Towards universal synthetic heterotrophy using a metabolic coordinator.

Engineering the utilization of non-native substrates, or synthetic heterotrophy, in proven industrial microbes such as Saccharomyces cerevisiae represents an opportunity to valorize plentiful and renewable sources of carbon and energy as inputs to bioprocesses. We previously demonstrated that activation of the galactose (GAL) regulon, a regulatory structure used by this yeast to coordinate substrate utilization with biomass formation during growth on galactose, during growth on the non-native substrate xylose results in a vastly altered gene expression profile and faster growth compared with constitutive overexpression of the same heterologous catabolic pathway. However, this effort involved the creation of a xylose-inducible variant of Gal3p (Gal3pSyn4.1), the sensor protein of the GAL regulon, preventing this semi-synthetic regulon approach from being easily adapted to additional non-native substrates. Here, we report the construction of a variant Gal3pMC (metabolic coordinator) that exhibits robust GAL regulon activation in the presence of structurally diverse substrates and recapitulates the dynamics of the native system. Multiple molecular modeling studies suggest that Gal3pMC occupies conformational states corresponding to galactose-bound Gal3p in an inducer-independent manner. Using Gal3pMC to test a regulon approach to the assimilation of the non-native lignocellulosic sugars xylose, arabinose, and cellobiose yields higher growth rates and final cell densities when compared with a constitutive overexpression of the same set of catabolic genes. The subsequent demonstration of rapid and complete co-utilization of all three non-native substrates suggests that Gal3pMC-mediated dynamic global gene expression changes by GAL regulon activation may be universally beneficial for engineering synthetic heterotrophy.

Flow cytometric evaluation of yeast-bacterial cell-cell interactions.

Synthetic cell-cell interaction systems can be useful for understanding multicellular communities or for screening binding molecules. We adapt a previously characterized set of synthetic cognate nanobody-antigen pairs to a yeast-bacteria coincubation format and use flow cytometry to evaluate cell-cell interactions mediated by binding between surface-displayed molecules. We further use fluorescence-activated cell sorting to enrich a specific yeast-displayed nanobody within a mixed yeast-display population. Finally, we demonstrate that this system supports the characterization of a therapeutically relevant nanobody-antigen interaction: a previously discovered nanobody that binds to the intimin protein expressed on the surface of enterohemorrhagic Escherichia coli. Overall, our findings indicate that the yeast-bacteria format supports efficient evaluation of ligand-target interactions. With further development, this format may facilitate systematic characterization and high-throughput discovery of bacterial surface-binding molecules.

A Silk-Based Platform to Stabilize Phenylalanine Ammonia-lyase for Orally Administered Enzyme Replacement Therapy.

Phenylalanine ammonia-lyase (PAL) has gained attention in recent years for the treatment of phenylketonuria (PKU), a genetic disorder that affects ∼1 in 15 000 individuals globally. However, the enzyme is easily degraded by proteases, unstable at room temperature, and currently administered in PKU patients as daily subcutaneous injections. We report here the stabilization of the PAL from Anabaena variabilis, which is currently used to formulate pegvaliase, through incorporation in a silk fibroin matrix. The combination with silk stabilizes PAL at 37 °C. In addition, in vitro studies showed that inclusion in a silk matrix preserves the biological activity of the enzyme in simulated intestinal fluid, which will enable oral administration of PAL to treat PKU.

Compartmentalization of the Carbaryl Degradation Pathway: Molecular Characterization of Inducible Periplasmic Carbaryl Hydrolase from Pseudomonas spp.

Pseudomonas sp. strains C5pp and C7 degrade carbaryl as the sole carbon source. Carbaryl hydrolase (CH) catalyzes the hydrolysis of carbaryl to 1-naphthol and methylamine. Bioinformatic analysis of mcbA, encoding CH, in C5pp predicted it to have a transmembrane domain (Tmd) and a signal peptide (Sp). In these isolates, the activity of CH was found to be 4- to 6-fold higher in the periplasm than in the cytoplasm. The recombinant CH (rCH) showed 4-fold-higher activity in the periplasm of Escherichia coli The deletion of Tmd showed activity in the cytoplasmic fraction, while deletion of both Tmd and Sp (Tmd+Sp) resulted in expression of the inactive protein. Confocal microscopic analysis of E. coli expressing a (Tmd+Sp)-green fluorescent protein (GFP) fusion protein revealed the localization of GFP into the periplasm. Altogether, these results indicate that Tmd probably helps in anchoring of polypeptide to the inner membrane, while Sp assists folding and release of CH in the periplasm. The N-terminal sequence of the mature periplasmic CH confirms the absence of the Tmd+Sp region and confirms the signal peptidase cleavage site as Ala-Leu-Ala. CH purified from strains C5pp, C7, and rCHΔ(Tmd)a were found to be monomeric with molecular mass of ∼68 to 76 kDa and to catalyze hydrolysis of the ester bond with an apparent Km and Vmax in the range of 98 to 111 µM and 69 to 73 µmol · min-1 · mg-1, respectively. The presence of low-affinity CH in the periplasm and 1-naphthol-metabolizing enzymes in the cytoplasm of Pseudomonas spp. suggests the compartmentalization of the metabolic pathway as a strategy for efficient degradation of carbaryl at higher concentrations without cellular toxicity of 1-naphthol.IMPORTANCE Proteins in the periplasmic space of bacteria play an important role in various cellular processes, such as solute transport, nutrient binding, antibiotic resistance, substrate hydrolysis, and detoxification of xenobiotics. Carbaryl is one of the most widely used carbamate pesticides. Carbaryl hydrolase (CH), the first enzyme of the degradation pathway which converts carbaryl to 1-naphthol, was found to be localized in the periplasm of Pseudomonas spp. Predicted transmembrane domain and signal peptide sequences of Pseudomonas were found to be functional in Escherichia coli and to translocate CH and GFP into the periplasm. The localization of low-affinity CH into the periplasm indicates controlled formation of toxic and recalcitrant 1-naphthol, thus minimizing its accumulation and interaction with various cellular components and thereby reducing the cellular toxicity. This study highlights the significance of compartmentalization of metabolic pathway enzymes for efficient removal of toxic compounds.

Insights into metabolism and sodium chloride adaptability of carbaryl degrading halotolerant Pseudomonas sp. strain C7.

Pseudomonas sp. strain C7 isolated from sediment of Thane creek near Mumbai, India, showed the ability to grow on glucose and carbaryl in the presence of 7.5 and 3.5% of NaCl, respectively. It also showed good growth in the absence of NaCl indicating the strain to be halotolerant. Increasing salt concentration impacted the growth on carbaryl; however, the specific activity of various enzymes involved in the metabolism remained unaffected. Among various enzymes, 1-naphthol 2-hydroxylase was found to be sensitive to chloride as compared to carbaryl hydrolase and gentisate 1,2-dioxygenase. The intracellular concentration of Cl- ions remained constant (6-8 mM) for cells grown on carbaryl either in the presence or absence of NaCl. Thus the ability to adapt to the increasing concentration of NaCl is probably by employing chloride efflux pump and/or increase in the concentration of osmolytes as mechanism for halotolerance. The halotolerant nature of the strain will be beneficial to remediate carbaryl from saline agriculture fields, ecosystems and wastewaters.

Metabolic regulation and chromosomal localization of carbaryl degradation pathway in Pseudomonas sp. strains C4, C5 and C6.

Pseudomonas sp. strains C4, C5 and C6 degrade carbaryl (1-naphthyl N-methylcarbamate) via 1-naphthol, 1,2-dihydroxynaphthalene, salicylate and gentisate. Carbon source-dependent metabolic studies suggest that enzymes responsible for carbaryl degradation are probably organized into 'upper' (carbaryl to salicylate), 'middle' (salicylate to gentisate) and 'lower' (gentisate to TCA cycle) pathway. Carbaryl and 1-naphthol were found to induce all carbaryl pathway enzymes, while salicylate and gentisate induce middle and lower pathway enzymes. The strains were found to harbor plasmid(s), and carbaryl degradation property was found to be stable. Genes encoding enzymes of the degradative pathway such as 1-naphthol 2-hydroxylase, salicylaldehyde dehydrogenase, salicylate 5-hydroxylase and gentisate 1,2-dioxygenase were amplified from chromosomal DNA of these strains. The gene-specific PCR products were sequenced from strain C6, and phylogenetic tree was constructed. Southern hybridization and PCR analysis using gel eluted DNA as template supported the presence of pathway genes onto the chromosome and not on the plasmid(s).

Integration of metabolism and regulation reveals rapid adaptability to growth on non-native substrates.

Engineering synthetic heterotrophy is a key to the efficient bio-based valorization of renewable and waste substrates. Among these, engineering hemicellulosic pentose utilization has been well-explored in Saccharomyces cerevisiae (yeast) over several decades-yet the answer to what makes their utilization inherently recalcitrant remains elusive. Through implementation of a semi-synthetic regulon, we find that harmonizing cellular and engineering objectives are a key to obtaining highest growth rates and yields with minimal metabolic engineering effort. Concurrently, results indicate that "extrinsic" factors-specifically, upstream genes that direct flux of pentoses into central carbon metabolism-are rate-limiting. We also reveal that yeast metabolism is innately highly adaptable to rapid growth on non-native substrates and that systems metabolic engineering (i.e., functional genomics, network modeling, etc.) is largely unnecessary. Overall, this work provides an alternate, novel, holistic (and yet minimalistic) approach based on integrating non-native metabolic genes with a native regulon system.

Cheating the Cheater: Suppressing False-Positive Enrichment during Biosensor-Guided Biocatalyst Engineering.

Transcription factor (TF)-based biosensors are very desirable reagents for high-throughput enzyme and strain engineering campaigns. Despite their potential, they are often difficult to deploy effectively as the small molecules being detected can leak out of high-producer cells, into low-producer cells, and activate the biosensor therein. This crosstalk leads to the overrepresentation of false-positive/cheater cells in the enriched population. While the host cell can be engineered to minimize crosstalk (e.g., by deleting responsible transporters), this is not easily applicable to all molecules of interest, particularly those that can diffuse passively. One such biosensor recently reported for trans-cinnamic acid (tCA) suffers from crosstalk when used for phenylalanine ammonia-lyase (PAL) enzyme engineering by directed evolution. We report that desensitizing the biosensor (i.e., increasing the limit of detection) suppresses cheater population enrichment. Furthermore, we show that, if we couple the biosensor-based screen with an orthogonal prescreen that eliminates a large fraction of true negatives, we can successfully reduce the cheater population during the fluorescence-activated cell sorting. Using the approach developed here, we were successfully able to isolate PAL variants with ∼70% higher kcat after a single sort. These mutants have tremendous potential in phenylketonuria (PKU) treatment and flavonoid production.

In-depth Sequence-Function Characterization Reveals Multiple Pathways to Enhance Enzymatic Activity.

Deep mutational scanning (DMS) has recently emerged as a powerful method to study protein sequence-function relationships but it has not been well-explored as a guide to enzyme engineering and identifying pathways by which their catalytic cycle may be improved. We report such a demonstration in this work using a Phenylalanine ammonia-lyase (PAL), which deaminates L-phenylalanine to trans-cinnamic acid and has widespread application in chemo-enzymatic synthesis, agriculture, and medicine. In particular, the PAL from Anabaena variabilis (AvPAL*) has garnered significant attention as the active ingredient in Pegvaliase®, the only FDA-approved drug treating classical Phenylketonuria (PKU). Although an extensive body of literature exists on the structure, substrate-specificity, and catalytic cycle, protein-wide sequence determinants of function remain unknown, as do intermediate reaction steps that limit turnover frequency, all of which has hindered rational engineering of these enzymes. Here, we created a detailed sequence-function landscape of AvPAL* by performing DMS and revealed 112 mutations at 79 functionally relevant sites that affect a positive change in enzyme fitness. Using fitness values and structure-function analysis, we picked a subset of positions for comprehensive single- and multi-site saturation mutagenesis and identified combinations of mutations that led to improved reaction kinetics in cell-free and cellular contexts. We then performed QM/MM and MD to understand the mechanistic role of the most beneficial mutations and observed that different mutants confer improvements via different mechanisms, including stabilizing transition and intermediate states, improving substrate diffusion into the active site, and decreasing product inhibition. This work demonstrates how DMS can be combined with computational analysis to effectively identify significant mutations that enhance enzyme activity along with the underlying mechanisms by which these mutations confer their benefit.

Directed evolution of Anabaena variabilis phenylalanine ammonia-lyase (PAL) identifies mutants with enhanced activities.

There is broad interest in engineering phenylalanine ammonia-lyase (PAL) for its biocatalytic applications in industry and medicine. While site-specific mutagenesis has been employed to improve PAL stability or substrate specificity, combinatorial techniques are poorly explored. Here, we report development of a directed evolution technique to engineer PAL enzymes. Central to this approach is a high-throughput enrichment that couples E. coli growth to PAL activity. Starting with the PAL used in the formulation of pegvaliase for PKU therapy, we report previously unidentified mutations that increase turnover frequency almost twofold after only a single round of engineering.

Nitrogen-dependent induction of atrazine degradation pathway in Pseudomonas sp. strain AKN5.

Soil isolate Pseudomonas sp. strain AKN5 degrades atrazine as the sole source of nitrogen. The strain showed expeditious growth on medium containing citrate as the carbon source and ammonium chloride as the nitrogen source as compared to citrate plus atrazine or cyanuric acid. Biochemical and nitrogen-source-dependent enzyme induction studies revealed that atrazine is metabolized through hydrolytic pathway and has two segments: the upper segment converts atrazine into cyanuric acid while the lower segment metabolizes cyanuric acid to CO2 and ammonia. Bioinformatics and co-transcriptional analyses suggest that atzA, atzB and atzC were transcribed as three independent transcripts while atzDEF were found to be transcribed as a single polycistronic mRNA indicating operonic arrangement. Transcriptional analysis showed inducible expression of atzA/B/C/DEF from atrazine grown cells while cyanuric acid grown cells showed significantly higher expression of atzDEF. Interestingly, growth profiles and enzyme activity measurements suggests that strain utilizes a simple nitrogen source (ammonium chloride) over the complex (atrazine or cyanuric acid) when grown on dual nitrogen source. These results suggest that atrazine degradation genes were up-regulated in the presence of atrazine but repressed in the presence of simple nitrogen source like ammonium chloride.

Draft Genome Sequence of Carbaryl-Degrading Soil Isolate Pseudomonas sp. Strain C5pp.

We report the draft genome sequence of carbaryl-degrading Pseudomonas sp. strain C5pp. Genes encoding salicylate and gentisate metabolism, large amounts of oxygenase, nitrogen metabolism, and heavy metal tolerance were identified. The sequence will provide further insight into the biochemical and evolutionary aspects of carbaryl degradation.

Insights into functional and evolutionary analysis of carbaryl metabolic pathway from Pseudomonas sp. strain C5pp.

Carbaryl (1-naphthyl N-methylcarbamate) is a most widely used carbamate pesticide in the agriculture field. Soil isolate, Pseudomonas sp. strain C5pp mineralizes carbaryl via 1-naphthol, salicylate and gentisate, however the genetic organization and evolutionary events of acquisition and assembly of pathway have not yet been studied. The draft genome analysis of strain C5pp reveals that the carbaryl catabolic genes are organized into three putative operons, 'upper', 'middle' and 'lower'. The sequence and functional analysis led to identification of new genes encoding: i) hitherto unidentified 1-naphthol 2-hydroxylase, sharing a common ancestry with 2,4-dichlorophenol monooxygenase; ii) carbaryl hydrolase, a member of a new family of esterase; and iii) 1,2-dihydroxy naphthalene dioxygenase, uncharacterized type-II extradiol dioxygenase. The 'upper' pathway genes were present as a part of a integron while the 'middle' and 'lower' pathway genes were present as two distinct class-I composite transposons. These findings suggest the role of horizontal gene transfer event(s) in the acquisition and evolution of the carbaryl degradation pathway in strain C5pp. The study presents an example of assembly of degradation pathway for carbaryl.

Analysis of preference for carbon source utilization among three strains of aromatic compounds degrading Pseudomonas.

Soil isolates Pseudomonas putida CSV86, Pseudomonas aeruginosa PP4 and Pseudomonas sp. C5pp degrade naphthalene, phthalate isomers and carbaryl, respectively. Strain CSV86 displayed a diauxic growth pattern on phenylpropanoid compounds (veratraldehyde, ferulic acid, vanillin or vanillic acid) plus glucose with a distinct second lag-phase. The glucose concentration in the medium remained constant with higher cell respiration rates on aromatics and maximum protocatechuate 3,4-dioxygenase activity in the first log-phase, which gradually decreased in the second log-phase with concomitant depletion of the glucose. In strains PP4 and C5pp, growth profile and metabolic studies suggest that glucose is utilized in the first log-phase with the repression of utilization of aromatics (phthalate or carbaryl). All three strains utilize benzoate via the catechol 'ortho' ring-cleavage pathway. On benzoate plus glucose, strain CSV86 showed preference for benzoate over glucose in contrast to strains PP4 and C5pp. Additionally, organic acids like succinate were preferred over aromatics in strains PP4 and C5pp, whereas strain CSV86 co-metabolizes them. Preferential utilization of aromatics over glucose and co-metabolism of organic acids and aromatics are found to be unique properties of P. putida CSV86 as compared with strains PP4 and C5pp and this property of strain CSV86 can be exploited for effective bioremediation.

Kinetic and spectroscopic characterization of 1-naphthol 2-hydroxylase from Pseudomonas sp. strain C5.

1-Naphthol 2-hydroxylase (1-NH) catalyzes the conversion of 1-naphthol to 1,2-dihydroxynaphthalene. 1-NH from carbaryl degrading Pseudomonas strain C5 was purified and characterized for its kinetic and spectroscopic properties. The enzyme was found to be NAD(P)H-dependent external flavin monooxygenase. Though the kinetic parameters of 1-NH from strain C5 appear to be similar to 1-NH enzyme from strains C4 and C6, however, they differ in their N-terminal sequences, mole content of flavin adenine dinucleotide (FAD), reconstitution of apoenzyme, and K i. 1-NH showed narrow substrate specificity with comparable hydroxylation efficiency on 1-naphthol and 5-amino 1-naphthol (~30 %) followed by 4-chloro 1-naphthol (~9 %). Salicylate was found to be the nonsubstrate effector. The flavin fluorescence of 1-NH was found to increase in the presence of 1-naphthol (K d = 11.3 µM) and salicylate (K d = 1027 µM). The circular dichroism (CD) spectra showed significant perturbations in the presence of NAD(P)H, whereas no changes were observed in the presence of 1-naphthol. Naphthalene, 1-chloronaphthalene, 2-napthol, and 2-naphthoic acid were found to be the mixed inhibitors. Chemical modification studies showed the probable involvement of His, Cys, and Tyr in the binding of 1-naphthol, whereas Trp was found to be involved in the binding of NAD(P)H.

Purification and characterization of NAD+ -dependent salicylaldehyde dehydrogenase from carbaryl-degrading Pseudomonas sp. strain C6.

NAD+-dependent salicylaldehyde dehydrogenase (SALDH) which catalyzes the oxidation of salicylaldehyde to salicylate was purified form carbaryl-degrading Pseudomonas sp. strain C6. The enzyme was found to be a functional homotrimer (150 kDa) with subunit molecular mass of 50 kDa and contained calcium (1.8 mol/mol of enzyme). These properties were found to be unique. External addition of metal ions showed no effect on the activity and addition of chelators showed moderate inhibition of the activity. Potassium ions were found to enhance the activity significantly. SALDH showed higher affinity for salicylaldehyde (Km = 4.5 µM) and accepts mono- as well as di-aromatic aldehydes; however it showed poor activity on aliphatic aldehydes. Chloro-/nitro-substituted benzaldehydes were potent substrate inhibitors as compared to benzaldehyde and 3-hydroxybenzaldehyde, while 2-naphthaldehyde and salicylaldehyde were moderate. The kinetic data revealed that SALDH, though having broad specificity, is more efficient for the oxidation of salicylaldehyde as compared to other aromatic aldehyde dehydrogenases which gives an advantage for Pseudomonas sp. strain C6 to bioremediate carbaryl and other aromatic aldehydes efficiently.