An In Vitro Hybrid Biocatalytic System Enabled by a Combination of Surface-Displayed, Purified, and Cell-Free Expressed Enzymes.

Enzymatic cascades have become a green and sustainable approach for the synthesis of valuable chemicals and pharmaceuticals. Using sequential enzymes to construct a multienzyme complex is an effective way to enhance the overall performance of biosynthetic routes. Here we report the design of an efficient in vitro hybrid biocatalytic system by assembling three enzymes that can convert styrene to (S)-1-phenyl-1,2-ethanediol. Specifically, we prepared the three enzymes in different ways, which were cell surface-displayed, purified, and cell-free expressed. To assemble them, we fused two orthogonal peptide-protein pairs (i.e., SpyTag/SpyCatcher and SnoopTag/SnoopCatcher) to the three enzymes, allowing their spatial organization by covalent assembly. By doing this, we constructed a multienzyme complex, which could enhance the production of (S)-1-phenyl-1,2-ethanediol by 3 times compared to the free-floating enzyme system without assembly. After optimization of the reaction system, the final product yield reached 234.6 µM with a substrate conversion rate of 46.9% (based on 0.5 mM styrene). Taken together, our strategy integrates the merits of advanced biochemical engineering techniques, including cellular surface display, spatial enzyme organization, and cell-free expression, which offers a new solution for chemical biosynthesis by enzymatic cascade biotransformation. We, therefore, anticipate that our approach will hold great potential for designing and constructing highly efficient systems to synthesize chemicals of agricultural, industrial, and pharmaceutical significance.

Engineering styrene biosynthesis: designing a functional trans-cinnamic acid decarboxylase in Pseudomonas.

We are interested in converting second generation feedstocks into styrene, a valuable chemical compound, using the solvent-tolerant Pseudomonas putida DOT-T1E as a chassis. Styrene biosynthesis takes place from L-phenylalanine in two steps: firstly, L-phenylalanine is converted into trans-cinnamic acid (tCA) by PAL enzymes and secondly, a decarboxylase yields styrene. This study focuses on designing and synthesizing a functional trans-cinnamic acid decarboxylase in Pseudomonas putida. To achieve this, we utilized the "wholesale" method, involving deriving two consensus sequences from multi-alignments of homologous yeast ferulate decarboxylase FDC1 sequences with > 60% and > 50% identity, respectively. These consensus sequences were used to design Pseudomonas codon-optimized genes named psc1 and psd1 and assays were conducted to test the activity in P. putida. Our results show that the PSC1 enzyme effectively decarboxylates tCA into styrene, whilst the PSD1 enzyme does not. The optimal conditions for the PSC1 enzyme, including pH and temperature were determined. The L-phenylalanine DOT-T1E derivative Pseudomonas putida CM12-5 that overproduces L-phenylalanine was used as the host for expression of pal/psc1 genes to efficiently convert L-phenylalanine into tCA, and the aromatic carboxylic acid into styrene. The highest styrene production was achieved when the pal and psc1 genes were co-expressed as an operon in P. putida CM12-5. This construction yielded styrene production exceeding 220 mg L-1. This study serves as a successful demonstration of our strategy to tailor functional enzymes for novel host organisms, thereby broadening their metabolic capabilities. This breakthrough opens the doors to the synthesis of aromatic hydrocarbons using Pseudomonas putida as a versatile biofactory.

Oxidative stress and inflammation cause auditory system damage via glial cell activation and dysregulated expression of gap junction proteins in an experimental model of styrene-induced oto/neurotoxicity.

BACKGROUND: Redox imbalance and inflammation have been proposed as the principal mechanisms of damage in the auditory system, resulting in functional alterations and hearing loss. Microglia and astrocytes play a crucial role in mediating oxidative/inflammatory injury in the central nervous system; however, the role of glial cells in the auditory damage is still elusive. OBJECTIVES: Here we investigated glial-mediated responses to toxic injury in peripheral and central structures of the auditory pathway, i.e., the cochlea and the auditory cortex (ACx), in rats exposed to styrene, a volatile compound with well-known oto/neurotoxic properties. METHODS: Male adult Wistar rats were treated with styrene (400 mg/kg daily for 3 weeks, 5/days a week). Electrophysiological, morphological, immunofluorescence and molecular analyses were performed in both the cochlea and the ACx to evaluate the mechanisms underlying styrene-induced oto/neurotoxicity in the auditory system. RESULTS: We showed that the oto/neurotoxic insult induced by styrene increases oxidative stress in both cochlea and ACx. This was associated with macrophages and glial cell activation, increased expression of inflammatory markers (i.e., pro-inflammatory cytokines and chemokine receptors) and alterations in connexin (Cxs) and pannexin (Panx) expression, likely responsible for dysregulation of the microglia/astrocyte network. Specifically, we found downregulation of Cx26 and Cx30 in the cochlea, and high level of Cx43 and Panx1 in the ACx. CONCLUSIONS: Collectively, our results provide novel evidence on the role of immune and glial cell activation in the oxidative/inflammatory damage induced by styrene in the auditory system at both peripheral and central levels, also involving alterations of gap junction networks. Our data suggest that targeting glial cells and connexin/pannexin expression might be useful to attenuate oxidative/inflammatory damage in the auditory system.

Expression of the two-component regulator StyS/StyR enhanced transcription of the styrene monooxygenase gene styAB and indigo biosynthesis in Escherichia coli.

Indigo, an economically important dye, could be biosynthesized from indole by catalysis of the styrene monooxygenase StyAB. To enhance indigo biosynthesis, the styAB gene and its transcription regulator gene styS/styR in styrene catabolism were cloned from Pseudomonas putida and coexpressed in Escherichia coli. The presence of the intact regulator gene styS/styR dramatically increased the transcriptional levels of styA and styB by approximately 120-fold in the recombinant strain SRAB2 with coexpression of styS/styR and styAB compared to the control strain ABST with solo expression of styAB. A yield of 67.6 mg/L indigo was detected in strain SRAB2 after 24 h of fermentation with 120 µg/mL indole, which was approximately 14-fold higher than that in the control strain ABST. The maximum yield of indigo was produced from 160 µg/mL indole in fermentation of strain SRAB2. However, the addition of styrene to the media significantly inhibited the transcription of styA and styB and consequent indigo biosynthesis in recombinant E. coli strains. Furthermore, the substitution of indole with tryptophan as the fermentation substrate remarkably boosted indigo production, and the maximal yield of 565.6 mg/L was detected in strain SRAB2 in fermentation with 1.2 mg/mL tryptophan. The results revealed that the regulation of styAB transcription by the two-component regulator StyS/StyR in styrene catabolism in P. putida was effective in E. coli, which provided a new strategy for the development of engineered E. coli strains with the capacity for highly efficient indigo production.

Effect of ionic strength on the assembly of simian vacuolating virus capsid protein around poly(styrene sulfonate).

Virus-like particles (VLPs) are noninfectious nanocapsules that can be used for drug delivery or vaccine applications. VLPs can be assembled from virus capsid proteins around a condensing agent, such as RNA, DNA, or a charged polymer. Electrostatic interactions play an important role in the assembly reaction. VLPs assemble from many copies of capsid protein, with a combinatorial number of intermediates. Hence, the mechanism of the reaction is poorly understood. In this paper, we combined solution small-angle X-ray scattering (SAXS), cryo-transmission electron microscopy (TEM), and computational modeling to determine the effect of ionic strength on the assembly of Simian Vacuolating Virus 40 (SV40)-like particles. We mixed poly(styrene sulfonate) with SV40 capsid protein pentamers at different ionic strengths. We then characterized the assembly product by SAXS and cryo-TEM. To analyze the data, we performed Langevin dynamics simulations using a coarse-grained model that revealed incomplete, asymmetric VLP structures consistent with the experimental data. We found that close to physiological ionic strength, [Formula: see text] VLPs coexisted with VP1 pentamers. At lower or higher ionic strengths, incomplete particles coexisted with pentamers and [Formula: see text] particles. Including the simulated structures was essential to explain the SAXS data in a manner that is consistent with the cryo-TEM images.

Three-dimensional cultured ampullae from rats as a screening tool for vestibulotoxicity: Proof of concept using styrene.

Numerous ototoxic drugs, such as some antibiotics and chemotherapeutics, are both cochleotoxic and vestibulotoxic (causing hearing loss and vestibular disorders). However, the impact of some industrial cochleotoxic compounds on the vestibular receptor, if any, remains unknown. As in vivo studies are long and expensive, there is considerable need for predictive and cost-effective in vitro models to test ototoxicity. Here, we present an organotypic model of cultured ampullae harvested from rat neonates. When cultured in a gelatinous matrix, ampulla explants form an enclosed compartment that progressively fills with a high-potassium (K+) endolymph-like fluid. Morphological analyses confirmed the presence of a number of cell types, sensory epithelium, secretory cells, and canalar cells. Treatments with inhibitors of potassium transporters demonstrated that the potassium homeostasis mechanisms were functional. To assess the potential of this model to reveal the toxic effects of chemicals, explants were exposed for either 2 or 72 h to styrene at a range of concentrations (0.5-1 mM). In the 2-h exposure condition, K+ concentration was significantly reduced, but ATP levels remained stable, and no histological damage was visible. After 72 h exposure, variations in K+ concentration were associated with histological damage and decreased ATP levels. This in vitro 3D neonatal rat ampulla model therefore represents a reliable and rapid means to assess the toxic properties of industrial compounds on this vestibular tissue, and can be used to investigate the specific underlying mechanisms.

Conformations of Human Immunodeficiency Virus Envelope Glycoproteins in Detergents and Styrene-Maleic Acid Lipid Particles.

The mature human immunodeficiency virus (HIV) envelope glycoprotein (Env) trimer, which consists of noncovalently associated gp120 exterior and gp41 transmembrane subunits, mediates virus entry into cells. The pretriggered (State-1) Env conformation is the major target for broadly neutralizing antibodies (bNAbs), whereas receptor-induced downstream Env conformations elicit immunodominant, poorly neutralizing antibody (pNAb) responses. To examine the contribution of membrane anchorage to the maintenance of the metastable pretriggered Env conformation, we compared wild-type and State-1-stabilized Envs solubilized in detergents or in styrene-maleic acid (SMA) copolymers. SMA directly incorporates membrane lipids and resident membrane proteins into lipid nanoparticles (styrene-maleic acid lipid particles [SMALPs]). The integrity of the Env trimer in SMALPs was maintained at both 4°C and room temperature. In contrast, Envs solubilized in Cymal-5, a nonionic detergent, were unstable at room temperature, although their stability was improved at 4°C and/or after incubation with the entry inhibitor BMS-806. Envs solubilized in ionic detergents were relatively unstable at either temperature. Comparison of Envs solubilized in Cymal-5 and SMA at 4°C revealed subtle differences in bNAb binding to the gp41 membrane-proximal external region, consistent with these distinct modes of Env solubilization. Otherwise, the antigenicity of the Cymal-5- and SMA-solubilized Envs was remarkably similar, both in the absence and in the presence of BMS-806. However, both solubilized Envs were recognized differently from the mature membrane Env by specific bNAbs and pNAbs. Thus, detergent-based and detergent-free solubilization at 4°C alters the pretriggered membrane Env conformation in consistent ways, suggesting that Env assumes default conformations when its association with the membrane is disrupted. IMPORTANCE The human immunodeficiency virus (HIV) envelope glycoproteins (Envs) in the viral membrane mediate virus entry into the host cell and are targeted by neutralizing antibodies elicited by natural infection or vaccines. Detailed studies of membrane proteins rely on purification procedures that allow the proteins to maintain their natural conformation. In this study, we show that a styrene-maleic acid (SMA) copolymer can extract HIV-1 Env from a membrane without the use of detergents. The Env in SMA is more stable at room temperature than Env in detergents. The purified Env in SMA maintains many but not all of the characteristics expected of the natural membrane Env. Our results underscore the importance of the membrane environment to the native conformation of HIV-1 Env. Purification methods that bypass the need for detergents could be useful tools for future studies of HIV-1 Env structure and its interaction with receptors and antibodies.

Multi-omic based production strain improvement (MOBpsi) for bio-manufacturing of toxic chemicals.

Robust systematic approaches for the metabolic engineering of cell factories remain elusive. The available models for predicting phenotypical responses and mechanisms are incomplete, particularly within the context of compound toxicity that can be a significant impediment to achieving high yields of a target product. This study describes a Multi-Omic Based Production Strain Improvement (MOBpsi) strategy that is distinguished by integrated time-resolved systems analyses of fed-batch fermentations. As a case study, MOBpsi was applied to improve the performance of an Escherichia coli cell factory producing the commodity chemical styrene. Styrene can be bio-manufactured from phenylalanine via an engineered pathway comprised of the enzymes phenylalanine ammonia lyase and ferulic acid decarboxylase. The toxicity, hydrophobicity, and volatility of styrene combine to make bio-production challenging. Previous attempts to create styrene tolerant E. coli strains by targeted genetic interventions have met with modest success. Application of MOBpsi identified new potential targets for improving performance, resulting in two host strains (E. coli NST74ΔaaeA and NST74ΔaaeA cpxPo) with increased styrene production. The best performing re-engineered chassis, NST74ΔaaeA cpxPo, produced ∼3 × more styrene and exhibited increased viability in fed-batch fermentations. Thus, this case study demonstrates the utility of MOBpsi as a systematic tool for improving the bio-manufacturing of toxic chemicals.

Production of Enantiopure Chiral Epoxides with E. coli Expressing Styrene Monooxygenase.

Styrene monooxygenases are a group of highly selective enzymes able to catalyse the epoxidation of alkenes to corresponding chiral epoxides in excellent enantiopurity. Chiral compounds containing oxirane ring or products of their hydrolysis represent key building blocks and precursors in organic synthesis in the pharmaceutical industry, and many of them are produced on an industrial scale. Two-component recombinant styrene monooxygenase (SMO) from Marinobacterium litorale was expressed as a fused protein (StyAL2StyB) in Escherichia coli BL21(DE3). By high cell density fermentation, 35 gDCW/L of biomass with overexpressed SMO was produced. SMO exhibited excellent stability, broad substrate specificity, and enantioselectivity, as it remained active for months and converted a group of alkenes to corresponding chiral epoxides in high enantiomeric excess (˃95-99% ee). Optically pure (S)-4-chlorostyrene oxide, (S)-allylbenzene oxide, (2R,5R)-1,2:5,6-diepoxyhexane, 2-(3-bromopropyl)oxirane, and (S)-4-(oxiran-2-yl)butan-1-ol were prepared by whole-cell SMO.

Biochemical Characterization of Phenylacetaldehyde Dehydrogenases from Styrene-degrading Soil Bacteria.

Four phenylacetaldehyde dehydrogenases (designated as FeaB or StyD) originating from styrene-degrading soil bacteria were biochemically investigated. In this study, we focused on the Michaelis-Menten kinetics towards the presumed native substrate phenylacetaldehyde and the obviously preferred co-substrate NAD+. Furthermore, the substrate specificity on four substituted phenylacetaldehydes and the co-substrate preference were studied. Moreover, these enzymes were characterized with respect to their temperature as well as long-term stability. Since aldehyde dehydrogenases are known to show often dehydrogenase as well as esterase activity, we tested this capacity, too. Almost all results showed clearly different characteristics between the FeaB and StyD enzymes. Furthermore, FeaB from Sphingopyxis fribergensis Kp5.2 turned out to be the most active enzyme with an apparent specific activity of 17.8 ± 2.1 U mg-1. Compared with that, both StyDs showed only activities less than 0.2 U mg-1 except the overwhelming esterase activity of StyD-CWB2 (1.4 ± 0.1 U mg-1). The clustering of both FeaB and StyD enzymes with respect to their characteristics could also be mirrored in the phylogenetic analysis of twelve dehydrogenases originating from different soil bacteria.

Enhanced cis- and enantioselective cyclopropanation of styrene catalysed by cytochrome P450BM3 using decoy molecules.

We report the enhanced cis- and enantioselective cyclopropanation of styrene catalysed by cytochrome P450BM3 in the presence of dummy substrates, i.e. decoy molecules. With the aid of the decoy molecule R-Ibu-Phe, diastereoselectivity for the cis diastereomers reached 91%, and the enantiomeric ratio for the (1S,2R) isomer reached 94%. Molecular dynamics simulations underpin the experimental data, revealing the mechanism of how enantioselectivity is controlled by the addition of decoy molecules.

Snow microbiome functional analyses reveal novel aspects of microbial metabolism of complex organic compounds.

Microbes active in extreme cold are not as well explored as those of other extreme environments. Studies have revealed a substantial microbial diversity and identified cold-specific microbiome molecular functions. We analyzed the metagenomes and metatranscriptomes of 20 snow samples collected in early and late spring in Svalbard, Norway using mi-faser, our read-based computational microbiome function annotation tool. Our results reveal a more diverse microbiome functional capacity and activity in the early- vs. late-spring samples. We also find that functional dissimilarity between the same-sample metagenomes and metatranscriptomes is significantly higher in early than late spring samples. These findings suggest that early spring samples may contain a larger fraction of DNA of dormant (or dead) organisms, while late spring samples reflect a new, metabolically active community. We further show that the abundance of sequencing reads mapping to the fatty acid synthesis-related microbial pathways in late spring metagenomes and metatranscriptomes is significantly correlated with the organic acid levels measured in these samples. Similarly, the organic acid levels correlate with the pathway read abundances of geraniol degradation and inversely correlate with those of styrene degradation, suggesting a possible nutrient change. Our study thus highlights the activity of microbial degradation pathways of complex organic compounds previously unreported at low temperatures.

Production of Substituted Styrene Bioproducts from Lignin and Lignocellulose Using Engineered Pseudomonas putida KT2440.

Ferulic acid is a renewable chemical found in lignocellulose from grasses such as wheat straw and sugarcane. Pseudomonas putida is able to liberate and metabolize ferulic acid from plant biomass. Deletion of the hydroxycinnamoyl-CoA hydratase-lyase gene (ech) produced a strain of P. putida unable to utilize ferulic and p-coumaric acid, which is able to accumulate ferulic acid and p-coumaric acid from wheat straw or sugar cane bagasse. Further engineering of this strain saw the replacement of ech with the phenolic acid decarboxylase padC, which converts p-coumaric and ferulic acid into 4-vinylphenol and the flavor agent 4-vinylguaiacol, respectively. The engineered strain containing padC is able to generate 4-vinylguaiacol and 4-vinylphenol from media containing lignocellulose or Green Value Protobind lignin as feedstock, and does not require the addition of an exogenous inducer molecule. Biopolymerization of 4-vinylguaiacol and 4-vinylcatechol styrene products is also carried out, using Trametes versicolor laccase, to generate "biopolystyrene" materials on small scale.

Cell-free styrene biosynthesis at high titers.

Styrene is an important petroleum-derived molecule that is polymerized to make versatile plastics, including disposable silverware and foamed packaging materials. Finding more sustainable methods, such as biosynthesis, for producing styrene is essential due to the increasing severity of climate change as well as the limited supply of fossil fuels. Recent metabolic engineering efforts have enabled the biological production of styrene in Escherichia coli, but styrene toxicity and volatility limit biosynthesis in cells. To address these limitations, we have developed a cell-free styrene biosynthesis platform. The cell-free system provides an open reaction environment without cell viability constraints, which allows exquisite control over reaction conditions and greater carbon flux toward product formation rather than cell growth. The two biosynthetic enzymes required for styrene production were generated via cell-free protein synthesis and mixed in defined ratios with supplemented L-phenylalanine and buffer. By altering the time, temperature, pH, and enzyme concentrations in the reaction, this approach increased the cell-free titer of styrene from 5.36 ± 0.63 mM to 40.33 ± 1.03 mM, the highest amount achieved using biosynthesis without process modifications and product removal strategies. Cell-free systems offer a complimentary approach to cellular synthesis of small molecules, which can provide particular benefits for producing toxic molecules.

Simultaneous biodegradation of methane and styrene in biofilters packed with inorganic supports: Experimental and macrokinetic study.

Four upflow 0.018 m3 biofilters (3 beds), B-ME, B-200, B-500 and B-700, all packed with inorganic materials, were operated at a constant air flow rate of 0.18 m3 h-1 to eliminate methane (CH4), a harmful greenhouse gas (GHG), and styrene (C8H8), a carcinogenic volatile organic compound (VOC). The biofilters were irrigated with 0.001 m3 of recycled nutrient solution (NS) every day (flow rate of 60 × 10-3 m3 h-1). Styrene inlet load (IL) was kept constant in each biofilter. Different CH4-ILs varying in the range of 7-60 gCH4 m-3 h-1 were examined in B-ME (IL of 0 gC8H8 m-3 h-1), B-200 (IL of 9 gC8H8 m-3 h-1), B-500 (IL of 22 gC8H8 m-3 h-1) and B-700 (IL of 32 gC8H8 m-3 h-1). Finally, the effect of C8H8 on the macrokinetic parameters of CH4 biofiltration was studied based on the Michaelis-Menten model. Average C8H8 removal efficiencies (RE) varying between 64 and 100% were obtained at CH4-ILs increasing from 7 to 60 gCH4 m-3 h-1 and for C8H8-ILs range of 0-32 gC8H8 m-3 h-1. More than 90% of C8H8 was removed in the bottom and middle beds of the biofilters. By increasing C8H8-IL from 0 to 32 gC8H8 m-3 h-1, maximal EC in Michaelis-Menten model and macrokinetic saturation constant declined from 311 to 39 g m-3 h-1 and from 19 to 2.3 g m-3, respectively, which confirmed that an uncompetitive inhibition occurred during CH4 biofiltration in the presence of C8H8.

Benzoic acid production via cascade biotransformation and coupled fermentation-biotransformation.

As an important bulk chemical, benzoic acid is currently manufactured from nonrenewable feedstocks under harsh conditions. Although there are natural pathways for biosynthesis of benzoic acid, they are often inefficient and subjected to complex regulation. Here we develop a nonnatural enzyme cascade to efficiently produce benzoic acid from styrene or biogenic L-phenylalanine under mild conditions. By using a modular approach, two whole-cell catalysts Escherichia coli LZ305 and LZ325 are engineered for coexpressing seven and nine enzymes for production of 133-146 mM benzoic acid (16.2-17.8 g/Laq ) with 88-97% conversion via seven- and nine-step cascade biotransformation of styrene and L-phenylalanine, respectively. The seven-step cascade represents a formal high-yielding biocatalytic oxidative cleavage of styrene, and the nine-step cascade showcases the high efficiency of extended nonnatural enzyme cascades. Moreover, to achieve benzoic acid production directly from low-cost renewable glycerol, a novel coupled fermentation-biotransformation process was developed by integration of fermentative production of L-phenylalanine with in situ biotransformation to give 63-70 mM benzoic acid (7.6-8.6 g/Laq ), which is around 20 times higher than the reported value via a natural pathway. The coupled fermentation-biotransformation process could be generally applicable to microbial production of growth-inhibitory or toxic chemicals in high concentrations.

Production of styrene oxide from styrene by a recombinant Escherichia coli with enhanced AcrAB-TolC efflux pump level in an aqueous-organic solvent two-phase system.

The AcrAB-TolC efflux pump is involved in the organic solvent tolerance of Escherichia coli. Most E. coli strains are highly sensitive to organic solvents such as n-hexane and cyclohexane. Here, a recombinant E. coli transformed with an expression plasmid containing acrAB and tolC became tolerant to n-hexane and cyclohexane. The levels of AcrA, AcrB, and TolC in the recombinant increased by 3- to 5-fold compared to those in the control strain without the plasmid for acrAB or tolC. To investigate the usability of the recombinant as a biocatalyst in an aqueous-organic solvent two-phase system, we further introduced xylMA xylene monooxygenase genes from Pseudomonas putida mt-2 into the recombinant and examined the production of styrene oxide from styrene. The resulting recombinant produced 1.8 mg and 1.0 mg styrene oxide mL-1 of medium in a medium overlaid with a 25% volume of n-hexane and cyclohexane containing 10% (wt vol-1) styrene, respectively.

Contribution of bacterial biodiversity on the operational performance of a styrene biotrickling filter.

Long-term operational stability of biotrickling filters (BTFs) degrading volatile organic compounds (VOCs) is dependent on both physicochemical as well as biological properties. Effects of increasingly stressful levels of air pollutants on the microbial structure of biofilms within BTFs are not well understood, especially for VOCs such as styrene. To investigate the relationship between biofilm biodiversity and operational stability, the temporal dynamics of a biofilm from a biotrickling filter subjected to stepwise increasing levels of air polluted with styrene was investigated using 16S rDNA pyrosequencing and PCR-denaturing gradient gel electrophoresis (PCR-DGGE). As styrene contaminant loads were increased, microbial community composition was distinctly altered and diversity was initially reduced in early stages but gradually stabilized and increased diversity in later stages, suggesting a recovery and acclimatization period within the microbial community during incremental exposure of the pollutant. Although temporary reductions in known styrene-degrading bacterial genera (Pseudomonas and Rhodococcus) occurred under increased styrene loads, stable BTF performance was maintained due to functional redundancy. New candidate genera for styrene degradation (Azoarcus, Dokdonella) were identified in conditions of high styrene loads, and may have supported the observed stable BTF performance throughout the experiment. Styrene inlet load was found to be important modulator of community composition and may have been partly responsible for the observed temporary reductions of Pseudomonas. Notable differences between dominant genera detected via pyrosequencing compared to species detected by PCR-DGGE suggests that simultaneous implementation of both techniques is valuable for fully characterizing dynamic microbial communities.

Genome engineering of E. coli for improved styrene production.

Microbial production of exogenous organic compounds is challenging as biosynthetic pathways are often complex and produce metabolites that are toxic to the hosts. Biogenic styrene is an example of this problem, which if addressed could result in a more sustainable supply of this important component of the plastics industry. In this study, we engineered Escherichia coli for the production of styrene. We systematically optimized the production capability by first screening different pathway expression levels in E. coli strains. We then further designed and constructed a transcription regulator library targeting 54 genes with 85,420 mutations, and tested this library for increased styrene resistance and production. A series of tolerant mutants not only exhibited improved styrene tolerance but also produced higher styrene concentrations compared to the parent strain. The best producing mutant, ST05 LexA_E45I, produced a 3.45-fold increase in styrene compared to the parent strain. The produced styrene was extracted via gas stripping into dodecane and used in a direct free radical synthesis of polystyrene.

Enhanced biodegradation of styrene vapors in the biotrickling filter inoculated with biosurfactant-generating bacteria under H2O2 stimulation.

Biotrickling filters (BTFs) applied to hydrophobic volatile organic compounds (VOCs) suffer from limited mass transfer. Phase transfer kinetic and equilibrium effects limit the biodegradation of hydrophobic VOCs especially at high concentrations. This study evaluates two strategies for overcoming the problem. First, a natural process was used to enhance the aqueous availability of styrene, a hydrophobic VOC model, by inoculating the BTF with a mixture of biosurfactant-generating bacteria. This method achieved a maximum elimination capacity (ECmax) of 139 g m-3h-1 in the BTF at an empty bed residence time (EBRT) of 60s. The highest concentrations of the biosurfactants surfactin and rhamnolipid were 205 and 86 mg L-1, respectively, in this step. Sequencing 16S rRNA confirmed the presence of biosurfactant-producing bacteria capable of biodegrading styrene in the BTF including Bacillus sonorensis, Bacillus subtilis, Lysinibacillus sphaericus, Lysinibacillus fusiformis, Alcaligenes feacalis, Arthrobacter creatinolyticus, and Kocuria rosea. Second, the effect of adding H2O2 to the recycle liquid on the BTF performance was determined. The biodegradation and mineralization of styrene in the BTF operated at a loading rate of 266 g m-3h-1 and H2O2/styrene molar ratio of 0.05 with EBRT as short as 15 s were 94% and 53%, respectively, with the EC of 250 g m-3h-1. High concentrations of antioxidant enzymes (peroxidase and catalase: 56 and 7 U gbiomass-1, respectively) were produced and biosurfactant generation was increased in this step, contributing to enhanced styrene biodegradation and mineralization. The styrene biodegradation and mineralization values in the BTF in the last day operated under similar conditions but without H2O2 were 11.4% and 5.3%, respectively. The bacterial population had no considerable change in the BTF after adding H2O2. Accordingly, stimulating the BTF inoculated with biosurfactant-generating bacteria with H2O2 is a promising strategy for improving the biodegradation of hydrophobic VOCs.