11 ± 1 μM). The presence of p-coumaroyl–CoA, but neither p-coumarate nor CoASH, abrogated CouR's binding to its operator DNA in vitro. The crystal structures of ligand-free CouR and its p-coumaroyl–CoA-bound form showed no significant conformational differences, in contrast to other MarR regulators. The CouR–p-coumaroyl–CoA structure revealed two ligand molecules bound to the CouR dimer with their phenolic moieties occupying equivalent hydrophobic pockets in each protomer and their CoA moieties adopting non-equivalent positions to mask the regulator's predicted DNA-binding surface. More specifically, the CoA phosphates formed salt bridges with predicted DNA-binding residues Arg36 and Arg38, changing the overall charge of the DNA-binding surface. The substitution of either arginine with alanine completely abrogated the ability of CouR to bind DNA. By contrast, the R36A/R38A double variant retained a relatively high affinity for p-coumaroyl–CoA (Kd = 89 ± 6 μM). Together, our data point to a novel mechanism of action in which the ligand abrogates the repressor's ability to bind DNA by steric occlusion of key DNA-binding residues and charge repulsion of the DNA backbone.

The actinomycete Rhodococcus jostii RHA1 contains a multitude of oxygenase enzymes, consonant with its remarkable activities in the catabolism of hydrophobic xenobiotic compounds. In the interests of identifying activities for the transformation of drug molecules, we have cloned genes encoding 23 cytochrome P450 heme domains from R. jostii and expressed them as fusions with the P450 reductase domain (RhfRED) of cytochrome P450Rhf from Rhodococcus sp. NCIMB 9784. Fifteen of the fusions were expressed in the soluble fraction of Escherichia coli Rosetta (DE3) cells. Strains expressing the fusions of RhfRED with genes ro02604, ro04667, ro11069, ro11320, ro11277, ro08984 and ro04671 were challenged with 48 commercially available drugs revealing many different activities commensurate with P450-catalyzed hydroxylation and demethylation reactions. One recombinant strain, expressing the fusion of P450 gene ro11069 (CYP257A1) with RhfRED, and named Ro07-RhfRED, catalyzed the N-demethylation of diltiazem and imipramine. This observation was in accord with previous reports of this enzyme's activity as a demethylase of alkaloid substrates. Ro07-RhfRED was purified and characterised, and applied in cell-free biotransformations of imipramine (7 μM) giving a 63% conversion to the N-desmethyl product.

Dye-decolorizing peroxidases (DyPs; EC 1.11.1.19) are heme enzymes that comprise a family of the dimeric α + β barrel structural superfamily of proteins. The first DyP, identified relatively recently in the fungus Bjerkandera adusta, was characterized for its ability to catalyze the decolorization of anthraquinone-based industrial dyes. These enzymes are now known to be present in all three domains of life, but do not appear to occur in plants or animals. They are involved in a range of physiological processes, although in many cases their roles remain unknown. This has not prevented the development of their biocatalytic potential, which includes the transformation of lignin. This review highlights the functional diversity of DyPs in the light of phylogenetic, structural and biochemical data. The phylogenetic analysis reveals the existence of at least five classes of DyPs. Their potential physiological roles are discussed based in part on synteny analyses. Finally, the considerable biotechnological potential of DyPs is summarized.

Many rhodococci are oleaginous and, as such, have considerable potential for the sustainable production of lipid-based commodity chemicals. Herein, we demonstrated that Rhodococcus jostii RHA1, a soil bacterium that catabolizes a wide range of organic compounds, produced wax esters (WEs) up to 0.0002% of its cellular dry weight during exponential growth on glucose. These WEs were fully saturated and contained primarily 31 to 34 carbon atoms. Moreover, they were present at higher levels during exponential growth than under lipid-accumulating conditions. Bioinformatics analyses revealed that RHA1 contains a gene encoding a putative fatty acyl coenzyme A (acyl-CoA) reductase (FcrA). The purified enzyme catalyzed the NADPH-dependent transformation of stearoyl-CoA to stearyl alcohol with a specific activity of 45 ± 3 nmol/mg · min and dodecanal to dodecanol with a specific activity of 5,300 ± 300 nmol/mg · min. Deletion of fcrA did not affect WE accumulation when grown in either carbon- or nitrogen-limited medium. However, the Δ fcrA mutant accumulated less than 20% of the amount of WEs as the wild-type strain under conditions of nitric oxide stress. A strain of RHA1 overproducing FcrA accumulated WEs to ∼13% cellular dry weight under lipid-accumulating conditions, and their acyl moieties had longer average chain lengths than those in wild-type cells (C 17 versus C 16 ). The results provide insight into the biosynthesis of WEs in rhodococci and facilitate the development of this genus for the production of high-value neutral lipids. IMPORTANCE Among the best-studied oleaginous bacteria, rhodococci have considerable potential for the sustainable production of lipid-based commodity chemicals, such as wax esters. However, many aspects of lipid synthesis in these bacteria are poorly understood. The current study identifies a key enzyme in wax ester synthesis in rhodococci and exploits it to significantly improve the yield of wax esters in bacteria. In so doing, this work contributes to the development of novel bioprocesses for an important class of oleochemicals that may ultimately allow us to phase out their unsustainable production from sources such as petroleum and palm oil.

Strain SYK-6 of the bacterium Sphingobium sp. catabolizes lignin-derived biphenyl via a meta-cleavage pathway. In this pathway, LigY is proposed to catalyze the hydrolysis of the meta-cleavage product (MCP) 4,11-dicarboxy-8-hydroxy-9-methoxy-2-hydroxy-6-oxo-6-phenyl-hexa-2,4-dienoate. Here, we validated this reaction by identifying 5-carboxyvanillate and 4-carboxy-2-hydroxypenta-2,4-dienoate as the products and determined the kcat and kcat/Km values as 9.3 ± 0.6 s−1 and 2.5 ± 0.2 × 107 m−1 s−1, respectively. Sequence analyses and a 1.9 Å resolution crystal structure established that LigY belongs to the amidohydrolase superfamily, unlike previously characterized MCP hydrolases, which are serine-dependent enzymes of the α/β-hydrolase superfamily. The active-site architecture of LigY resembled that of α-amino-β-carboxymuconic-ϵ-semialdehyde decarboxylase, a class III amidohydrolase, with a single zinc ion coordinated by His-6, His-8, His-179, and Glu-282. Interestingly, we found that LigY lacks the acidic residue proposed to activate water for hydrolysis in other class III amidohydrolases. Moreover, substitution of His-223, a conserved residue proposed to activate water in other amidohydrolases, reduced the kcat to a much lesser extent than what has been reported for other amidohydrolases, suggesting that His-223 has a different role in LigY. Substitution of Arg-72, Tyr-190, Arg-234, or Glu-282 reduced LigY activity over 100-fold. On the basis of these results, we propose a catalytic mechanism involving substrate tautomerization, substrate-assisted activation of water for hydrolysis, and formation of a gem-diol intermediate. This last step diverges from what occurs in serine-dependent MCP hydrolases. This study provides insight into C–C–hydrolyzing enzymes and expands the known range of reactions catalyzed by the amidohydrolase superfamily.

Most mycolic acid-containing actinobacteria and some proteobacteria use steroids as growth substrates, but the catabolism of the last two steroid rings has yet to be elucidated. In Mycobacterium tuberculosis, this pathway includes virulence determinants and has been proposed to be encoded by the KstR2-regulated genes, which include a predicted coenzyme A (CoA) transferase gene (ipdAB) and an acyl-CoA reductase gene (ipdC). In the presence of cholesterol, ΔipdC and ΔipdAB mutants of either M. tuberculosis or Rhodococcus jostii strain RHA1 accumulated previously undescribed metabolites: 3aα-H-4α(carboxyl-CoA)-5-hydroxy-7aβ-methylhexahydro-1-indanone (5-OH HIC-CoA) and (R)-2-(2-carboxyethyl)-3-methyl-6-oxocyclohex-1-ene-1-carboxyl-CoA (COCHEA-CoA), respectively. A ΔfadE32 mutant of Mycobacterium smegmatis accumulated 4-methyl-5-oxo-octanedioic acid (MOODA). Incubation of synthetic 5-OH HIC-CoA with purified IpdF, IpdC, and enoyl-CoA hydratase 20 (EchA20), a crotonase superfamily member, yielded COCHEA-CoA and, upon further incubation with IpdAB and a CoA thiolase, yielded MOODA-CoA. Based on these studies, we propose a pathway for the final steps of steroid catabolism in which the 5-member ring is hydrolyzed by EchA20, followed by hydrolysis of the 6-member ring by IpdAB. Metabolites accumulated by ΔipdF and ΔechA20 mutants support the model. The conservation of these genes in known steroid-degrading bacteria suggests that the pathway is shared. This pathway further predicts that cholesterol catabolism yields four propionyl-CoAs, four acetyl-CoAs, one pyruvate, and one succinyl-CoA. Finally, a ΔipdAB M. tuberculosis mutant did not survive in macrophages and displayed severely depleted CoASH levels that correlated with a cholesterol-dependent toxicity. Our results together with the developed tools provide a basis for further elucidating bacterial steroid catabolism and virulence determinants in M. tuberculosis., IMPORTANCE Bacteria are the only known steroid degraders, but the pathway responsible for degrading the last two steroid rings has yet to be elucidated. In Mycobacterium tuberculosis, this pathway includes virulence determinants. Using a series of mutants in M. tuberculosis and related bacteria, we identified a number of novel CoA thioesters as pathway intermediates. Analysis of the metabolites combined with enzymological studies establishes how the last two steroid rings are hydrolytically opened by enzymes encoded by the KstR2 regulon. Our results provide experimental evidence for novel ring-degrading enzymes, significantly advance our understanding of bacterial steroid catabolism, and identify a previously uncharacterized cholesterol-dependent toxicity that may facilitate the development of novel tuberculosis therapeutics.