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1.
JACS Au ; 1(1): 8-12, 2021 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-34467267

RESUMO

Chemical upcycling of waste polyolefins via hydrogenolysis offers unique opportunities for selective depolymerization compared to high temperature thermal deconstruction. Here, we demonstrate the hydrogenolysis of polyethylene into liquid alkanes under mild conditions using ruthenium nanoparticles supported on carbon (Ru/C). Reactivity studies on a model n-octadecane substrate showed that Ru/C catalysts are highly active and selective for the hydrogenolysis of C(sp3)-C(sp3) bonds at temperatures ranging from 200 to 250 °C. Under optimal conditions of 200 °C in 20 bar H2, polyethylene (average M w ∼ 4000 Da) was converted into liquid n-alkanes with yields of up to 45% by mass after 16 h using a 5 wt % Ru/C catalyst with the remaining products comprising light alkane gases (C1-C6). At 250 °C, nearly stoichiometric yields of CH4 were obtained from polyethylene over the catalyst. The hydrogenolysis of long chain, low-density polyethylene (LDPE) and a postconsumer LDPE plastic bottle to produce C7-C45 alkanes was also achieved over Ru/C, demonstrating the feasibility of this reaction for the valorization of realistic postconsumer plastic waste. By identifying Ru-based catalysts as a class of active materials for the hydrogenolysis of polyethylene, this study elucidates promising avenues for the valorization of plastic waste under mild conditions.

2.
JACS Au ; 1(3): 252-261, 2021 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-34467290

RESUMO

Biological funneling of lignin-derived aromatic compounds is a promising approach for valorizing its catalytic depolymerization products. Industrial processes for aromatic bioconversion will require efficient enzymes for key reactions, including demethylation of O-methoxy-aryl groups, an essential and often rate-limiting step. The recently characterized GcoAB cytochrome P450 system comprises a coupled monoxygenase (GcoA) and reductase (GcoB) that catalyzes oxidative demethylation of the O-methoxy-aryl group in guaiacol. Here, we evaluate a series of engineered GcoA variants for their ability to demethylate o-and p-vanillin, which are abundant lignin depolymerization products. Two rationally designed, single amino acid substitutions, F169S and T296S, are required to convert GcoA into an efficient catalyst toward the o- and p-isomers of vanillin, respectively. Gain-of-function in each case is explained in light of an extensive series of enzyme-ligand structures, kinetic data, and molecular dynamics simulations. Using strains of Pseudomonas putida KT2440 already optimized for p-vanillin production from ferulate, we demonstrate demethylation by the T296S variant in vivo. This work expands the known aromatic O-demethylation capacity of cytochrome P450 enzymes toward important lignin-derived aromatic monomers.

3.
ChemSusChem ; 2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34587366

RESUMO

There is keen interest to develop new technologies to recycle the plastic poly(ethylene terephthalate) (PET). To this end, the use of PET-hydrolyzing enzymes has shown promise for PET deconstruction to its monomers, terephthalate (TPA) and ethylene glycol (EG). Here, the Ideonella sakaiensis PETase wild-type enzyme was compared to a previously reported improved variant (W159H/S238F). The thermostability of each enzyme was compared and a 1.45 Šresolution structure of the mutant was described, highlighting changes in the substrate binding cleft compared to the wild-type enzyme. Subsequently, the performance of the wild-type and variant enzyme was compared as a function of temperature, substrate morphology, and reaction mixture composition. These studies showed that reaction temperature had the strongest influence on performance between the two enzymes. It was also shown that both enzymes achieved higher levels of PET conversion for substrates with moderate crystallinity relative to amorphous substrates. Finally, the impact of product accumulation on reaction progress was assessed for the hydrolysis of both PET and bis(2-hydroxyethyl) terephthalate (BHET). Each enzyme displayed different inhibition profiles to mono(2-hydroxyethyl) terephthalate (MHET) and TPA, while both were sensitive to inhibition by EG. Overall, this study highlights the importance of reaction conditions, substrate selection, and product accumulation for catalytic performance of PET-hydrolyzing enzymes, which have implications for enzyme screening in the development of enzyme-based polyester recycling.

4.
Metab Eng ; 68: 14-25, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34438073

RESUMO

While biomass-derived carbohydrates have been predominant substrates for biological production of renewable fuels, chemicals, and materials, organic waste streams are growing in prominence as potential alternative feedstocks to improve the sustainability of manufacturing processes. Catalytic fast pyrolysis (CFP) is a promising approach to generate biofuels from lignocellulosic biomass, but it generates a complex, carbon-rich, and toxic wastewater stream that is challenging to process catalytically but could be biologically upgraded to valuable co-products. In this work, we implemented modular, heterologous catabolic pathways in the Pseudomonas putida KT2440-derived EM42 strain along with the overexpression of native toxicity tolerance machinery to enable utilization of 89% (w/w) of carbon in CFP wastewater. The dmp monooxygenase and meta-cleavage pathway from Pseudomonas putida CF600 were constitutively expressed to enable utilization of phenol, cresols, 2- and 3-ethyl phenol, and methyl catechols, and the native chaperones clpB, groES, and groEL were overexpressed to improve toxicity tolerance to diverse aromatic substrates. Next, heterologous furfural and acetone utilization pathways were incorporated, and a native alcohol dehydrogenase was overexpressed to improve methanol utilization, generating reducing equivalents. All pathways (encoded by genes totaling ~30 kilobases of DNA) were combined into a single strain that can catabolize a mock CFP wastewater stream as a sole carbon source. Further engineering enabled conversion of all aromatic compounds in the mock wastewater stream to (methyl)muconates with a ~90% (mol/mol) yield. Biological upgrading of CFP wastewater as outlined in this work provides a roadmap for future applications in valorizing other heterogeneous waste streams.


Assuntos
Pseudomonas putida , Acetona , Furaldeído , Pseudomonas putida/genética , Pirólise , Ácido Sórbico/análogos & derivados , Águas Residuárias
5.
J Biol Chem ; 297(2): 100931, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34216620

RESUMO

Family 7 glycoside hydrolases (GH7) are among the principal enzymes for cellulose degradation in nature and industrially. These enzymes are often bimodular, including a catalytic domain and carbohydrate-binding module (CBM) attached via a flexible linker, and exhibit an active site that binds cello-oligomers of up to ten glucosyl moieties. GH7 cellulases consist of two major subtypes: cellobiohydrolases (CBH) and endoglucanases (EG). Despite the critical importance of GH7 enzymes, there remain gaps in our understanding of how GH7 sequence and structure relate to function. Here, we employed machine learning to gain data-driven insights into relationships between sequence, structure, and function across the GH7 family. Machine-learning models, trained only on the number of residues in the active-site loops as features, were able to discriminate GH7 CBHs and EGs with up to 99% accuracy, demonstrating that the lengths of loops A4, B2, B3, and B4 strongly correlate with functional subtype across the GH7 family. Classification rules were derived such that specific residues at 42 different sequence positions each predicted the functional subtype with accuracies surpassing 87%. A random forest model trained on residues at 19 positions in the catalytic domain predicted the presence of a CBM with 89.5% accuracy. Our machine learning results recapitulate, as top-performing features, a substantial number of the sequence positions determined by previous experimental studies to play vital roles in GH7 activity. We surmise that the yet-to-be-explored sequence positions among the top-performing features also contribute to GH7 functional variation and may be exploited to understand and manipulate function.


Assuntos
Glicosídeo Hidrolases , Aprendizado de Máquina , Domínio Catalítico , Celulose/metabolismo , Cinética , Simulação de Dinâmica Molecular
6.
Metab Eng ; 67: 250-261, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34265401

RESUMO

Poly(ethylene terephthalate) (PET) is the most abundantly consumed synthetic polyester and accordingly a major source of plastic waste. The development of chemocatalytic approaches for PET depolymerization to monomers offers new options for open-loop upcycling of PET, which can leverage biological transformations to higher-value products. To that end, here we perform four sequential metabolic engineering efforts in Pseudomonas putida KT2440 to enable the conversion of PET glycolysis products via: (i) ethylene glycol utilization by constitutive expression of native genes, (ii) terephthalate (TPA) catabolism by expression of tphA2IIA3IIBIIA1II from Comamonas and tpaK from Rhodococcus jostii, (iii) bis(2-hydroxyethyl) terephthalate (BHET) hydrolysis to TPA by expression of PETase and MHETase from Ideonella sakaiensis, and (iv) BHET conversion to a performance-advantaged bioproduct, ß-ketoadipic acid (ßKA) by deletion of pcaIJ. Using this strain, we demonstrate production of 15.1 g/L ßKA from BHET at 76% molar yield in bioreactors and conversion of catalytically depolymerized PET to ßKA. Overall, this work highlights the potential of tandem catalytic deconstruction and biological conversion as a means to upcycle waste PET.


Assuntos
Polietilenotereftalatos , Pseudomonas putida , Adipatos , Burkholderiales , Etilenos , Hidrolases , Ácidos Ftálicos , Pseudomonas putida/genética , Rhodococcus
7.
Curr Opin Biotechnol ; 73: 1-13, 2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34242853

RESUMO

Significant developments in the understanding and manipulation of microbial metabolism have enabled the use of engineered biological systems toward a more sustainable energy and materials economy. While developments in metabolic engineering have primarily focused on the conversion of carbohydrates, substantial opportunities exist for using these same principles to extract value from more heterogeneous and toxic waste streams, such as those derived from lignin, biomass pyrolysis, or industrial waste. Funneling heterogeneous substrates from these streams toward valuable products, termed biological funneling, presents new challenges in balancing multiple catabolic pathways competing for shared cellular resources and engineering against perturbation from toxic substrates. Solutions to many of these challenges have been explored within the field of lignin valorization. This perspective aims to extend beyond lignin to highlight the challenges and discuss opportunities for use of biological systems to upgrade previously inaccessible waste streams.

9.
J Biol Chem ; 296: 100758, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33965373

RESUMO

Lignostilbene-α,ß-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 ß-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 Å 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.


Assuntos
Proteínas de Bactérias/metabolismo , Dioxigenases/metabolismo , Sphingomonadaceae/metabolismo , Proteínas de Bactérias/química , Cristalografia por Raios X , Dioxigenases/química , Lignina/metabolismo , Modelos Moleculares , Conformação Proteica , Sphingomonadaceae/química , Especificidade por Substrato
10.
Nat Commun ; 12(1): 2261, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33859194

RESUMO

Expanding the portfolio of products that can be made from lignin will be critical to enabling a viable bio-based economy. Here, we engineer Pseudomonas putida for high-yield production of the tricarboxylic acid cycle-derived building block chemical, itaconic acid, from model aromatic compounds and aromatics derived from lignin. We develop a nitrogen starvation-detecting biosensor for dynamic two-stage bioproduction in which itaconic acid is produced during a non-growth associated production phase. Through the use of two distinct itaconic acid production pathways, the tuning of TCA cycle gene expression, deletion of competing pathways, and dynamic regulation, we achieve an overall maximum yield of 56% (mol/mol) and titer of 1.3 g/L from p-coumarate, and 1.4 g/L titer from monomeric aromatic compounds produced from alkali-treated lignin. This work illustrates a proof-of-principle that using dynamic metabolic control to reroute carbon after it enters central metabolism enables production of valuable chemicals from lignin at high yields by relieving the burden of constitutively expressing toxic heterologous pathways.


Assuntos
Lignina/metabolismo , Engenharia Metabólica/métodos , Pseudomonas putida/metabolismo , Succinatos/metabolismo , Álcalis/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Basidiomycota/enzimologia , Basidiomycota/genética , Técnicas Biossensoriais , Burkholderia/enzimologia , Burkholderia/genética , Carbono/metabolismo , Ciclo do Ácido Cítrico/genética , Ácidos Cumáricos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Microbiologia Industrial/métodos , Lignina/química , Estudo de Prova de Conceito , Pseudomonas putida/genética
11.
Metab Eng ; 65: 111-122, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33741529

RESUMO

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.


Assuntos
Pseudomonas putida , Lignina , Pseudomonas putida/genética , Pironas
12.
J Phys Chem B ; 125(8): 2018-2030, 2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33616402

RESUMO

Serine hydrolases cleave peptide and ester bonds and are ubiquitous in nature, with applications in biotechnology, in materials, and as drug targets. The serine hydrolase two-step mechanism employs a serine-histidine-aspartate/glutamate catalytic triad, where the histidine residue acts as a base to activate poor nucleophiles (a serine residue or a water molecule) and as an acid to allow the dissociation of poor leaving groups. This mechanism has been the subject of debate regarding how histidine shuttles the proton from the nucleophile to the leaving group. To elucidate the reaction mechanism of serine hydrolases, we employ quantum mechanics/molecular mechanics-based transition path sampling to obtain the reaction coordinate using the Aspergillus niger feruloyl esterase A (AnFaeA) as a model enzyme. The optimal reaction coordinates include terms involving nucleophilic attack on the carbonyl carbon and proton transfer to, and dissociation of, the leaving group. During the reaction, the histidine residue undergoes a reorientation on the time scale of hundreds of femtoseconds that supports the "moving histidine" mechanism, thus calling into question the "ring flip" mechanism. We find a concerted mechanism, where the transition state coincides with the tetrahedral intermediate with the histidine residue pointed between the nucleophile and the leaving group. Moreover, motions of the catalytic aspartate toward the histidine occur concertedly with proton abstraction by the catalytic histidine and help stabilize the transition state, thus partially explaining how serine hydrolases enable poor nucleophiles to attack the substrate carbonyl carbon. Rate calculations indicate that the second step (deacylation) is rate-determining, with a calculated rate constant of 66 s-1. Overall, these results reveal the pivotal role of active-site dynamics in the catalytic mechanism of AnFaeA, which is likely similar in other serine hydrolases.


Assuntos
Hidrolases de Éster Carboxílico , Hidrolases , Hidrolases de Éster Carboxílico/genética , Catálise , Amostragem
13.
Metab Eng ; 64: 167-179, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33549838

RESUMO

Pseudomonas putida KT2440 (hereafter KT2440) is a well-studied platform bacterium for the production of industrially valuable chemicals from heterogeneous mixtures of aromatic compounds obtained from lignin depolymerization. KT2440 can grow on lignin-related monomers, such as ferulate (FA), 4-coumarate (4CA), vanillate (VA), 4-hydroxybenzoate (4HBA), and protocatechuate (PCA). Genes associated with their catabolism are known, but knowledge about the uptake systems remains limited. In this work, we studied the KT2440 transporters of lignin-related monomers and their substrate selectivity. Based on the inhibition by protonophores, we focused on five genes encoding aromatic acid/H+ symporter family transporters categorized into major facilitator superfamily that uses the proton motive force. The mutants of PP_1376 (pcaK) and PP_3349 (hcnK) exhibited significantly reduced growth on PCA/4HBA and FA/4CA, respectively, while no change was observed on VA for any of the five gene mutants. At pH 9.0, the conversion of these compounds by hcnK mutant (FA/4CA) and vanK mutant (VA) was dramatically reduced, revealing that these transporters are crucial for the uptake of the anionic substrates at high pH. Uptake assays using 14C-labeled substrates in Escherichia coli and biosensor-based assays confirmed that PcaK, HcnK, and VanK have ability to take up PCA, FA/4CA, and VA/PCA, respectively. Additionally, analyses of the predicted protein structures suggest that the size and hydropathic properties of the substrate-binding sites of these transporters determine their substrate preferences. Overall, this study reveals that at physiological pH, PcaK and HcnK have a major role in the uptake of PCA/4HBA and FA/4CA, respectively, and VanK is a VA/PCA transporter. This information can contribute to the engineering of strains for the efficient conversion of lignin-related monomers to value-added chemicals.


Assuntos
Pseudomonas putida , Simportadores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Lignina/metabolismo , Prótons , Pseudomonas putida/genética , Pseudomonas putida/metabolismo
14.
Metab Eng ; 65: 1-10, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33636323

RESUMO

Lignin biosynthesis typically results in a polymer with several inter-monomer bond linkages, and the heterogeneity of linkages presents a challenge for depolymerization processes. While several enzyme classes have been shown to cleave common dimer linkages in lignin, the pathway of bacterial ß-1 spirodienone linkage cleavage has not been elucidated. Here, we identified a pathway for cleavage of 1,2-diguaiacylpropane-1,3-diol (DGPD), a ß-1 linked biaryl representative of a ring-opened spirodienone linkage, in Novosphingobium aromaticivorans DSM12444. In vitro assays using cell lysates demonstrated that RS14230 (LsdE) converts DGPD to a lignostilbene intermediate, which the carotenoid oxygenase, LsdA, then converts to vanillin. A Pseudomonas putida KT2440 strain engineered with lsdEA expression catabolizes erythro-DGPD, but not threo-DGPD. We further engineered P. putida to convert DGPD to a product, cis,cis-muconic acid. Overall, this work demonstrates the potential to identify new enzymatic reactions in N. aromaticivorans and expands the biological funnel of P. putida for microbial lignin valorization.


Assuntos
Pseudomonas putida , Sphingomonadaceae , Lignina , Pseudomonas putida/genética
16.
Sci Adv ; 6(46)2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33188025

RESUMO

Vascular plant pathogens travel long distances through host veins, leading to life-threatening, systemic infections. In contrast, nonvascular pathogens remain restricted to infection sites, triggering localized symptom development. The contrasting features of vascular and nonvascular diseases suggest distinct etiologies, but the basis for each remains unclear. Here, we show that the hydrolase CbsA acts as a phenotypic switch between vascular and nonvascular plant pathogenesis. cbsA was enriched in genomes of vascular phytopathogenic bacteria in the family Xanthomonadaceae and absent in most nonvascular species. CbsA expression allowed nonvascular Xanthomonas to cause vascular blight, while cbsA mutagenesis resulted in reduction of vascular or enhanced nonvascular symptom development. Phylogenetic hypothesis testing further revealed that cbsA was lost in multiple nonvascular lineages and more recently gained by some vascular subgroups, suggesting that vascular pathogenesis is ancestral. Our results overall demonstrate how the gain and loss of single loci can facilitate the evolution of complex ecological traits.

17.
Metab Eng ; 62: 260-274, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32979486

RESUMO

Microbial terephthalic acid (TPA) catabolic pathways are conserved among the few bacteria known to turnover this xenobiotic aromatic compound. However, to date there are few reported cases in which this pathway has been successfully expressed in heterologous hosts to impart efficient utilization of TPA as a sole carbon source. In this work, we aimed to engineer TPA conversion in Acinetobacter baylyi ADP1 via the heterologous expression of catabolic and transporter genes from a native TPA-utilizing bacterium. Specifically, we obtained ADP1-derived strains capable of growing on TPA as the sole carbon source using chromosomal insertion and targeted amplification of the tph catabolic operon from Comamonas sp. E6. Adaptive laboratory evolution was then used to improve growth on this substrate. TPA consumption rates of the evolved strains, which retained multiple copies of the tph genes, were ~0.2 g/L/h (or ~1 g TPA/g cells/h), similar to that of Comamonas sp. E6 and almost 2-fold higher than that of Rhodococcus jostii RHA1, another native TPA-utilizing strain. To evaluate TPA transport in the evolved ADP1 strains, we engineered a TPA biosensor consisting of the transcription factor TphR and a fluorescent reporter. In combination with whole-genome sequencing, the TPA biosensor revealed that transport of TPA was not mediated by the heterologous proteins from Comamonas sp. E6. Instead, the endogenous ADP1 muconate transporter MucK, a member of the major facilitator superfamily, was responsible for TPA transport in several evolved strains in which MucK variants were found to enhance TPA uptake. Furthermore, the IclR-type transcriptional regulator DcaS was identified as a repressor of mucK expression. Overall, this work presents an unexpected function of a native protein identified through gene amplification, adaptive laboratory evolution, and a combination of screening methods. This study also provides a TPA biosensor for application in ADP1 and identifies transporter variants for use in metabolic engineering applications focused on plastic upcycling of polyesters.

18.
Proc Natl Acad Sci U S A ; 117(41): 25771-25778, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-32989155

RESUMO

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 GcoAEP4'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.


Assuntos
Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Guaiacol/metabolismo , Lignina/metabolismo , Rhodococcus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Biodegradação Ambiental , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Guaiacol/química , Cinética , Lignina/química , Rhodococcus/química , Rhodococcus/genética , Rhodococcus/metabolismo , Especificidade por Substrato
19.
Proc Natl Acad Sci U S A ; 117(41): 25476-25485, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-32989159

RESUMO

Plastics pollution represents a global environmental crisis. In response, microbes are evolving the capacity to utilize synthetic polymers as carbon and energy sources. Recently, Ideonella sakaiensis was reported to secrete a two-enzyme system to deconstruct polyethylene terephthalate (PET) to its constituent monomers. Specifically, the I. sakaiensis PETase depolymerizes PET, liberating soluble products, including mono(2-hydroxyethyl) terephthalate (MHET), which is cleaved to terephthalic acid and ethylene glycol by MHETase. Here, we report a 1.6 Å resolution MHETase structure, illustrating that the MHETase core domain is similar to PETase, capped by a lid domain. Simulations of the catalytic itinerary predict that MHETase follows the canonical two-step serine hydrolase mechanism. Bioinformatics analysis suggests that MHETase evolved from ferulic acid esterases, and two homologous enzymes are shown to exhibit MHET turnover. Analysis of the two homologous enzymes and the MHETase S131G mutant demonstrates the importance of this residue for accommodation of MHET in the active site. We also demonstrate that the MHETase lid is crucial for hydrolysis of MHET and, furthermore, that MHETase does not turnover mono(2-hydroxyethyl)-furanoate or mono(2-hydroxyethyl)-isophthalate. A highly synergistic relationship between PETase and MHETase was observed for the conversion of amorphous PET film to monomers across all nonzero MHETase concentrations tested. Finally, we compare the performance of MHETase:PETase chimeric proteins of varying linker lengths, which all exhibit improved PET and MHET turnover relative to the free enzymes. Together, these results offer insights into the two-enzyme PET depolymerization system and will inform future efforts in the biological deconstruction and upcycling of mixed plastics.


Assuntos
Proteínas de Bactérias/metabolismo , Burkholderiales/enzimologia , Plásticos/metabolismo , Engenharia de Proteínas/métodos , Modelos Moleculares , Mutação , Plásticos/química , Polietilenotereftalatos/química , Polietilenotereftalatos/metabolismo , Conformação Proteica , Domínios Proteicos , Especificidade por Substrato
20.
Metab Eng Commun ; 11: e00143, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32963959

RESUMO

Pseudomonas putida KT2440 is a promising bacterial chassis for the conversion of lignin-derived aromatic compound mixtures to biofuels and bioproducts. Despite the inherent robustness of this strain, further improvements to aromatic catabolism and toxicity tolerance of P. putida will be required to achieve industrial relevance. Here, tolerance adaptive laboratory evolution (TALE) was employed with increasing concentrations of the hydroxycinnamic acids p-coumaric acid (pCA) and ferulic acid (FA) individually and in combination (pCA â€‹+ â€‹FA). The TALE experiments led to evolved P. putida strains with increased tolerance to the targeted acids as compared to wild type. Specifically, a 37 â€‹h decrease in lag phase in 20 â€‹g/L pCA and a 2.4-fold increase in growth rate in 30 â€‹g/L FA was observed. Whole genome sequencing of intermediate and endpoint evolved P. putida populations revealed several expected and non-intuitive genetic targets underlying these aromatic catabolic and toxicity tolerance enhancements. PP_3350 and ttgB were among the most frequently mutated genes, and the beneficial contributions of these mutations were verified via gene knockouts. Deletion of PP_3350, encoding a hypothetical protein, recapitulated improved toxicity tolerance to high concentrations of pCA, but not an improved growth rate in high concentrations of FA. Deletion of ttgB, part of the TtgABC efflux pump, severely inhibited growth in pCA â€‹+ â€‹FA TALE-derived strains but did not affect growth in pCA â€‹+ â€‹FA in a wild type background, suggesting epistatic interactions. Genes involved in flagellar movement and transcriptional regulation were often mutated in the TALE experiments on multiple substrates, reinforcing ideas of a minimal and deregulated cell as optimal for domesticated growth. Overall, this work demonstrates increased tolerance towards and growth rate at the expense of hydroxycinnamic acids and presents new targets for improving P. putida for microbial lignin valorization.

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