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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.

Summary To drive innovation in chemical and material applications beyond what has been afforded by the mature petrochemical industry, new molecules that possess diverse chemical functionality are needed. One source of such molecules lies in the varied metabolic pathways that soil microbes utilize to catabolize aromatic compounds generated during plant decomposition. Here, we have engineered Pseudomonas putida KT2440 to convert these aromatic compounds to 15 catabolic intermediates that exhibit substantial chemical diversity. Bioreactor cultivations, analytical methods, and bench-scale separations were developed to enable production (up to 58 g/L), detection, and purification of each target molecule. We further engineered strains for production of a subset of these molecules from glucose, achieving a 41% molar yield of muconic acid. Finally, we produce materials from three compounds to illustrate the potential for realizing performance-advantaged properties relative to petroleum-derived analogs.

Product inhibition is a frequent bottleneck in industrial enzymes, and testing mutations to alleviate product inhibition via traditional methods remains challenging as many variants need to be tested against multiple substrate and product concentrations. Further, traditional screening methods are conducted in vitro, and resulting enzyme variants may perform differently in vivo in the context of whole-cell metabolism and regulation. In this study, we address these two problems by establishing a high-throughput screening method to alleviate product inhibition in an industrially relevant enzyme, chorismate pyruvate-lyase (UbiC). First, we engineered a highly specific, genetically encoded biosensor for 4-hydroxybenzoate (4HB) in an industrially relevant host, Pseudomonas putida KT2440. We subsequently applied the biosensor to detect the activity of a heterologously expressed UbiC that converts chorismate into 4HB and pyruvate. By using benzoate as a product surrogate that inhibits UbiC without activating the biosensor, we were able to efficiently create and screen a diversified library for UbiC variants with reduced product inhibition. Introduction of the improved UbiC enzyme variant into an experimental production strain for the industrial precursor cis,cis-muconic acid (muconate), enabled a >2-fold yield improvement for glucose to muconate conversion when the new UbiC variant was expressed from a plasmid and a 60% yield increase when the same UbiC variant was genomically integrated into the strain. Overall, this work demonstrates that by coupling a library of enzyme variants to whole-cell catalysis and biosensing, variants with reduced product inhibition can be identified, and that this improved enzyme can result in increased titers of a downstream molecule of interest.

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.

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, β-ketoadipic acid (βKA) by deletion of pcaIJ. Using this strain, we demonstrate production of 15.1 g/L βKA from BHET at 76% molar yield in bioreactors and conversion of catalytically depolymerized PET to βKA. Overall, this work highlights the potential of tandem catalytic deconstruction and biological conversion as a means to upcycle waste PET.

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.

Significance Deconstruction of recalcitrant polymers, such as cellulose or chitin, is accomplished in nature by synergistic enzyme cocktails that evolved over millions of years. In these systems, soluble dimeric or oligomeric intermediates are typically released via interfacial biocatalysis, and additional enzymes often process the soluble intermediates into monomers for microbial uptake. The recent discovery of a two-enzyme system for polyethylene terephthalate (PET) deconstruction, which employs one enzyme to convert the polymer into soluble intermediates and another enzyme to produce the constituent PET monomers (MHETase), suggests that nature may be evolving similar deconstruction strategies for synthetic plastics. This study on the characterization of the MHETase enzyme and synergy of the two-enzyme PET depolymerization system may inform enzyme cocktail-based strategies for plastics upcycling., 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.

Experimental evolution is a critical tool in many disciplines, including metabolic engineering and synthetic biology. However, current methods rely on the chance occurrence of a key step that can dramatically accelerate evolution in natural systems, namely increased gene dosage. Our studies sought to induce the targeted amplification of chromosomal segments to facilitate rapid evolution. Since increased gene dosage confers novel phenotypes and genetic redundancy, we developed a method, Evolution by Amplification and Synthetic Biology (EASy), to create tandem arrays of chromosomal regions. In Acinetobacter baylyi, EASy was demonstrated on an important bioenergy problem, the catabolism of lignin-derived aromatic compounds. The initial focus on guaiacol (2-methoxyphenol), a common lignin degradation product, led to the discovery of Amycolatopsis genes (gcoAB) encoding a cytochrome P450 enzyme that converts guaiacol to catechol. However, chromosomal integration of gcoAB in Pseudomonas putida or A. baylyi did not enable guaiacol to be used as the sole carbon source despite catechol being a growth substrate. In ∼1,000 generations, EASy yielded alleles that in single chromosomal copy confer growth on guaiacol. Different variants emerged, including fusions between GcoA and CatA (catechol 1,2-dioxygenase). This study illustrates the power of harnessing chromosomal gene amplification to accelerate the evolution of desirable traits.

Robust fluorescence-based biosensors are emerging as critical tools for high-throughput strain improvement in synthetic biology. Many biosensors are developed in model organisms where sophisticated synthetic biology tools are also well established. However, industrial biochemical production often employs microbes with phenotypes that are advantageous for a target process, and biosensors may fail to directly transition outside the host in which they are developed. In particular, losses in sensitivity and dynamic range of sensing often occur, limiting the application of a biosensor across hosts. Here we demonstrate the optimization of an Escherichia coli-based biosensor in a robust microbial strain for the catabolism of aromatic compounds, Pseudomonas putida KT2440, through a generalizable approach of modulating interactions at the protein-DNA interface in the promoter and the protein-protein dimer interface. The high-throughput biosensor optimization approach demonstrated here is readily applicable towards other allosteric regulators. Keywords: Whole cell biosensor, Aromatic catabolism, Transcription factor, PcaU, Shikimate

Muconic acid (MA) is a bio-based platform chemical that can be converted into the commodity petrochemical building blocks adipic acid or terephthalic acid, or used in emerging, performance-advantaged materials. MA is a metabolic intermediate in the β-ketoadipate pathway, and can be produced from carbohydrates or other traditional carbon sources via the shikimate pathway. MA can also be produced from lignin-derived aromatic compounds with high atom efficiency through aromatic-catabolic pathways. Metabolic engineering efforts to date have developed efficient muconic acid-producing strains of the aromatic-catabolic microbe Pseudomonas putida KT2440, but the titers, productivities, and yields from aromatic compounds in most cases remain below the thresholds needed for industrially-relevant bioreactor cultivations. To that end, this work presents further process and host development towards improving MA titers, yields, and productivities, using the hydroxycinnamic acids, p-coumaric acid and ferulic acid, as model aromatic compounds. Coupling strain engineering and bioprocess development enabled the discovery of new bottlenecks in P. putida that hinder MA production from these compounds. A combination of gene overexpression and removal of a global catabolic regulator resulted in high-yielding strains (100% molar yield). Maximum MA titers of 50 g L−1, which is near the lethal toxicity limit in this bacterium, and productivities over 0.5 g L−1 h−1 were achieved in separate process configurations. Additionally, a high-pH feeding strategy, which could potentially reduce the salt load and enable higher titers by decreasing product dilution, was tested with model compounds and lignin-rich streams from corn stover and a complete conversion of the primary monomeric aromatic compounds to MA was demonstrated, obtaining a titer of 4 g L−1. Overall, this study presents a step forward for the production of value-added chemicals from lignin and highlights critical needs for further strain improvement and bioprocess development that can be applied in the biological valorization of lignin.

The sugar dehydration products, furfural and 5-(hydroxymethyl)furfural (HMF), are commonly formed during high-temperature processing of lignocellulose, most often in thermochemical pretreatment, liquefaction, or pyrolysis. Typically, these two aldehydes are considered major inhibitors in microbial conversion processes. Many microbes can convert these compounds to their less toxic, dead-end alcohol counterparts, furfuryl alcohol and 5-(hydroxymethyl)furfuryl alcohol. Recently, the genes responsible for aerobic catabolism of furfural and HMF were discovered in Cupriavidus basilensis HMF14 to enable complete conversion of these compounds to the TCA cycle intermediate, 2-oxo-glutarate. In this work, we engineer the robust soil microbe, Pseudomonas putida KT2440, to utilize furfural and HMF as sole carbon and energy sources via complete genomic integration of the 12 kB hmf gene cluster previously reported from Burkholderia phytofirmans. The common intermediate, 2-furoic acid, is shown to be a bottleneck for both furfural and HMF metabolism. When cultured on biomass hydrolysate containing representative amounts of furfural and HMF from dilute-acid pretreatment, the engineered strain outperforms the wild type microbe in terms of reduced lag time and enhanced growth rates due to catabolism of furfural and HMF. Overall, this study demonstrates that an approach for biological conversion of furfural and HMF, relative to the typical production of dead-end alcohols, enables both enhanced carbon conversion and substantially improves tolerance to hydrolysate inhibitors. This approach should find general utility both in emerging aerobic processes for the production of fuels and chemicals from biomass-derived sugars and in the biological conversion of high-temperature biomass streams from liquefaction or pyrolysis where furfural and HMF are much more abundant than in biomass hydrolysates from pretreatment., Highlights • HMF and furfural are common microbial inhibitors in biomass conversion. • HMF and furfural gene cluster was isolated from Burkholderia phytofirmans.. • We heterologously express the HMF/furfural gene cluster in Pseudomonas putida.. • Expression enables cultivation on HMF and furfural as a sole carbon source. • Expression also enables enhanced conversion on lignocellulosic hydrolysate.

Carbon catabolite repression refers to the preference of microbes to metabolize certain growth substrates over others in response to a variety of regulatory mechanisms. Such preferences are important for the fitness of organisms in their natural environments, but may hinder their performance as domesticated microbial cell factories. In a Pseudomonas putida KT2440 strain engineered to convert lignin-derived aromatic monomers such as p-coumarate and ferulate to muconate, a precursor to bio-based nylon and other chemicals, metabolic intermediates including 4-hydroxybenzoate and vanillate accumulate and subsequently reduce productivity. We hypothesized that these metabolic bottlenecks may be, at least in part, the effect of carbon catabolite repression caused by glucose or acetate, more preferred substrates that must be provided to the strain for supplementary energy and cell growth. Using mass spectrometry-based proteomics, we have identified the 4-hydroxybenzoate hydroxylase, PobA, and the vanillate demethylase, VanAB, as targets of the Catabolite Repression Control (Crc) protein, a global regulator of carbon catabolite repression. By deleting the gene encoding Crc from this strain, the accumulation of 4-hydroxybenzoate and vanillate are reduced and, as a result, muconate production is enhanced. In cultures grown on glucose, the yield of muconate produced from p-coumarate after 36 h was increased nearly 70% with deletion of the gene encoding Crc (94.6 ± 0.6% vs. 56.0 ± 3.0% (mol/mol)) while the yield from ferulate after 72 h was more than doubled (28.3 ± 3.3% vs. 12.0 ± 2.3% (mol/mol)). The effect of eliminating Crc was similar in cultures grown on acetate, with the yield from p-coumarate just slightly higher in the Crc deletion strain after 24 h (47.7 ± 0.6% vs. 40.7 ± 3.6% (mol/mol)) and the yield from ferulate increased more than 60% after 72 h (16.9 ± 1.4% vs. 10.3 ± 0.1% (mol/mol)). These results are an example of the benefit that reducing carbon catabolite repression can have on conversion of complex feedstocks by microbial cell factories, a concept we posit could be broadly considered as a strategy in metabolic engineering for conversion of renewable feedstocks to value-added chemicals., Highlights • Crc is a global regulator of carbon catabolite repression in pseudomonads. • The gene encoding Crc was deleted from muconate a producing P. putida strain. • Based on our proteomics data, expression of PobA and VanAB are regulated by Crc. • Deleting Crc improved conversion to muconate in the presence of glucose or acetate. • This may be a useful strategy toward developing pseudomonad cell factories.

SummaryThe biophysical and biochemical properties of membrane lipids can alter the conformation and function of membrane-spanning proteins, yet the specific, physiological consequence in vivo of changing the membrane milieu for a specific protein has been rarely investigated. Using various genetic approaches to eliminate expression of the membrane-associated protein Flotillin-1, we have found that the lipid environment of the dopamine transporter (DAT) is necessary for mice to respond to amphetamine but not cocaine, because the localization of DAT to cholesterol-rich membranes is required for a DAT conformation that is essential for reverse transport of dopamine. Furthermore, a conditional rather than constitutive loss-of-function approach was necessary to reveal this phenotype, indicating a broader role for membrane-protein interactions that are modulated by Flotillin-1. Taken together, these findings demonstrate how interaction of a transmembrane protein with its membrane environment can regulate distinct events in the vertebrate brain that give rise to specific behavioral outcomes.

Mammals maintain a nearly constant core body temperature (T(b)) by balancing heat production and heat dissipation. This comes at a high metabolic cost which is sustainable if adequate calorie intake is maintained. When nutrients are scarce or experimentally reduced such as during calorie restriction (CR), endotherms can reduce energy expenditure by lowering T(b) [1–6]. This adaptive response conserves energy, limiting the loss of body weight due to low calorie intake [7–10]. Here we show that this response is regulated by the kappa opioid receptor (KOR). CR associated with increased hypothalamic levels of the endogenous opioid leu-enkephalin; which is derived from the KOR agonist precursor, dynorphin [11]. Pharmacological inhibition of KOR, but not of the delta or the mu opioid receptor subtypes, fully blocked CR-induced hypothermia and increased weight loss during CR independently of calorie intake. Similar results were seen with diet-induced obese mice subjected to CR. In contrast, inhibiting KOR did not change T(b) in animals fed ad libitum. Chemogenetic inhibition of KOR neurons in the hypothalamic preoptic area reduced the CR-induced hypothermia, while chemogenetic activation of prodynorphin-expressing neurons in the arcuate or the parabrachial nuclei lowered T(b). These data indicate that KOR signaling is a pivotal regulator of energy homeostasis and can affect body weight during dieting by modulating T(b) and energy expenditure.

Significance Lignin is an abundant but underutilized heterogeneous polymer found in terrestrial plants. In current lignocellulosic biorefinery paradigms, lignin is primarily slated for incineration, but for a nonfood plant-based bioeconomy to be successful, lignin valorization is critical. An emerging concept to valorize lignin uses aromatic–catabolic pathways and microbes to funnel heterogeneous lignin-derived aromatic compounds to single high-value products. For this approach to be viable, the discovery and engineering of enzymes to conduct key reactions is critical. In this work, we have engineered a two-component cytochrome P450 enzyme system to conduct one of the most important reactions in biological lignin conversion: aromatic O-demethylation of syringol, the base aromatic unit of S-lignin, which is highly abundant in hardwoods and grasses., Microbial conversion of aromatic compounds is an emerging and promising strategy for valorization of the plant biopolymer lignin. A critical and often rate-limiting reaction in aromatic catabolism is O-aryl-demethylation of the abundant aromatic methoxy groups in lignin to form diols, which enables subsequent oxidative aromatic ring-opening. Recently, a cytochrome P450 system, GcoAB, was discovered to demethylate guaiacol (2-methoxyphenol), which can be produced from coniferyl alcohol-derived lignin, to form catechol. However, native GcoAB has minimal ability to demethylate syringol (2,6-dimethoxyphenol), the analogous compound that can be produced from sinapyl alcohol-derived lignin. Despite the abundance of sinapyl alcohol-based lignin in plants, no pathway for syringol catabolism has been reported to date. Here we used structure-guided protein engineering to enable microbial syringol utilization with GcoAB. Specifically, a phenylalanine residue (GcoA-F169) interferes with the binding of syringol in the active site, and on mutation to smaller amino acids, efficient syringol O-demethylation is achieved. Crystallography indicates that syringol adopts a productive binding pose in the variant, which molecular dynamics simulations trace to the elimination of steric clash between the highly flexible side chain of GcoA-F169 and the additional methoxy group of syringol. Finally, we demonstrate in vivo syringol turnover in Pseudomonas putida KT2440 with the GcoA-F169A variant. Taken together, our findings highlight the significant potential and plasticity of cytochrome P450 aromatic O-demethylases in the biological conversion of lignin-derived aromatic compounds.

Correction for ‘Thermochemical wastewater valorization via enhanced microbial toxicity tolerance’ by Lahiru N. Jayakody et al., Energy Environ. Sci., 2018, 11, 1625–1638, DOI: 10.1039/C8EE00460A.

Summary: Hot flushes are a sudden feeling of warmth commonly associated with the decline of gonadal hormones at menopause. Neurons in the arcuate nucleus of the hypothalamus that express kisspeptin and neurokinin B (Kiss1ARH neurons) are candidates for mediating hot flushes because they are negatively regulated by sex hormones. We used a combination of genetic and viral technologies in mice to demonstrate that artificial activation of Kiss1ARH neurons evokes a heat-dissipation response resulting in vasodilation (flushing) and a corresponding reduction of core-body temperature in both females and males. This response is sensitized by ovariectomy. Brief activation of Kiss1ARH axon terminals in the preoptic area of the hypothalamus recapitulates this response, while pharmacological blockade of neurokinin B (NkB) receptors in the same brain region abolishes it. We conclude that transient activation of Kiss1ARH neurons following sex-hormone withdrawal contributes to the occurrence of hot flushes via NkB release in the rostral preoptic area. : The underlying cause of hot flushes is poorly understood. Padilla et al. provide evidence that a subpopulation of hypothalamic neurons can generate hot flush symptoms in mice. Establishing the mechanism of hot flush generation may allow for the development of therapies. Keywords: hot flashes, temperature regulation, neurokinin B, kisspeptin, menopause, estrogen, optogenetics, chemogenetics

Microbial aromatic catabolism offers a promising approach to convert lignin, a vast source of renewable carbon, into useful products. Aryl-O-demethylation is an essential biochemical reaction to ultimately catabolize coniferyl and sinapyl lignin-derived aromatic compounds, and is often a key bottleneck for both native and engineered bioconversion pathways. Here, we report the comprehensive characterization of a promiscuous P450 aryl-O-demethylase, consisting of a cytochrome P450 protein from the family CYP255A (GcoA) and a three-domain reductase (GcoB) that together represent a new two-component P450 class. Though originally described as converting guaiacol to catechol, we show that this system efficiently demethylates both guaiacol and an unexpectedly wide variety of lignin-relevant monomers. Structural, biochemical, and computational studies of this novel two-component system elucidate the mechanism of its broad substrate specificity, presenting it as a new tool for a critical step in biological lignin conversion., Catabolizing lignin-derived aromatic compounds requires an aryl-O-demethylation step. Here the authors present the structures of GcoA and GcoB, a cytochrome P450-reductase pair that catalyzes aryl-O-demethylations and show that GcoA displays broad substrate specificity, which is of interest for biotechnology applications.

Significance Synthetic polymers are ubiquitous in the modern world but pose a global environmental problem. While plastics such as poly(ethylene terephthalate) (PET) are highly versatile, their resistance to natural degradation presents a serious, growing risk to fauna and flora, particularly in marine environments. Here, we have characterized the 3D structure of a newly discovered enzyme that can digest highly crystalline PET, the primary material used in the manufacture of single-use plastic beverage bottles, in some clothing, and in carpets. We engineer this enzyme for improved PET degradation capacity and further demonstrate that it can also degrade an important PET replacement, polyethylene-2,5-furandicarboxylate, providing new opportunities for biobased plastics recycling., Poly(ethylene terephthalate) (PET) is one of the most abundantly produced synthetic polymers and is accumulating in the environment at a staggering rate as discarded packaging and textiles. The properties that make PET so useful also endow it with an alarming resistance to biodegradation, likely lasting centuries in the environment. Our collective reliance on PET and other plastics means that this buildup will continue unless solutions are found. Recently, a newly discovered bacterium, Ideonella sakaiensis 201-F6, was shown to exhibit the rare ability to grow on PET as a major carbon and energy source. Central to its PET biodegradation capability is a secreted PETase (PET-digesting enzyme). Here, we present a 0.92 Å resolution X-ray crystal structure of PETase, which reveals features common to both cutinases and lipases. PETase retains the ancestral α/β-hydrolase fold but exhibits a more open active-site cleft than homologous cutinases. By narrowing the binding cleft via mutation of two active-site residues to conserved amino acids in cutinases, we surprisingly observe improved PET degradation, suggesting that PETase is not fully optimized for crystalline PET degradation, despite presumably evolving in a PET-rich environment. Additionally, we show that PETase degrades another semiaromatic polyester, polyethylene-2,5-furandicarboxylate (PEF), which is an emerging, bioderived PET replacement with improved barrier properties. In contrast, PETase does not degrade aliphatic polyesters, suggesting that it is generally an aromatic polyesterase. These findings suggest that additional protein engineering to increase PETase performance is realistic and highlight the need for further developments of structure/activity relationships for biodegradation of synthetic polyesters.

cis,cis-Muconic acid is a polyunsaturated dicarboxylic acid that can be produced renewably via the biological conversion of sugars and lignin-derived aromatic compounds. Subsequently, muconic acid can be catalytically converted to adipic acid – the most commercially significant dicarboxylic acid manufactured from petroleum. Nylon-6,6 is the major industrial application for adipic acid, consuming 85% of market demand; however, high purity adipic acid (99.8%) is required for polymer synthesis. As such, process technologies are needed to effectively separate and catalytically transform biologically derived muconic acid to adipic acid in high purity over stable catalytic materials. To that end, this study: (1) demonstrates bioreactor production of muconate at 34.5 g L−1 in an engineered strain of Pseudomonas putida KT2440, (2) examines the staged recovery of muconic acid from culture media, (3) screens platinum group metals (e.g., Pd, Pt, Rh, Ru) for activity and leaching stability on activated carbon (AC) and silica supports, (4) evaluates the time-on-stream performance of Rh/AC in a trickle bed reactor, and (5) demonstrates the polymerization of bio-adipic acid to nylon-6,6. Separation experiments confirmed AC effectively removed broth color compounds, but subsequent pH/temperature shift crystallization resulted in significant levels of Na, P, K, S and N in the crystallized product. Ethanol dissolution of muconic acid precipitated bulk salts, achieving a purity of 99.8%. Batch catalysis screening reactions determined that Rh and Pd were both highly active compared to Pt and Ru, but Pd leached significantly (1–9%) from both AC and silica supports. Testing of Rh/AC in a continuous trickle bed reactor for 100 h confirmed stable performance after 24 h, although organic adsorption resulted in reduced steady-state activity. Lastly, polymerization of bio-adipic acid with hexamethyldiamine produced nylon-6,6 with comparable properties to its petrochemical counterpart, thereby demonstrating a path towards bio-based nylon production via muconic acid.

Lignin represents an untapped feedstock for the production of fuels and chemicals, but its intrinsic heterogeneity makes lignin valorization a significant challenge. In nature, many aerobic organisms degrade lignin-derived aromatic molecules through conserved central intermediates including catechol and protocatechuate. Harnessing this microbial approach offers potential for lignin upgrading in modern biorefineries, but significant technical development is needed to achieve this end. Catechol and protocatechuate are subjected to aromatic ring cleavage by dioxygenase enzymes that, depending on the position, ortho or meta relative to adjacent hydroxyl groups, result in different products that are metabolized through parallel pathways for entry into the TCA cycle. These degradation pathways differ in the combination of succinate, acetyl-CoA, and pyruvate produced, the reducing equivalents regenerated, and the amount of carbon emitted as CO2-factors that will ultimately impact the yield of the targeted product. As shown here, the ring-cleavage pathways can be interchanged with one another, and such substitutions have a predictable and substantial impact on product yield. We demonstrate that replacement of the catechol ortho degradation pathway endogenous to Pseudomonas putida KT2440 with an exogenous meta-cleavage pathway from P. putida mt-2 increases yields of pyruvate produced from aromatic molecules in engineered strains. Even more dramatically, replacing the endogenous protocatechuate ortho pathway with a meta-cleavage pathway from Sphingobium sp. SYK-6 results in a nearly five-fold increase in pyruvate production. We further demonstrate the aerobic conversion of pyruvate to l-lactate with a yield of 41.1 ± 2.6% (wt/wt). Overall, this study illustrates how aromatic degradation pathways can be tuned to optimize the yield of a desired product in biological lignin upgrading.

Lignin is an alkyl-aromatic polymer present in plant cell walls for defense, structure, and water transport. Despite exhibiting a high-energy content, lignin is typically slated for combustion in modern biorefineries due to its inherent heterogeneity and recalcitrance, whereas cellulose and hemicellulose are converted to renewable fuels and chemicals. However, it is critical for the viability of third-generation biorefineries to valorize lignin alongside polysaccharides. To that end, we employ metabolic engineering, separations, and catalysis to convert lignin-derived species into cis,cis-muconic acid, for subsequent hydrogenation to adipic acid, the latter being the most widely produced dicarboxylic acid. First, Pseudomonas putida KT2440 was metabolically engineered to funnel lignin-derived aromatics to cis,cis-muconate, which is an atom-efficient biochemical transformation. This engineered strain was employed in fed-batch biological cultivation to demonstrate a cis,cis-muconate titer of 13.5 g L−1 in 78.5 h from a model lignin-derived compound. cis,cis-Muconic acid was recovered in high purity (>97%) and yield (74%) by activated carbon treatment and crystallization (5 °C, pH 2). Pd/C was identified as a highly active catalyst for cis,cis-muconic acid hydrogenation to adipic acid with high conversion (>97%) and selectivity (>97%). Under surface reaction controlling conditions (24 °C, 24 bar, ethanol solvent), purified cis,cis-muconic acid exhibits a turnover frequency of 23–30 s−1 over Pd/C, with an apparent activation energy of 70 kJ mol−1. Lastly, cis,cis-muconate was produced with engineered P. putida grown on a biomass-derived, lignin-enriched stream, demonstrating an integrated strategy towards lignin valorization to an important commodity chemical.

Lignin is an energy-dense, heterogeneous polymer comprised of phenylpropanoid monomers used by plants for structure, water transport, and defense, and it is the second most abundant biopolymer on Earth after cellulose. In production of fuels and chemicals from biomass, lignin is typically underused as a feedstock and burned for process heat because its inherent heterogeneity and recalcitrance make it difficult to selectively valorize. In nature, however, some organisms have evolved metabolic pathways that enable the utilization of lignin-derived aromatic molecules as carbon sources. Aromatic catabolism typically occurs via upper pathways that act as a "biological funnel" to convert heterogeneous substrates to central intermediates, such as protocatechuate or catechol. These intermediates undergo ring cleavage and are further converted via the β-ketoadipate pathway to central carbon metabolism. Here, we use a natural aromatic-catabolizing organism, Pseudomonas putida KT2440, to demonstrate that these aromatic metabolic pathways can be used to convert both aromatic model compounds and heterogeneous, lignin-enriched streams derived from pilot-scale biomass pretreatment into medium chain-length polyhydroxyalkanoates (mcl-PHAs). mcl-PHAs were then isolated from the cells and demonstrated to be similar in physicochemical properties to conventional carbohydrate-derived mcl-PHAs, which have applications as bioplastics. In a further demonstration of their utility, mcl-PHAs were catalytically converted to both chemical precursors and fuel-range hydrocarbons. Overall, this work demonstrates that the use of aromatic catabolic pathways enables an approach to valorize lignin by overcoming its inherent heterogeneity to produce fuels, chemicals, and materials.

Lipidomic analysis of the complex mixture of lipids isolated from biological systems can be a challenging process that often involves tandem mass spectrometry and interpretation of both precursor ions and product ions relative to the molecular structure of the lipids. Therefore, detailed understanding of the gas-phase ion chemistry occurring for each class of phospholipids is critically important for an accurate assignment of lipid structure. Some oxidized phosphatidylcholines are known to be biologically active and responsible for pathological events, and are therefore important targets for detection in lipidomic studies. Modification of fatty acyl chains by oxidation may, however, change the behavior of ion formation and decomposition in the mass spectrometer. In this study, we report on the mass-spectrometric behavior of 1-palmitoyl-2-(9'-oxononanoyl)-sn-glycero-3-phosphocholine, a bioactive product of phosphatidylcholine oxidation. In addition to [M-15](-) and the acetate adduct [M+59](-), three additional adduct ions, including [M-H](-), were present in significant abundance in the negative ion electrospray mass spectrum. It was found that this unexpected [M-H](-) ion was formed by the transfer of a methyl group from the choline residue on the polar head group to the aldehyde functionality of the sn-2 substituent, resulting in a 14-Da increase in the mass of the resulting sn-2 carboxylate anion formed by collisional activation of this ion. These results suggest additional rules for understanding the gas-phase ion chemistry of aldehydic phosphatidylcholine molecular species.

Platelet-activating factor (PAF) is an early product of the inflammatory environment, influencing development and resolution of inflammation. Its production is greater in neutrophils and macrophages, which predominantly synthesize 1-alkyl sn-2 acetyl glycerophosphocholine (GPC) than in non-granulocytes (B cells and endothelial cells), which lack a respiratory burst and synthesize 1-acyl sn-2 acetyl GPC as their major PAF species. This study investigated whether the respiratory burst was responsible for the quantitative and qualitative differences in sn-2 acetyl GPC species generation by neutrophils and macrophages versus those cells lacking the NADPH oxidase complex. The myeloid cell line PLB-985 (capable of differentiation into neutrophils) was used to test this hypothesis, since these cells had previously been generated with a non-functional respiratory burst (X-CGD PLB-985). Differentiated PLB-985 cells underwent a large respiratory burst in response to PMA (phorbol ester), and smaller respiratory bursts in response to A23187 (calcium ionophore), and the bacterial polypeptide fMLP (receptor mediated activation). Concurrently, treated cells were assessed for production of 1-hexadecyl and 1-palmitoyl sn-2 acetyl GPC species by gas chromatography/mass spectrometry. Neither cell type generated these lipid species in response to PMA, but both cell types generated equal levels of sn-2 acetyl GPC in response to A23187, with five times more 1-hexadecyl than 1-palmitoyl species. Upon fMLP activation, X-CGD PLB-985 cells produced significantly less 1-hexadecyl and 1-palmitoyl sn-2 acetyl GPC in comparison to the wild-type PLB-985 cells. These findings suggest phagocytic oxidant production by NADPH oxidase is not essential for sn-2 acetyl GPC generation, but appears important for optimal production of PAF in response to some stimuli.

Invertebrate and vertebrate vestigial (vg) and vestigial-like (VGLL) genes are involved in embryonic patterning and cell fate determination. These genes encode cofactors that interact with members of the Scalloped/TEAD family of transcription factors and modulate their activity. We have previously shown that, in mice, Vgll2 is differentially expressed in the developing facial prominences. In this study, we show that the zebrafish ortholog vgll2a is expressed in the pharyngeal endoderm and ectoderm surrounding the neural crest derived mesenchyme of the pharyngeal arches. Moreover, both the FGF and retinoic acid (RA) signaling pathways, which are critical components of the hierarchy controlling craniofacial patterning, regulate this domain of vgll2a expression. Consistent with these observations, vgll2a is required within the pharyngeal endoderm for NCC survival and pharyngeal cartilage development. Specifically, knockdown of Vgll2a in zebrafish embryos using Morpholino injection results in increased cell death within the pharyngeal arches, aberrant endodermal pouch morphogenesis, and hypoplastic cranial cartilages. Overall, our data reveal a novel non-cell autonomous role for Vgll2a in development of the NCC-derived vertebrate craniofacial skeleton.

Chronic granulomatous disease is the manifestation of genetic defects of the leukocyte NADPH oxidase resulting in the absence of a respiratory burst. Patients with chronic granulomatous disease can develop chronic granulomas in many locations of the body, including the skin. Using an established murine model of X-linked chronic granulomatous disease (X-CGD) created by homologous recombinant disruption of the gene encoding the gp91phox component of the NADPH oxidase, in this study we examined cutaneous reactivity to sterile Aspergillus fumigatus hyphae. Injection of Aspergillus fumigatus into the dorsal ears of X-CGD mice resulted in an enhanced inflammatory response by 24 h, consisting of neutrophils, which developed into suppurative granulomas by 10 d. Intradermal injection of Aspergillus fumigatus into wild-type mice only resulted in a transient inflammatory response that resolved by 10 d. Injection of Aspergillus fumigatus into female carrier mice resulted in an acute inflammatory response that was similar to that of wild-type mice, but, at higher doses of Aspergillus fumigatus, many carriers subsequently developed granulomatous lesions that were qualitatively similar but smaller than those seen in X-CGD mice by 30 d. Consistent with the ability of X-CGD mice to mount an enhanced neutrophil-rich inflammatory response to Aspergillus fumigatus, significant levels of the potent neutrophil activator/chemoattractant leukotriene B4 were measured by mass spectrometry in skin biopsies at 24 and 72 h. In contrast to the exaggerated inflammatory response to intradermal Aspergillus fumigatus in X-CGD mice compared to their wild-type counterparts, similar levels of inflammation were seen in a model of delayed-type hypersensitivity using 2,4-dinitrofluorobenzene. This study represents the first report of a cutaneous granuloma model in mice with X-CGD, which may also prove useful as a functional test to evaluate the efficacy of gene therapy protocols being developed for chronic granulomatous disease.

Unilateral ureteral obstruction (UUO) is characterized by decreases in renal function and increases in interstitial fibrosis. Previous studies have indicated that pharmacologic manipulations that increase nitric oxide (NO) are beneficial to the obstructed kidneys. NO is produced from arginine by nitric oxide synthase (NOS), an enzyme that exists in both constitutive and inducible (iNOS) forms. To determine the role of the inducible form of NOS in UUO, we used mice with a targeted deletion of iNOS (iNOS -/- mice) and compared them with wild-type (WT) mice. Kidneys were obstructed for 2 weeks in both WT and iNOS -/- mice, and were then removed and bisected. Half of the kidney was embedded in paraffin and tissue sections were examined for interstitial volume or the presence of macrophages. The remainder was flash-frozen and samples were used to measure tissue collagen (hydroxyproline) or transforming growth factor-beta (TGF-beta). This study demonstrates that both cortex and medulla of obstructed kidneys of iNOS -/- mice exhibit significantly increased interstitial volume and interstitial macrophages as compared with their WT counterparts. Furthermore tissue collagen was increased to 9.2+/-1.3 microg/mg tissue in WT obstructed kidneys, whereas in iNOS -/- kidneys, collagen was increased to 13.2+/-0.8 microg/mg tissue. The profibrotic cytokine TGF-beta was also significantly increased in obstructed kidneys of iNOS -/- mice, as compared with WT mice. No differences were noted between the unobstructed kidneys of iNOS -/- mice compared with WT mice in any of the parameters examined. These results demonstrate that targeted deletion of the iNOS results in exacerbation of fibrotic events in the obstructed kidney. These results confirm previous pharmacologic studies, and suggest that NO produced via the inducible NOS normally serves a protective function in UUO.

Evidence is accumulating suggesting that platelet-activating factor plays a role in inflammatory dermatoses. Mass spectrometric methods were used to examine the molecular species of sn-2 acetyl glycerophosphocholines (GPC) synthesized by primary cultures of human neonatal foreskin-derived keratinocytes. Ionophore-stimulated keratinocytes synthesize both 1-alkyl and 1-acyl sn-2 acetyl-GPC, and the relative amounts were as follows: hexadecyl > palmitoyl > octadecyl > stearoyl at the sn-1 position. PAF synthesis in the keratinocyte-derived cell line HaCaT was inhibited by dexamethasone, suggesting that the anti-inflammatory effects of glucocorticosteroids in inflammatory dermatoses might be in part related to the inhibition of the synthesis of mediators such as PAF.

Lignin is a primary component of lignocellulosic biomass that is an underutilized feedstock in the growing biofuels industry. Despite the fact that lignin depolymerization has long been studied, the intrinsic heterogeneity of lignin typically leads to heterogeneous streams of aromatic compounds, which in turn present significant technical challenges when attempting to produce lignin-derived chemicals where purity is often a concern. In Nature, microorganisms often encounter this same problem during biomass turnover wherein powerful oxidative enzymes produce heterogeneous slates of aromatics compounds. Some microbes have evolved metabolic pathways to convert these aromatic species via ‘upper pathways’ into central intermediates, which can then be funneled through ‘lower pathways’ into central carbon metabolism in a process we dubbed ‘biological funneling’. This funneling approach offers a direct, biological solution to overcome heterogeneity problems in lignin valorization for the modern biorefinery. Coupled to targeted separations and downstream chemical catalysis, this concept offers the ability to produce a wide range of molecules from lignin. This perspective describes research opportunities and challenges ahead for this new field of research, which holds significant promise towards a biorefinery concept wherein polysaccharides and lignin are treated as equally valuable feedstocks. In particular, we discuss tailoring the lignin substrate for microbial utilization, host selection for biological funneling, ligninolytic enzyme–microbe synergy, metabolic engineering, expanding substrate specificity for biological funneling, and process integration, each of which presents key challenges. Ultimately, for biological solutions to lignin valorization to be viable, multiple questions in each of these areas will need to be addressed, making biological lignin valorization a multidisciplinary, co-design problem.