High‐Titer Bio‐Styrene Production Afforded by Whole‐Cell Cascade Biotransformation

Biosynthetic routes based on cost‐efficient, eco‐friendly, and sustainable platforms for compounds such as styrene are urgently needed. The biosynthesis of styrene from L‐phenylalanine (L‐Phe) via trans‐cinnamate has long been established, but styrene toxicity limits yields. This study demonstrates that whole‐cell cascade biotransformation employing an E. coli consortium expressing respectively phenylalanine ammonia‐lyase and ferulic acid decarboxylase negates both the issue of styrene toxicity and the need for enzyme purification. Using resting or lyophilised cells, efficient conversion of L‐Phe to styrene (up to ∼24 g/L) is readily achieved and combined with robust extraction methods. The use of L‐Phe enriched biomass with the E. coli consortium yields an equally robust and rapid production and isolation of renewable styrene. This study establishes an improved strategy for industrial bio‐production of styrene, and by extension other toxic or reactive chemicals from corresponding bio‐compatible precursors.


Introduction
Styrene is a large-volume commodity petrochemical with a diverse range of commercial applications in the synthesis of polymers and other fine chemicals and pharmaceutics. Styrene is extensively used for producing a wide selection of industrial polymer and co-polymers such as polystyrene, acrylonitrilebutadiene-styrene, styrene-acrylonitrile and styrene-butadiene rubber. Recent applications of polystyrene include its use in the synthesis of nanobeads in the lateral flow assay for SARS-CoV-2 antibody detection, and in the polystyrene-toner centrifugal microfluidic device for rapid molecular diagnosis of COVID-19. [1,2] Due to the widespread applications of styrene polymers and functional co-polymers, there is an increasing demand for styrene production. The global market for styrene is valued at 56.51 billion USD in 2022 and is anticipated to reach 77.92 billion USD by 2028. [3] Conventional production of styrene depends on chemical catalysis, an energetically demanding route that relies mainly on fossil fuel resources. [4] Fortunately, styrene production occurs naturally in various organisms such as bacteria, yeast and certain plant species, making bio-production of styrene feasible. [5][6][7] The biosynthesis of styrene from the naturally occurring amino acid L-phenylalanine (L-Phe) occurs via transcinnamate as shown in Scheme 1. Non-oxidative deamination of L-Phe is catalysed by phenylalanine ammonia-lyase (PAL) to trans-cinnamate. [8][9][10] This is followed by a decarboxylation reaction catalysed by ferulic acid decarboxylase (Fdc1) to produce styrene. [11,12] A technoeconomic evaluation has demonstrated that production of styrene by biosynthetic routes could be competitive with the existing petroleum-based synthetic route [13] and several recombinant hosts including Escherichia coli (E. coli) and Saccharomyces cerevisiae have been engineered to produce styrene in order to accomplish this goal. [5,[14][15][16][17][18] However, despite sustained efforts to produce styrene in microbial hosts, the titers, productivities and yield are not adequate to meet commercial-scale production.
One major issue that currently hinders high-titer production is styrene toxicity on growing cells, with 350 mg styrene per litre the highest achieved so far in vivo in E. coli. [16] Other approaches attempted to improve this titer via genome engineering, in vitro cell-free styrene biosynthesis and strain improvement for biomanufacturing of toxic compounds, have achieved 4.2-5.3 g/L of styrene. [17,[19][20][21] Nevertheless, these approaches are not (yet) economically viable, and the challenging task of developing a cost-effective eco-friendly renewable bioprocess to meet the commercial demand of styrene remains. Table 1 summarises the previous reported titers of styrene bioproduction.
To avoid the issue of styrene toxicity, we here present a two-step approach to styrene production, accumulating the biocompatible precursor L-Phe prior to a whole-cell based cascade biotransformation for robust production of bio-styrene.

Results and Discussion
A panel of four PAL enzymes (PbPAL from Planctomyces brasiliensis, DdPAL from Dictyostelium discoideum, RgPAL from Rhodotorula glutinis, AvPAL from Anabaena variabilis) as well as Aspergillus niger Fdc1 (AnFdc1) were each heterologously produced in E. coli BL21 (DE3) cells ( Figure S1) as described in the methods section, Supporting Information. The Fdc1 construct (AnFdc1/EcUbiX) includes expression of the flavin prenyltransferase (UbiX) that yields the prenylated FMN (prFMN) cofactor required for the Fdc1 decarboxylation activity. [11] Resting cells were prepared for each of the PALs and AnFdc1/ EcUbiX, and employed in the biotransformation reactions as described in the experimental section. The reactions were analysed by HPLC analysis (as described in the experimental section) to monitor levels of the L-Phe substrate, the transcinnamate intermediate and the styrene product. To quantify the bio-styrene production, quantitative HPLC analyses were performed utilising styrene standard curves ( Figure S7) prepared as described in the experimental section. The styrene standards were prepared in the reaction buffer and in control (empty) cell cultures of E. coli BL21(DE3) to mimic the conditions of the biotransformation, sample preparation and analysis. Analysis of styrene standards added to cell culture were identical compared to those added to buffer ( Figure S7), indicating the analysis protocol used to quantify styrene is appropriate.
Of the four PALs tested, both RgPAL and AvPAL showed complete consumption of L-Phe (10 mM) when combined with AnFdc1/EcUbiX producing styrene, with RgPAL revealing the best biotransformation efficiency under the conditions used ( Figure 1). RgPAL performs so much better than other PALs tested, in line with the fact RgPAL has the highest catalytic capacity among the reported PALs, with k cat /K m of 1.9 × 10 4 mM À 1 s À 1 for L-Phe ammonia elimination. [22] Figure S4 illustrates the multiple sequence alignment of the four PAL tested in this study.
The efficiency of the whole-cell biotransformation was further optimised as described in the experimental section. Initial optimisation indicated that variation of the pH from 6 to 8 has no significant effect on the efficiency of the biotransformation reaction catalysed by RgPAL and AnFdc1/EcUbiX under the conditions used ( Figure S5). Therefore pH 7.0 was chosen to conduct the biotransformation reactions at 30°C, a value previously reported to be optimal for the combined PAL/Fdc1 reaction. [19] Initial amount of cell pellet used in the reaction was 30 g cdw/L, followed by testing lower amounts to determine the minimum amount required with a view of cost efficient industrial/commercial application. Varying the amount of the resting-cell biocatalysts (10-30 g cdw/L) in the reaction revealed that L-Phe is fully converted to styrene within 1-4 h, with the time point at which full conversion is obtained linked to the quantity of the biocatalysts included ( Figure 2).
To ensure the approach used is robust, we prepared replicate RgPAL and AnFdc1/EcUbiX cells, and tested the biotransformation protocol with distinct cell preparations (Figure 3). This reveals different combinations of various preparations of RgPAL and AnFdc1/EcUbiX yield very similar results, validating the robustness of the biotransformation protocol under the conditions used.
We have also tested the efficiency of the biotransformation protocol using various quantities of lyophilised cell biocatalysts (1-20 g/L), prepared as described in the experimental section. The lyophilised biocatalysts were employed at those levels as the lyophilised cell weight is approximately 20-25 % of the corresponding resting cell weight. Up to 20 g/L were initially tried to test the performance and stability of the lyophilised biocatalysts. Our results demonstrate that employing modest quantities of lyophilised biocatalysts (as small as 1 g per litre reaction) are able to produce styrene from L-Phe under the conditions used ( Figure 4).
The biotransformation protocol employing either resting cells (20 g cdw/L) or lyophilised cells (5 g/L) of RgPAL and AnFdc1/EcUbiX biocatalysts was examined for efficiency of transformation with concentrations of up to 100 mM L-Phe (16.52 g/L) as well as a saturated solution and L-Phe suspensions (Table 2). In case of concentrations up to 100 mM, complete consumption of L-Phe was mostly achieved within 2 h. For example, reactions containing 100 mM L-Phe produce 9.60 � 0.03 g/L styrene under the conditions used. Figure S8 illustrates some examples and Table 2 presents a summary of the styrene titers achieved under the conditions used. Styrene Cell-free biosynthesis [20] 5.3 g/L Engineered E. coli strain and in situ product recovery (ISPR) with n-dodecane and gas stripping [17] product levels were found to be 91 � 1 % of the expected titers. This is likely due in part to the volatile nature of styrene, which can be easily lost during sample preparation, dilution, and analysis. It is noted that no trans-cinnamate accumulation was observed with L-Phe concentrations of < 100 mM. For higher L-Phe concentrations (� 100 mM), a small amount of transcinnamate (1-2.5 mM) was occasionally observed, accounting for~1-2.5 % of the L-Phe concentration under the conditions used only when the reaction time exceeds 12 h. This suggests that at higher styrene and CO 2 concentrations accumulated under the closed volume reaction conditions used, the Fdc1 mediated reaction does not go to completion.
The solubility of L-Phe does not extend much beyond 100 mM at room temperature (25°C), but higher styrene titres were obtained when (super)saturated L-Phe solutions or L-Phe suspensions were used in the biotransformation reactions at 30°C. This yielded a production of up to 18.3 g/L styrene and demonstrates L-Phe availability as one of the main limiting factors, offering a promising green route towards industrial bioproduction of styrene. To further improve this strategy for industrial application, the styrene product was captured from the biotransformation reactions (at 37°C) by use of a cold trap as described in the experimental section. Up to 23.9 � 1.1 g/L (230 � 11 mM) highly pure styrene was captured from L-Phe (264.9 � 5.2 mM) in 4 h ( Figure S9).
Having established L-Phe can be readily converted to styrene, we sought to prove this could be readily applied to L-Phe rich biomass. The potential use of protein-rich biomass, agro-industrial and food waste as L-Phe sources for bio-styrene production is a promising route. We started with testing casamino acids. Casamino acids were dissolved in water to   biotransformation is not inhibited by other amino acids. We then examined blue green spirulina algae and pumpkin seed powders (containing approximately 65-70 % and 60-65 % protein content respectively) as model sources of L-Phe, after acid hydrolysis treatment and neutralisation as described in the experimental section. The biotransformation reaction for each preparation was prepared by adding 20 mg each of RgPAL and AnFdc1/EcUbiX resting cells to 1 mL volume. For Spirulina powder, L-Phe content in the biotransformation reaction was 9.6 � 0.8 mM, the styrene product was 8.9 � 0.6 mM after 2 h, accounting for 93 % of the expected titer. In case of pumpkin seeds powder, L-Phe content in the reaction was 1.6 � 0.2 mM and the produced styrene was 1.3 � 0.2 mM after 2 h, accounting for 83 % of the expected titer. The results reveals that the developed protocol could be widely applied to produce styrene from various L-Phe containing sources and is not inhibited by other biomass components.
Several microbial strains capable of producing L-Phe from cheap carbon sources have been reported, which can be readily coupled to the established biotransformation protocol in a twostep procedure. The E. coli NST74 cells (as an example of L-Phe producing strains) [23,24] were grown in shaking flasks and in a small laboratory fermenter as described in the methods section, Supporting Information. NST74 cells were initially grown in shaking flasks at 37°C ( Figure S2) demonstrating L-Phe production (Table S1). The NST74 cells were grown in small fed-batch fermentation at 33°C in MM12 media for fermentative production of L-Phe, with a production of 10.7 mM of L-Phe (1.8 g/L) under non-optimised conditions ( Figure S3, Table S2). Coupling the NST74 L-Phe production with the biotransformation was achieved by suspending the RgPAL and AnFdc1/EcUbiX biocatalysts (20 cdw or 5 g lyophilised cells/L reaction) into the NST74 culture following complete fermentation. Full consumption of the fermentative L-Phe product was achieved within 2 h ( Figure S10). HPLC analysis revealed production of 0.81-0.91 g/L of styrene, equivalent to 77.9-87.5 % of the expected styrene titer. Extraction of styrene produced from L-Phe rich biomass via a cold trap as described in the method section, Supporting Information, reveals no impurities were detected, and confirm pure styrene is efficiently obtained from biomass using this protocol ( Figure S11).

Conclusion
The biotransformation approach described in this laboratory scale study thus demonstrates a promising route towards costefficient, eco-friendly, and sustainable production of biostyrene, employing a combination of resting cells expressing RgPAL and AnFdc1/EcUbiX. Our data showed the two-step biotransformation accomplished efficient styrene production from L-Phe and L-Phe rich sources by employing resting E. coli cells expressing the biocatalysts. For whole-cell biotransformation to proceed, the substrates and products must be transported across the cell membrane. Cells normally have transport systems to move compounds in and out as the cell needs them and amino acids and L-Phe specific transport systems have been identified in E. coli. [25][26][27][28] Based on the result achieved in this study, E. coli cell membrane does not present a barrier to the substrates and products of both biocatalysts (L-Phe, transcinnamic acid and styrene). Employing whole-cell biotransformation with resting cells as opposed to purified enzymes negates the costs of enzyme purification and/or cell lysis. Furthermore, the cells could be used in lyophilised form, and were found to be stable when stored for 9 months ( Figure S12) bypassing the need for refrigerated storage. Crucially, this twostep approach does not rely on cell growth during the styrene production step. Therefore, it does not suffer from the adverse effects of styrene reactivity/toxicity. Furthermore, separating the individual enzyme activities avoids premature styrene production in vivo. While AnFdc1 has been previously used for in vivo styrene production, [5,[16][17][18]20] the AnFdc1/EcUbiX construct used here co-expresses the endogenous E. coli UbiX gene to provide sufficient levels of the prenylated flavin (Fdc1 cofactor) and thus maximise enzyme activity. The RgPAL employed in this study possess the highest catalytic activity amongst the reported PALs. Our results reveal that bio-production of 9.6 g styrene/L is readily achievable from 100 mM L-Phe within 1-4 h time scale, depending mainly on the amount of biocatalyst used at pH 7.0, 30°C, and up to 18.3 g/L styrene is readily achievable from an L-Phe suspension in 4 h. The styrene titer was further improved to~24 g/L through use of a cold trap. To our knowledge, this is the highest reported titer achieved so far for bio-styrene production with the extraction procedure offering a robust method to isolate highly pure product. In this study, the six-fold improvement in styrene titer over the earlier published studies is likely due to a combination of several factors, including the co-expression of EcUbiX (yielding active Fdc1), overcoming the toxicity of styrene on living cells (by using resting E. coli cells), use of higher L-Phe concentrations in the biotransformation reactions, maintaining the pH over the reaction time and the continuous capture of styrene from the reaction by cold trap. The method is compatible with a range of L-Phe sources, such as casamino acids, blue green spirulina algae or pumpkin seed powders. Crucially, L-Phe can be produced from cheap renewable sources using engineered microbial strains reported to produce up to 72.9 g L-Phe/L. [29] Indeed, as proof of principle, combination of the L-Phe producing E. coli strain NST74 with the cascade biotransformation was shown to be an efficient route to produce pure styrene from renewable sources. The protocol presented here presents a starting point for further increase of the styrene titer to develop a promising industrial platform for styrene bioproduction.
The platform and strategy developed in this study could be applied to access other toxic and reactive compounds from corresponding bio-compatible precursors. For example, the ubiquitous ammonia-lyase enzymes can catalyse deamination of other aromatic amino acids such as tyrosine, tryptophan, and histidine which, when combined with Fdc1 or suitable variants, could provide renewable biological routes to other styrene-like compounds. Other routes employing Fdc1 variants to yield isobutene or butadiene in vivo have also been reported. [30,31] In each case, a two-step procedure accumulating the biocompatible acid precursor prior to production of the volatile hydrocarbon using Fdc1-resting cells might offer substantial improvement in titer and product handling.

Experimental Section
Cloning and expression of AnFdc1/EcUbiX construct, expression of PALs for the production of phenylalanine ammonia-lyases (PALs) in E. coli cells and the production of L-Phe from E. coli NST74 strain were described in the Supporting Information.

Optimisation of the biotransformation reaction
Optimising the biotransformation reaction of L-Phe to produce styrene from pure L-Phe (10 mM) was performed utilising the cell pellets of both biocatalysts (a PAL and AnFdc1/EcUbiX) with various amount of cell dry weight of each biocatalyst (10, 20 or 30 g cdw/L) in 50 mM sodium phosphate buffer at pH 7.0 in 1 mL reaction volumes. Reactions were incubated at 30°C, 180 rpm in 7 mL sealed vials and were analysed by HPLC at various time intervals usually between 0-4 h unless otherwise stated. Control reactions were always run in parallel to the biotransformation. Reactions were performed in replicates inside a fume hood.

Biotransformation protocol using resting cells as biocatalysts
Biotransformation was performed at the established optimal condition of 20 g cdw/L of RgPAL or AnFdc1/EcUbiX, 50 mM sodium phosphate buffer pH 7.0, 30°C at 180 rpm with L-Phe or L-Phe sources such as grown cultures of NST74 strain (fermentative L-Phe production as described in Supporting Information), L-Phe in E. coli biomass (grown E. coli culture supplemented with L-Phe), casamino acids, blue green algae spirulina powder, and defatted pumpkin seeds powder. For the spirulina powder and the defatted pumpkin seeds powder an acid hydrolysis step with 6N HCl was performed (50 ml acid for every g) under reflux for 24 h to hydrolyse the proteins into amino acids followed by a neutralisation step with NaOH to pH 7.0. The preparations were then concentrated by vacuum evaporation. Casamino acids powder was dissolved in water and used directly. The reactions were carried out in tightly sealed vials for at least 4 h and in some cases for overnight to monitor any changes in the product profile. All biotransformation reactions were performed inside a fume hood and analysed by HPLC analysis as described below. All reactions were performed in biological and analytical replicates. At least four preparations of each biocatalyst were tested during 6 months of storage and were shown to be stable.

Biotransformation protocol using lyophilised biocatalysts
Biotransformation reactions were also performed using the lyophilised resting cells of RgPAL and AnFdc1/EcUbiX biocatalysts as described for the resting cells. The harvested cells of each biocatalyst were washed in 100 mM phosphate buffered saline (PBS) buffer pH 7.4, instantly flash frozen in liquid nitrogen followed by freeze-drying under high vacuum for~24-48 h to yield a powdered formulation. The lyophilised powder was stored at room temperature (25°C) in sealed falcon tubes (under moisture free conditions) until usage and was tested over 9 months and shown to be stable.

Capture of styrene product by cold trapping
Styrene product was captured from the biotransformation reactions by cold trapping inside a fume hood. The biotransformation reactions were either carried out in flasks or in a controlled chemical reactor attached to a customised cold trap system and the styrene product was collected in acetonitrile at À 20°C. Reactions carried out in flasks do not reach completion due to an increase in pH (uncontrolled pH beyond the buffer capacity) therefore reactions (100 mL volumes) were carried out in an EasyMax 102, METTLER TOLEDO chemical reactor that contains the desired concentration of L-Phe, 200 μl of 10 % antifoam 204 and 1 g of each lyophilised biocatalyst in 50 mM sodium phosphate buffer pH 7.0 for 4 h at 100 rpm as in Table 2. The Temperature was controlled at 37°C and the pH was maintained at pH 7.0 by the addition of 3 M HCl. An air pump was connected to the biotransformation reaction in the chemical reactor to purge air onto the bioreactor vessel and the styrene product was passed on dried CaCl 2 and then captured by the cold trap inside a fume hood. The cold trap containing acetonitrile (100 mL) was immersed in cold isopropanol that was cooled to À 20°C by a Julaba FT902 immersion cooler. Samples from the biotransformation reaction, and the captured product were analysed by HPLC.

High Performance Liquid Chromatography (HPLC) analysis
HPLC methodology was established to monitor and analyse the biotransformation reactions of L-Phe to styrene. Consumption of L-Phe was monitored at 254 nm, while production of styrene and transient accumulation of the trans-cinnamic acid intermediate were observed at 245 nm and at 273 nm respectively ( Figure S6). Samples from the biotransformation reactions were prepared for analysis by the addition of acetonitrile (HPLC grade) to stop the reaction at a volume ratio of 1 : 1 and then centrifuged at 14000 rpm for 10 min. Supernatants were filtered through 0.22 μm PDVP filters and transferred to amber glass HPLC vials and sealed with a Teflon-lined cap. Reverse phase HPLC analysis was carried out using an Agilent 1260 Infinity II LC system with UV detector. An Eclipse XDB-C18 column (5 μm, 4.6 × 150 mm) from Agilent was used. Mobile phase: A (HPLC-grade water + 0.1 % trifluoroacetic acid, TFA) and B (HPLC-grade acetonitrile + 0.1 % TFA) at a total constant flow rate of 1.0 mL/min. The eluent was kept for the first 2 min as a mixture of 80 % solvent A and 20 % solvent B, then followed by linear gradient elution with 20 % solvent B to 100 % solvent B for 7 min, and then held constant with a mixture of 80 % solvent A and 20 % solvent B for 2 min. The column temperature was maintained at 35°C and the DAD detector was set to collect data at 254 nm (for L-Phe), 273 nm (for trans-cinnamic acid) and 245 nm (for styrene). Standard curves for L-Phe, trans-cinnamic acid and styrene were prepared for quantitative analysis under the same conditions. Standard curves were measured by preparing various concentrations of standard solutions by dissolving the analyte(s) in buffer(L-Phe) or methanol (trans-cinnamic acid & styrene) spiked into the biotransformation buffer, then extracted by acetonitrile (1 : 1 ratio) and treated as the samples. To validate the efficiency of the analysis method, standards were added/spiked in buffer as well as in grown E. coli cell culture of BL21(DE3) strain. For the analysis of the biotransformation reaction, 20 μL volumes of samples or standards were injected and the data were analysed by measuring the peak areas using the quantitative Agilent software. Samples required dilutions due to the detection limit of the peak areas of styrene and this was achieved by diluting the samples to 5-,10-, 20-, 50-, 100-and 200-fold with buffer/acetonitrile mixture (1 : 1) so that the measured peak areas fit within the linear range of the standard curves. All diluted samples as well as the undiluted samples were measured at the three wavelengths. L-Phe was measured in the "undiluted" samples whereas styrene was measured in "diluted" samples. Standard curves measured are shown in Figure S7, Supporting Information.