Carbon dioxide valorization into resveratrol via lithoautotrophic fermentation using engineered Cupriavidus necator H16.

BACKGROUND: Industrial biomanufacturing of value-added products using CO2 as a carbon source is considered more sustainable, cost-effective and resource-efficient than using common carbohydrate feedstocks. Cupriavidus necator H16 is a representative H2-oxidizing lithoautotrophic bacterium that can be utilized to valorize CO2 into valuable chemicals and has recently gained much attention as a promising platform host for versatile C1-based biomanufacturing. Since this microbial platform is genetically tractable and has a high-flux carbon storage pathway, it has been engineered to produce a variety of valuable compounds from renewable carbon sources. In this study, the bacterium was engineered to produce resveratrol autotrophically using an artificial phenylpropanoid pathway. RESULTS: The heterologous genes involved in the resveratrol biosynthetic pathway-tyrosine ammonia lyase (TAL), 4-coumaroyl CoA ligase (4CL), and stilbene synthase (STS) -were implemented in C. necator H16. The overexpression of acetyl-CoA carboxylase (ACC), disruption of the PHB synthetic pathway, and an increase in the copy number of STS genes enhanced resveratrol production. In particular, the increased copies of VvSTS derived from Vitis vinifera resulted a 2-fold improvement in resveratrol synthesis from fructose. The final engineered CR-5 strain produced 1.9 mg/L of resveratrol from CO2 and tyrosine via lithoautotrophic fermentation. CONCLUSIONS: To the best of our knowledge, this study is the first to describe the valorization of CO2 into polyphenolic compounds by engineering a phenylpropanoid pathway using the lithoautotrophic bacterium C. necator H16, demonstrating the potential of this strain a platform for sustainable chemical production.

The Biocatalytic Potential of Aromatic Ammonia-Lyase from Loktanella atrilutea.

Characterization of the aromatic ammonia-lyase from Loktanella atrilutea (LaAAL) revealed reduced activity towards canonical AAL substrates: l-Phe, l-Tyr, and l-His, contrasted by its pronounced efficiency towards 3,4-dimethoxy-l-phenylalanine. Assessing the optimal conditions, LaAAL exhibited maximal activity at pH 9.5 in the ammonia elimination reaction route, distinct from the typical pH ranges of most PALs and TALs. Within the exploration of the ammonia source for the opposite, synthetically valuable ammonia addition reaction, the stability of LaAAL exhibited a positive correlation with the ammonia concentration, with the highest stability in 4 M ammonium carbamate of unadjusted pH of ~9.5. While the enzyme activity increased with rising temperatures yet, the highest operational stability and highest stationary conversions of LaAAL were observed at 30 °C. The substrate scope analysis highlighted the catalytic adaptability of LaAAL in the hydroamination of diverse cinnamic acids, especially of meta-substituted and di-/multi-substituted analogues, with structural modelling exposing steric clashes between the substrates' ortho-substituents and catalytic site residues. LaAAL showed a predilection for ammonia elimination, while classifying as a tyrosine ammonia-lyase (TAL) among the natural AAL classes. However, its distinctive attributes, such as genomic context, unique substrate specificity and catalytic fingerprint, suggest a potential natural role beyond those of known AAL classes.

Methyl jasmonate redirects the dynamics of carbohydrates and amino acids toward the lignans accumulation in Linum album cells.

Linum album accumulates lignans e.g., podophyllotoxin (PTOX) and 6-methoxy podophyllotoxin (6MPTOX). This study was aimed to figure out how different concentrations of MeJA (0, 50, 100, 150, and 200 µM) by affecting on free sugars and amino acids contents induce lignans accumulation in L. album cells. Results revealed that hydrogen peroxide (H2O2) content increased at 50µM, while it decreased at the high levels of MeJA (150 and 200 µM). Also, increasing trend of nitric oxide (NO) and lipid peroxidation levels peaked at 200 µM MeJA. An increased antioxidant enzymes activity was also observed in the treated cells. Moreover, an increase in rhamnose/xylose, glucose, and mannose was detected at 150 and 200 µM MeJA compared to the control. These compounds provide energy source and carbon skeleton for amino acids biosynthesis. Our results emphasized variations in amino acids levels in the presence of MeJA, where Phe level shifts along with synthesizing phenolics. Likewise, MeJA treatment switch on phenyl-ammonia lyase (PAL) and tyrosine-ammonia lyase (TAL) activities that regenerate phenolic compounds. Changes in phenolic acids (cinnamic, coumaric, caffeic, ferulic, and salicylic acid) and flavonoids (catechin, vitexin, myricetin, and kaempferol) were observed under MeJA treatment. Eventually, MeJA induced lignans production except for lariciresinol (LARI), so that the highest amounts of PTOX and 6MPTOX were analyzed at 50 µM, which were 4 and 5 time of control, respectively. Conclusively, it can be suggested that MeJA-induced oxidative status change redirects free sugars and amino acids toward the production of phenolic compounds especially lignans in L. album cells.

Phenylalanine suppresses cell death caused by loss of fumarylacetoacetate hydrolase in Arabidopsis.

Fumarylacetoacetate hydrolase (FAH) catalyzes the final step of Tyrosine (Tyr) degradation pathway essential to animals and the deficiency of FAH causes an inborn lethal disease. In plants, a role of this pathway was unknown until we found that mutation of Short-day Sensitive Cell Death1 (SSCD1), encoding Arabidopsis FAH, results in cell death under short day. Phenylalanine (Phe) could be converted to Tyr and then degraded in both animals and plants. Phe ingestion in animals worsens the disease caused by FAH defect. However, in this study we found that Phe represses cell death caused by FAH defect in plants. Phe treatment promoted chlorophyll biosynthesis and suppressed the up-regulation of reactive oxygen species marker genes in the sscd1 mutant. Furthermore, the repression of sscd1 cell death by Phe could be reduced by α-aminooxi-ß-phenylpropionic acid but increased by methyl jasmonate, which inhibits or activates Phe ammonia-lyase catalyzing the first step of phenylpropanoid pathway, respectively. In addition, we found that jasmonate signaling up-regulates Phe ammonia-lyase 1 and mediates the methyl jasmonate enhanced repression of sscd1 cell death by Phe. These results uncovered the relation between chlorophyll biosynthesis, phenylpropanoid pathway and jasmonate signaling in regulating the cell death resulting from loss of FAH in plants.

Programmable Polyproteams of Tyrosine Ammonia Lyases as Cross-Linked Enzymes for Synthesizing p-Coumaric Acid.

Ideal immobilization with enhanced biocatalyst activity and thermostability enables natural enzymes to serve as a powerful tool to yield synthetically useful chemicals in industry. Such an enzymatic method strategy becomes easier and more convenient with the use of genetic and protein engineering. Here, we developed a covalent programmable polyproteam of tyrosine ammonia lyases (TAL-CLEs) by fusing SpyTag and SpyCatcher peptides into the N-terminal and C-terminal of the TAL, respectively. The resulting circular enzymes were clear after the spontaneous isopeptide bonds formed between the SpyTag and SpyCatcher. Furthermore, the catalytic performance of the TAL-CLEs was measured via a synthesis sample of p-Coumaric acid. Our TAL-CLEs showed excellent catalytic efficiency, with 98.31 ± 1.14% yield of the target product-which is 4.15 ± 0.08 times higher than that of traditional glutaraldehyde-mediated enzyme aggregates. They also showed over four times as much enzyme-activity as wild-type TAL does and demonstrated good reusability, and so may become a good candidate for industrial enzymes.

Assessment of the Role of PAL in Lignin Accumulation in Wheat (Tríticum aestívum L.) at the Early Stage of Ontogenesis.

The current study evaluates the role of phenylalanine ammonia-lyase (PAL) and the associated metabolic complex in the accumulation of lignin in common wheat plants (Tríticum aestívum L.) at the early stages of ontogenesis. The data analysis was performed using plant samples that had reached Phases 4 and 5 on the Feekes scale-these phases are characterized by a transition to the formation of axial (stem) structures in cereal plants. We have shown that the substrate stimulation of PAL with key substrates, such as L-phenylalanine and L-tyrosine, leads to a significant increase in lignin by an average of 20% in experimental plants compared to control plants. In addition, the presence of these compounds in the nutrient medium led to an increase in the number of gene transcripts associated with lignin synthesis (PAL6, C4H1, 4CL1, C3H1). Inhibition was the main tool of the study. Potential competitive inhibitors of PAL were used: the optical isomer of L-phenylalanine-D-phenylalanine-and the hydroxylamine equivalent of phenylalanine-O-Benzylhydroxylamine. As a result, plants incubated on a medium supplemented with O-Benzylhydroxylamine were characterized by reduced PAL activity (almost one third). The lignin content of the cell wall in plants treated with O-Benzylhydroxylamine was almost halved. In contrast, D-phenylalanine did not lead to significant changes in the lignin-associated metabolic complex, and its effect was similar to that of specific substrates.

Structure and Mechanism of d-Glucosaminate-6-phosphate Ammonia-lyase: A Novel Octameric Assembly for a Pyridoxal 5'-Phosphate-Dependent Enzyme, and Unprecedented Stereochemical Inversion in the Elimination Reaction of a d-Amino Acid.

d-Glucosaminate-6-phosphate ammonia-lyase (DGL) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that produces 2-keto-3-deoxygluconate 6-phosphate (KDG-6-P) in the metabolism of d-glucosaminic acid by Salmonella enterica serovar typhimurium. We have determined the crystal structure of DGL by SAD phasing with selenomethionine to a resolution of 2.58 Å. The sequence has very low identity with most other members of the aminotransferase (AT) superfamily. The structure forms an octameric assembly as a tetramer of dimers that has not been observed previously in the AT superfamily. PLP is covalently bound as a Schiff base to Lys-213 in the catalytic dimer at the interface of two monomers. The structure lacks the conserved arginine that binds the α-carboxylate of the substrate in most members of the AT superfamily. However, there is a cluster of arginines in the small domain that likely serves as a binding site for the phosphate of the substrate. The deamination reaction performed in D2O gives a KDG-6-P product stereospecifically deuterated at C3; thus, the mechanism must involve an enamine intermediate that is protonated by the enzyme before product release. Nuclear magnetic resonance (NMR) analysis demonstrates that the deuterium is located in the pro-R position in the product, showing that the elimination of water takes place with inversion of configuration at C3, which is unprecedented for a PLP-dependent dehydratase/deaminase. On the basis of the crystal structure and the NMR data, a reaction mechanism for DGL is proposed.

Discovery of a New, Recurrent Enzyme in Bacterial Phosphonate Degradation: (R)-1-Hydroxy-2-aminoethylphosphonate Ammonia-lyase.

Phosphonates represent an important source of bioavailable phosphorus in certain environments. Accordingly, many microorganisms (particularly marine bacteria) possess catabolic pathways to degrade these molecules. One example is the widespread hydrolytic route for the breakdown of 2-aminoethylphosphonate (AEP, the most common biogenic phosphonate). In this pathway, the aminotransferase PhnW initially converts AEP into phosphonoacetaldehyde (PAA), which is then cleaved by the hydrolase PhnX to yield acetaldehyde and phosphate. This work focuses on a pyridoxal 5'-phosphate-dependent enzyme that is encoded in >13% of the bacterial gene clusters containing the phnW-phnX combination. This enzyme (which we termed PbfA) is annotated as a transaminase, but there is no obvious need for an additional transamination reaction in the established AEP degradation pathway. We report here that PbfA from the marine bacterium Vibrio splendidus catalyzes an elimination reaction on the naturally occurring compound (R)-1-hydroxy-2-aminoethylphosphonate (R-HAEP). The reaction releases ammonia and generates PAA, which can be then hydrolyzed by PhnX. In contrast, PbfA is not active toward the S enantiomer of HAEP or other HAEP-related compounds such as ethanolamine and d,l-isoserine, indicating a very high substrate specificity. We also show that R-HAEP (despite being structurally similar to AEP) is not processed efficiently by the PhnW-PhnX couple in the absence of PbfA. In summary, the reaction catalyzed by PbfA serves to funnel R-HAEP into the hydrolytic pathway for AEP degradation, expanding the scope and the usefulness of the pathway itself.

p97 and p47 function in membrane tethering in cooperation with FTCD during mitotic Golgi reassembly.

p97ATPase-mediated membrane fusion is required for the biogenesis of the Golgi complex. p97 and its cofactor p47 function in soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) priming, but the tethering complex for p97/p47-mediated membrane fusion remains unknown. In this study, we identified formiminotransferase cyclodeaminase (FTCD) as a novel p47-binding protein. FTCD mainly localizes to the Golgi complex and binds to either p47 or p97 via its association with their polyglutamate motifs. FTCD functions in p97/p47-mediated Golgi reassembly at mitosis in vivo and in vitro via its binding to p47 and to p97. We also showed that FTCD, p47, and p97 form a big FTCD-p97/p47-FTCD tethering complex. In vivo tethering assay revealed that FTCD that was designed to localize to mitochondria caused mitochondria aggregation at mitosis by forming a complex with endogenous p97 and p47, which support a role for FTCD in tethering biological membranes in cooperation with the p97/p47 complex. Therefore, FTCD is thought to act as a tethering factor by forming the FTCD-p97/p47-FTCD complex in p97/p47-mediated Golgi membrane fusion.

Introducing a delivery system for melanogenesis inhibition in melanoma B16F10 cells mediated by the conjugation of tyrosine ammonia-lyase and a TAT-penetrating peptide.

Hyperpigmentation disorders negatively influence an individual's quality of life and may cause emotional distress. Over the years, various melanogenesis inhibitors (mainly tyrosinase inhibitors) have been developed, most of which with low efficacy or high toxicity. Although metabolic engineering by deviation in the flux of substrate is of considerable interest, trials to develop a melanogenesis inhibitor based on L-tyrosine (L-Tyr) restriction are missing. We propose a novel proteinaceous melanogenesis inhibitor called tyrosine ammonia-lyase (TAL), an enzyme that catalyzes the conversion of L-Tyr to p-coumaric acid and ammonia. Since the cell membrane can act as a barrier for intracellular protein delivery, we have covalently conjugated a recombinant TAL enzyme from Rhodobacter sphaeroides (RsTAL) to a trans-activator of transcription (TAT) cell-penetrating peptide (CPP) to afford the intracellular delivery. The heterologously expressed TAT-RsTAL fusion protein was delivered successfully into B16F10 melanocytes as confirmed by the direct fluorescence microscopy with increased intensity from 30 to 180 min. TAT-RsTAL showed sufficient intracellular activity of about 0.83 ± 0.04 and 0.34 ± 0.03 nmol•mg-1 •s-1 for the native and inclusion body-extracted conjugates, respectively. The conjugate inhibited melanin biosynthesis in B16F10 cells in a time-dependent manner. Melanin accumulation was inhibited by 12.7 ± 6.2%, 28.2 ± 5.7%, and 33.9 ± 2.9% compared to the nontreated control groups after 24, 48, and 72 hr of incubation, respectively. L-Tyr restriction had no significant effect on the cell viability up to a concentration of 100 µgml-1 even after 72 hr. According to the observed hypopigmentary effect of the conjugate in this study, TAT-RsTAL can be suggested as a melanogenesis inhibitor for further investigations.

Beneficial impact of exogenous arginine, cysteine and methionine on postharvest senescence of broccoli.

This study examined the ability of l-arginine, l-cysteine and l-methionine, to inhibit postharvest senescence of broccoli. Florets were dipped in aqueous solutions of the amino acids at concentrations from 1.0 to 100 mM and stored at 10 °C. A 5 mM dip was found to be optimal in delaying senescence as measured by retention of green colour, vitamin C and antioxidant activity, and a lower level of ethylene production, respiration, weight loss, phenylalanine ammonia lyase (PAL) activity and ion leakage with the benefits being similar for all three amino acids. Arginine, cysteine and methionine have Generally Recognised As Safe (GRAS) status and should have few impediments in obtaining regulatory approval for commercial use if similar effects were found for other leafy vegetables.

Enhancement of pipecolic acid production by the expression of multiple lysine cyclodeaminase in the Escherichia coli whole-cell system.

Pipecolic acid, a non-proteinogenic amino acid, is a metabolite in lysine metabolism and a key chiral precursor in local anesthesia and macrolide antibiotics. To replace the environmentally unfriendly chemical production or preparation procedure of pipecolic acid, many biological synthetic routes have been studied for a long time. Among them, synthesis by lysine cyclodeaminase (LCD), encoded by pipA, has several advantages, including stability of enzyme activity and NAD+ self-regeneration. Thus, we selected this enzyme for pipecolic acid biosynthesis in a whole-cell bioconversion. To construct a robust pipecolic acid production system, we investigated important conditions including expression vector, strain, culture conditions, and other reaction parameters. The most important factor was the introduction of multiple pipA genes into the whole-cell system. As a result, we produced 724 mM pipecolic acid (72.4 % conversion), and the productivity was 0.78 g/L/h from 1 M l-lysine after 5 days. This is the highest production reported to date.

Independent evolution of transcript abundance and gene regulatory dynamics.

Changes in gene expression drive novel phenotypes, raising interest in how gene expression evolves. In contrast to the static genome, cells modulate gene expression in response to changing environments. Previous comparative studies focused on specific conditions, describing interspecies variation in expression levels, but providing limited information about variation across different conditions. To close this gap, we profiled mRNA levels of two related yeast species in hundreds of conditions and used coexpression analysis to distinguish variation in the dynamic pattern of gene expression from variation in expression levels. The majority of genes whose expression varied between the species maintained a conserved dynamic pattern. Cases of diverged dynamic pattern correspond to genes that were induced under distinct subsets of conditions in the two species. Profiling the interspecific hybrid allowed us to distinguish between genes with predominantly cis- or trans-regulatory variation. We find that trans-varying alleles are dominantly inherited, and that cis-variations are often complemented by variations in trans Based on these results, we suggest that gene expression diverges primarily through changes in expression levels, but does not alter the pattern by which these levels are dynamically regulated.

De novo resveratrol production through modular engineering of an Escherichia coli-Saccharomyces cerevisiae co-culture.

BACKGROUND: Resveratrol is a plant secondary metabolite with diverse, potential health-promoting benefits. Due to its nutraceutical merit, bioproduction of resveratrol via microbial engineering has gained increasing attention and provides an alternative to unsustainable chemical synthesis and straight extraction from plants. However, many studies on microbial resveratrol production were implemented with the addition of water-insoluble phenylalanine or tyrosine-based precursors to the medium, limiting in the sustainable development of bioproduction. RESULTS: Here we present a novel coculture platform where two distinct metabolic background species were modularly engineered for the combined total and de novo biosynthesis of resveratrol. In this scenario, the upstream Escherichia coli module is capable of excreting p-coumaric acid into the surrounding culture media through constitutive overexpression of codon-optimized tyrosine ammonia lyase from Trichosporon cutaneum (TAL), feedback-inhibition-resistant 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase (aroGfbr) and chorismate mutase/prephenate dehydrogenase (tyrAfbr) in a transcriptional regulator tyrR knockout strain. Next, to enhance the precursor malonyl-CoA supply, an inactivation-resistant version of acetyl-CoA carboxylase (ACC1S659A,S1157A) was introduced into the downstream Saccharomyces cerevisiae module constitutively expressing codon-optimized 4-coumarate-CoA ligase from Arabidopsis thaliana (4CL) and resveratrol synthase from Vitis vinifera (STS), and thus further improve the conversion of p-coumaric acid-to-resveratrol. Upon optimization of the initial inoculation ratio of two populations, fermentation temperature, and culture time, this co-culture system yielded 28.5 mg/L resveratrol from glucose in flasks. In further optimization by increasing initial net cells density at a test tube scale, a final resveratrol titer of 36 mg/L was achieved. CONCLUSIONS: This is first study that demonstrates the use of a synthetic E. coli-S. cerevisiae consortium for de novo resveratrol biosynthesis, which highlights its potential for production of other p-coumaric-acid or resveratrol derived biochemicals.

Structure-function investigation of 3-methylaspartate ammonia lyase reveals substrate molecular determinants for the deamination reaction.

The enzymatic reactions leading to the deamination of ß-lysine, lysine, or 2-aminoadipic acid are of great interest for the metabolic conversion of lysine to adipic acid. Enzymes able to carry out these reactions are not known, however ammonia lyases (EC 4.3.1.-) perform deamination on a wide range of substrates. We have studied 3-methylaspartate ammonia lyase (MAL, EC 4.3.1.2) as a potential candidate for protein engineering to enable deamination towards ß-lysine, that we have shown to be a competitive inhibitor of MAL. We have characterized MAL activity, binding and inhibition properties on six different compounds that would allow to define the molecular determinants necessary for MAL to deaminate our substrate of interest. Docking calculations showed that ß-lysine as well as the other compounds investigated could fit spatially into MAL catalytic pocket, although they probably are weak or very transient binders and we identified molecular determinants involved in the binding of the substrate. The hydrophobic interactions formed by the methyl group of 3-methylaspartic acid, together with the presence of the amino group on carbon 2, play an essential role in the appropriate binding of the substrate. The results showed that ß-lysine is able to fit and bind in MAL catalytic pocket and can be potentially converted from inhibitor to substrate of MAL upon enzyme engineering. The characterization of the binding and inhibition properties of the substrates tested here provide the foundation for future and more extensive studies on engineering MAL that could lead to a MAL variant able to catalyse this challenging deamination reaction.

Structural and mutational analyses of the bifunctional arginine dihydrolase and ornithine cyclodeaminase AgrE from the cyanobacterium Anabaena.

In cyanobacteria, metabolic pathways that use the nitrogen-rich amino acid arginine play a pivotal role in nitrogen storage and mobilization. The N-terminal domains of two recently identified bacterial enzymes: ArgZ from Synechocystis and AgrE from Anabaena, have been found to contain an arginine dihydrolase. This enzyme provides catabolic activity that converts arginine to ornithine, resulting in concomitant release of CO2 and ammonia. In Synechocystis, the ArgZ-mediated ornithine-ammonia cycle plays a central role in nitrogen storage and remobilization. The C-terminal domain of AgrE contains an ornithine cyclodeaminase responsible for the formation of proline from ornithine and ammonia production, indicating that AgrE is a bifunctional enzyme catalyzing two sequential reactions in arginine catabolism. Here, the crystal structures of AgrE in three different ligation states revealed that it has a tetrameric conformation, possesses a binding site for the arginine dihydrolase substrate l-arginine and product l-ornithine, and contains a binding site for the coenzyme NAD(H) required for ornithine cyclodeaminase activity. Structure-function analyses indicated that the structure and catalytic mechanism of arginine dihydrolase in AgrE are highly homologous with those of a known bacterial arginine hydrolase. We found that in addition to other active-site residues, Asn-71 is essential for AgrE's dihydrolase activity. Further analysis suggested the presence of a passage for substrate channeling between the two distinct AgrE active sites, which are situated ∼45 Šapart. These results provide structural and functional insights into the bifunctional arginine dihydrolase-ornithine cyclodeaminase enzyme AgrE required for arginine catabolism in Anabaena.

Characterization of two new aromatic amino acid lyases from actinomycetes for highly efficient production of p-coumaric acid.

p-Coumaric acid (p-CA) is a bioactive natural product and an important industrial material for pharmaceuticals and nutraceuticals. It can be synthesized from deamination of L-tyrosine by tyrosine ammonia lyase (TAL). In this work, we discovered two aromatic amino acid lyase genes, Sas-tal and Sts-tal, from Saccharothrix sp. NRRL B-16348 and Streptomyces sp. NRRL F-4489, respectively, and expressed them in Escherichia coli BL21(DE3). The two enzymes were functionally characterized as TAL. The optimum reaction temperature for Sas-TAL and Sts-TAL is 55 °C and 50 °C, respectively; while, the optimum pH for both TALs is 11. Sas-TAL had a kcat/Km value of 6.2 µM-1 min-1, while Sts-TAL had a much higher efficiency with a kcat/Km value of 78.3 µM-1 min-1. Both Sts-TAL and Sas-TAL can also take L-phenylalanine as the substrate to yield trans-cinnamic acid, and Sas-TAL showed much higher phenylalanine ammonia lyase activity than Sts-TAL. Using E. coli/Sts-TAL as a whole-cell biocatalyst, the productivity of p-CA reached 2.88 ± 0.12 g (L h)-1, which represents the highest efficiency for microbial production of p-CA. Therefore, this work not only reports the identification of two new TALs from actinomycetes, but also provides an efficient way to produce the industrially valuable material p-CA.

Conformational gating in ammonia lyases.

BACKGROUND: Ammonia lyases are enzymes of industrial and biomedical interest. Knowledge of structure-dynamics-function relationship in ammonia lyases is instrumental for exploiting the potential of these enzymes in industrial or biomedical applications. METHODS: We investigated the conformational changes in the proximity of the catalytic pocket of a 3-methylaspartate ammonia lyase (MAL) as a model system. At this scope, we used microsecond all-atom molecular dynamics simulations, analyzed with dimensionality reduction techniques, as well as in terms of contact networks and correlated motions. RESULTS: We identify two regulatory elements in the MAL structure, i.e., the ß5-α2 loop and the helix-hairpin-loop subdomain. These regulatory elements undergo conformational changes switching from 'occluded' to 'open' states. The rearrangements are coupled to changes in the accessibility of the active site. The ß5-α2 loop and the helix-hairpin-loop subdomain modulate the formation of tunnels from the protein surface to the catalytic site, making the active site more accessible to the substrate when they are in an open state. CONCLUSIONS: Our work pinpoints a sequential mechanism, in which the helix-hairpin-loop subdomain of MAL needs to break a subset of intramolecular interactions first to favor the displacement of the ß5-α2 loop. The coupled conformational changes of these two elements contribute to modulate the accessibility of the catalytic site. GENERAL SIGNIFICANCE: Similar molecular mechanisms can have broad relevance in other ammonia lyases with similar regulatory loops. Our results also imply that it is important to account for protein dynamics in the design of variants of ammonia lyases for industrial and biomedical applications.

Heterologous caffeic acid biosynthesis in Escherichia coli is affected by choice of tyrosine ammonia lyase and redox partners for bacterial Cytochrome P450.

BACKGROUND: Caffeic acid is industrially recognized for its antioxidant activity and therefore its potential to be used as an anti-inflammatory, anticancer, antiviral, antidiabetic and antidepressive agent. It is traditionally isolated from lignified plant material under energy-intensive and harsh chemical extraction conditions. However, over the last decade bottom-up biosynthesis approaches in microbial cell factories have been established, that have the potential to allow for a more tailored and sustainable production. One of these approaches has been implemented in Escherichia coli and only requires a two-step conversion of supplemented L-tyrosine by the actions of a tyrosine ammonia lyase and a bacterial Cytochrome P450 monooxygenase. Although the feeding of intermediates demonstrated the great potential of this combination of heterologous enzymes compared to others, no de novo synthesis of caffeic acid from glucose has been achieved utilizing the bacterial Cytochrome P450 thus far. RESULTS: The herein described work aimed at improving the efficiency of this two-step conversion in order to establish de novo caffeic acid formation from glucose. We implemented alternative tyrosine ammonia lyases that were reported to display superior substrate binding affinity and selectivity, and increased the efficiency of the Cytochrome P450 by altering the electron-donating redox system. With this strategy we were able to achieve final titers of more than 300 µM or 47 mg/L caffeic acid over 96 h in an otherwise wild type E. coli MG1655(DE3) strain with glucose as the only carbon source. We observed that the choice and gene dose of the redox system strongly influenced the Cytochrome P450 catalysis. In addition, we were successful in applying a tethering strategy that rendered even a virtually unproductive Cytochrome P450/redox system combination productive. CONCLUSIONS: The caffeic acid titer achieved in this study is about 10% higher than titers reported for other heterologous caffeic acid pathways in wildtype E. coli without L-tyrosine supplementation. The tethering strategy applied to the Cytochrome P450 appears to be particularly useful for non-natural Cytochrome P450/redox partner combinations and could be useful for other recombinant pathways utilizing bacterial Cytochromes P450.

Development of a Quorum-Sensing Based Circuit for Control of Coculture Population Composition in a Naringenin Production System.

As synthetic biology and metabolic engineering tools improve, it is feasible to construct more complex microbial synthesis systems that may be limited by the machinery and resources available in an individual cell. Coculture fermentation is a promising strategy for overcoming these constraints by distributing objectives between subpopulations, but the primary method for controlling the composition of the coculture of production systems has been limited to control of the inoculum composition. We have developed a quorum sensing (QS)-based growth-regulation circuit that provides an additional parameter for regulating the composition of a coculture over the course of the fermentation. Implementation of this tool in a naringenin-producing coculture resulted in a 60% titer increase over a system that was optimized by varying inoculation ratios only. We additionally demonstrated that the growth control circuit can be implemented in combination with a communication module that couples transcription in one subpopulation to the cell-density of the other population for coordination of behavior, resulting in an additional 60% improvement in naringenin titer.