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A microbial process for the production of benzyl acetate – Nature Chemical Engineering

Strains and media

The bacterial strains used in this study are listed in Supplementary Table 1. E. coli DH5α was used for molecular cloning and plasmid propagation, while E. coli W3110, NST74 (ref. 29) or their derivatives were used for benzyl acetate production. For construction of plasmids and strains, E. coli cells were routinely cultured in the lysogeny broth (LB) medium (10 g l−1 tryptone, 5 g l−1 yeast extract and 5 g l−1 NaCl) or on LB-agar medium (LB medium containing 1.5% (w/v) agar). For benzyl acetate production and toxicity test, MR minimal medium containing (per liter) 6.67 g KH2PO4, 4 g (NH4)2HPO4, 0.8 g citric acid, 0.8 g MgSO4·7H2O and 5 ml of trace metal solution30 was used, and the pH was adjusted to 7.0 using solid NaOH. The trace metal solution (per liter) contained 10.0 g FeSO4·7H2O, 2.65 g CaCl2·2H2O, 2.2 g ZnSO4·7H2O, 0.58 g MnSO4·4H2O, 1.0 g CuSO4·5H2O, 0.1 g (NH4)6Mo7O24·4H2O, 0.02 g Na2B4O7·10H2O and 10 ml of fuming HCl aqueous solution. Stock solutions of d-glucose (500 g l−1) and MgSO4·7H2O (80 g l−1) were autoclaved separately and added to the MR medium after autoclaving the rest of the components together. Antibiotics were selectively supplemented at following concentrations when necessary: 100 μg ml−1 ampicillin; 50 μg ml−1 kanamycin; 50 μg ml−1 streptomycin; 34 μg ml−1 chloramphenicol.

Plasmid construction

Plasmids used in this study are listed in Supplementary Table 1. Oligonucleotide primers and template DNAs used for plasmid construction are listed in Supplementary Table 11 along with the construction schemes. The DNA sequences of the codon-optimized genes are listed in Supplementary Table 12. All DNA manipulations for plasmid construction were conducted according to the standard protocols31. The sequence of all constructed plasmids was verified by DNA sequencing.

Chromosomal gene knockout

The chromosomal ilvE gene was knocked out from the E. coli LLD6 strain17 to construct the LLD9 strain through the one-step homologous recombination protocol32,33 using a DNA fragment ilvE-KO-100 (Supplementary Table 13) and plasmids pKD46 and pJW168 (Supplementary Table 1).

Benzyl acetate toxicity test

To examine the inhibitory effect of benzyl acetate to E. coli, the wild-type E. coli W3110 strain was firstly inoculated to a test tube containing 5 ml LB medium and incubated at 37 °C and 200 rpm for 12 h. Next, 1 ml of the preculture was transferred to a 300-ml baffled flask containing 50 ml MR medium supplemented with 10 g l−1 glucose and 0–1 g l−1 benzyl acetate and cultured at 37 °C and 200 rpm. The inhibitory effect was evaluated by monitoring the cell growth based on the optical density at the wavelength of 600 nm (OD600).

Partition coefficient measurement

To determine the partition coefficients of benzyl acetate and benzyl alcohol between the aqueous and organic phases, benzyl acetate and benzyl alcohol were first dissolved in MR medium without glucose at 1 g l−1, and 800 μl of the resulting solutions were mixed with the equal volume of tributyrin (97 % (w/w), Thermo Scientific) in a 2-ml microcentrifuge tube. The mixtures were vortexed for 1 min and horizontally shaken at 200 rpm and 30 °C for 24 h. Subsequently, the mixtures were centrifuged at 16,000g for 20 min to separate the aqueous and organic phases, and the concentrations of benzyl acetate and benzyl alcohol in each phase were measured using high-performance liquid chromatography (HPLC) as described in ‘Analytical procedures’ below. Lastly, the partition coefficients were calculated by dividing the concentration in the organic phase by that in the aqueous phase.

Shake-flask culture conditions

Shake-flask cultures were conducted using the MR medium in 300-ml baffled flasks. Briefly, the precultures were prepared by inoculating test tubes containing 5 ml LB medium and incubating at 37 °C and 200 rpm for 12 h. Then, 1 ml of the precultures were transferred to baffled flasks containing 50 ml or 25 ml MR medium with 10 g l−1 glucose for single-phase benzyl alcohol production or two-phase benzyl acetate production, respectively, unless mentioned otherwise. For co-culture of the upstream and downstream strains, the 1 ml of inoculum was formulated by mixing the precultures of each strain according to the indicated volumetric ratio. For example, 0.75 ml of the upstream strain preculture and 0.25 ml of the downstream preculture were mixed to generate 1 ml inoculum with the upstream and downstream inoculum ratio of 3:1. The inoculated flasks were cultured in a shaking incubator at 30 °C and 200 rpm for 60 h. At 6 h when the OD600 reached 0.6–0.8, overexpression of the introduced genes was induced by supplementing 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). For benzyl acetate production through two-phase extractive culture, 5 ml of tributyrin was also added as an organic phase to 25 ml of the culture volume at this point (that is aqueous-to-organic phase volumetric ratio of 5:1). For benzoic acid conversion test, 1 ml of 59 g l−1 sodium benzoate was added to the two-phase culture broth composed of 25 ml of aqueous phase and 5 ml of organic phase (equivalent to the final benzoic acid concentration of 1.92 g l−1 in the aqueous phase) at 12 h. To overcome the auxotrophy generated by knocking out relevant genes, the following supplements were additionally supplied at the beginning of the flask culture unless indicated otherwise: 0.1 g l−1 l-tyrosine and 10 mg l−1 thiamine for the tyrA knockout; 3 g l−1 l-aspartic acid for the tyrB and aspC knockouts; 0.04 mg l−1 l-phenylalanine and 3 g l−1 yeast extract for the ilvE knockout.

Fed-batch fermentation conditions

Two-phase extractive fed-batch fermentations were conducted at 30 °C using a 6.6-l fermentor (Bioflo 320; New Brunswick Scientific) containing 2 l of MR medium supplemented with 20 g l−1 of glucose. Briefly, the seed culture was prepared by inoculating a test tube containing 5 ml of LB medium and incubating at 37 °C and 200 rpm for 12 h. Next, each of two 300-ml Erlenmeyer flasks containing 100 ml of LB medium were inoculated with 2 ml of the seed culture and then incubated at 37 °C and 200 rpm for 12 h. The resulting 200 ml of the preculture was transferred into the fermentor containing 1.8 l of medium containing components equivalent to 2 l of MR medium supplemented with 20 g l−1 glucose. For standard co-culture of the upstream and downstream strains, the 200 ml of inoculum was formulated by mixing the precultures of each strain according to the indicated volumetric ratio. For example, 150 ml of the upstream strain preculture and 50 ml of the downstream preculture were mixed to generate 200 ml inoculum with the upstream and downstream inoculum ratio of 3:1.

During fermentation, the pH was controlled at 7.0 using saturated ammonia solution (28%, w/w). The dissolved oxygen level was maintained at 40% of the air saturation by controlling the agitation speed within a range of 200–1,000 rpm and constantly supplying filtered air at 2.0 l min−1. Once the OD600 value reached 5 or more, 1 mM IPTG was supplemented to induce the expression of benzyl acetate biosynthesis genes and 0.4 l of tributyrin was supplied at 1.11 ml min−1. Antifoam 204 (Sigma-Aldrich) was used to repress the formation of foam during fermentation. After the initial glucose was depleted, a nutrient solution was supplied at 9.4 ml min−1 whenever pH value was above 7.02.

For benzyl acetate production through the benzoic acid-dependent pathway, 0.1 g l−1 l-tyrosine and 10 mg l−1 thiamine were supplemented to the fermentation media at the beginning, and 0.1 g l-tyrosine (in forms of powder or 50 g l−1 stock solution) was further supplemented at 24 h and 48 h points of the fermentation. The nutrient solution consisted of 700 g l−1 glucose, 8 g l−1 MgSO4·7H2O, 5 ml l−1 trace metal solution, 10 mg l−1 thiamine and 1 mM IPTG.

For benzyl acetate production through the benzoic acid-independent pathway, 0.1 g l−1 l-tyrosine, 0.1 g l−1 l-phenylalanine, 3 g l−1 l-aspartic acid, 3 g l−1 yeast extract and 10 mg l−1 thiamine were supplemented to the fermentation media at the beginning. The nutrient solution contained 700 g l−1 glucose, 8 g l−1 MgSO4·7H2O, 3 g l−1 yeast extract, 5 ml l−1 trace metal solution, 10 mg l−1 thiamine and 1 mM IPTG.

Delayed co-culture conditions

For delayed co-culture of the Bn1 and Bn-BnAc3 strains, the preculture of the Bn1 strain was prepared and transferred to a fermentor supplemented with 0.1 g l−1 l-tyrosine and 10 mg l−1 thiamine following the fed-batch fermentation procedures described above. Once the OD600 value reached 5 or more, however, only 1 mM IPTG was supplemented to induce the expression of benzyl acetate biosynthesis genes without supplying tributyrin. Meanwhile, the delayed inoculum of the Bn-BnAc3 strain was prepared by inoculating each of four or eight 300-ml baffled flasks that contain 50 ml of the MR medium supplemented with 20 g l−1 glucose, 0.1 g l−1 l-tyrosine and 10 mg l−1 thiamine with 1 ml of the Bn-BnAc3 strain seed culture (prepared as aforementioned) and culturing at 30 °C and 200 rpm for 12–15 h, depending on target cell density. Subsequently, 200 ml or 400 ml of the preculture was transferred to the fermentor at 48 h point (unless mentioned otherwise), followed by supply of 0.44 l or 0.48 l of tributyrin at 1.11 ml min−1, respectively.

The rest of the conditions followed those of standard fed-batch fermentation for producing benzyl acetate through the benzoic acid-dependent pathway. Briefly, the pH was controlled at 7.0 using saturated ammonia solution (28%, w/w). The dissolved oxygen level was maintained at 40% of the air saturation by controlling the agitation speed within a range of 200–1,000 rpm and constantly supplying filtered air at 2.0 l min−1. Antifoam 204 (Sigma-Aldrich) was used to repress the formation of foam during fermentation, and 0.1 g l-tyrosine (in forms of powder or 50 g l−1 stock solution) was further supplemented at the 24 h and 48 h points of the fermentation. After the initial glucose was depleted, a nutrient solution (700 g l−1 glucose, 8 g l−1 MgSO4·7H2O, 5 ml l−1 trace metal solution, 10 mg l−1 of thiamine and 1 mM IPTG) was supplied at 9.4 ml min−1 whenever pH value was above 7.02.

Analytical procedures

Cell density was monitored by measuring OD600 using an Ultrospec 3100 spectrophotometer (Amersham Biosciences).

The levels of residual d-glucose, organic acids, benzyl acetate and related metabolites (for example, intermediates) were analyzed using HPLC systems. To prepare samples for HPLC analysis, culture samples were first centrifuged at 16,000g for 20 min to separate the aqueous and organic phases. The aqueous phase samples were appropriately diluted with deionized water and filtered using 0.22-µm PVDF filters, and the organic phase samples were appropriately diluted with tributyrin and filtered using 0.22-µm PTFE filters. The residual d-glucose and organic acid levels during the two-phase fed-batch fermentations were analyzed using a HPLC system operated by the Breeze2 (Database version 6.20.00.00) software and equipped with a 1515 isocratic pump and 2414 refractive index detector (Waters). A MetaCarb 87H column (7.8 × 300 mm, Agilent) was eluted isocratically with 0.01 M H2SO4 at 35 °C at a flow rate of 0.5 ml min−1.

The concentrations of pathway metabolites were analyzed using 1260 Infinity II liquid chromatography system (Agilent) operated by the OpenLAB CDS (ChemStation Edition; version information unavailable) software and equipped with a diode array detector, monitoring absorbance at 210 nm (for l-phenylalanine, benzyl alcohol and benzyl acetate), 230 nm (for l-tyrosine, benzoic acid and cinnamyl acetate) and 255 nm (for benzaldehyde and trans-cinnamic acid). Poroshell 120 column (4.6 × 150 mm, Agilent) was eluted using mixed solutions of 0.1% (v/v) trifluoroacetic acid aqueous solution (buffer A) and pure acetonitrile (buffer B) flowing at 0.625 ml min−1. The ratio of the buffers A and B were controlled according to the following program: 0–1 min, 90:10; 1–10 min, a linear gradient from 90:10 to 30:70; 10–12 min, 30:70; 12-–14 min, a linear gradient from 30:70 to 90:10; 14–18 min, 90:10.

The overall product titers of the two-phase cultures were calculated with respect to the aqueous phase volume. Benzyl acetate, benzyl alcohol, benzaldehyde and cinnamyl acetate were mostly detected in the organic phase samples and the other metabolites were mostly detected in the aqueous phase samples. Nevertheless, we calculated the overall titers of the metabolites by dividing their concentrations in the organic phase sample by five (that is, the ratio of the aqueous phase volume to the organic phase volume) and adding to their concentrations in the aqueous phase sample.

Calculation of the theoretical maximum flux of benzyl acetate biosynthesis

Theoretical maximum fluxes of benzyl acetate biosynthesis through the benzoic acid-dependent and -independent pathways were analyzed using iML1515, a genome-scale metabolic model of E. coli34. Each biosynthetic pathway was added to the model by introducing the following reactions. For the upstream strain (strain 1) harboring the benzoic acid-dependent pathway, the following reactions were introduced: ‘l-phenylalanine → trans-cinnamic acid + NH4’, ‘trans-cinnamic acid + ATP + CoA → cinnamoyl-CoA + AMP + pyrophosphate’, ‘cinnamoyl-CoA + H2O → 3-hydroxyphenylpropionyl-CoA’, ‘3-hydroxyphenylpropionyl-CoA + NAD+ → 3-ketophenylpropionyl-CoA + NADH + H+’, ‘3-ketophenylpropionyl-CoA + H2O → benzoic acid + acetyl-CoA + H+’, transportation of benzoic acid to extracellular space and a benzoic acid exchange reaction. Also, the following reactions were introduced for the downstream strain (strain 2): a benzoic acid exchange reaction, transportation of benzoic acid to extracellular space, ‘benzoic acid + ATP + NADPH + H+ → benzaldehyde + AMP + pyrophosphate + NADP+’, ‘benzaldehyde + NADPH + H+ → benzyl alcohol + NADP+’, ‘acetyl-CoA + benzyl alcohol → CoA + benzyl acetate’, transportation of benzyl acetate to extracellular space and a benzyl acetate exchange reaction.

For the upstream strain (strain 1) harboring the benzoic acid-independent pathway, the following reactions were introduced: ‘phenylpyruvic acid + O2 → S-mandelic acid + CO2’, ‘S-mandelic acid + FMN → phenylglyoxylic acid + FMNH2’, ‘phenylglyoxylic acid + H+ → benzaldehyde + CO2’, ‘benzaldehyde + NADPH + H+ → benzyl alcohol + NADP+’, transportation of benzyl alcohol to extracellular space and a benzyl alcohol exchange reaction. Also, following the reactions were introduced for the downstream strain (strain 2): a benzyl alcohol exchange reaction, transportation of benzyl alcohol to extracellular space, ‘acetyl-CoA + benzyl alcohol → CoA + benzyl acetate’, transportation of benzyl acetate to extracellular space and a benzyl acetate exchange reaction.

For the integrated strain harboring the entire benzoic acid-dependent pathway, the following reactions were added: ‘l-phenylalanine → trans-cinnamic acid + NH4’, ‘trans-cinnamic acid + ATP + CoA → cinnamoyl-CoA + AMP + pyrophosphate’, ‘cinnamoyl-CoA + H2O → 3-hydroxyphenylpropionyl-CoA’, ‘3-hydroxyphenylpropionyl-CoA + NAD+ → 3-ketophenylpropionyl-CoA + NADH + H+’, ‘3-ketophenylpropionyl-CoA + H2O → benzoic acid + acetyl-CoA + H+’, ‘benzoic acid + ATP + NADPH + H+ → benzaldehyde + AMP + pyrophosphate + NADP+’, ‘benzaldehyde + NADPH + H+ → benzyl alcohol + NADP+’, ‘acetyl-CoA + benzyl alcohol → CoA + benzyl acetate’, transportation of benzyl acetate to extracellular space and a benzyl acetate exchange reaction.

Finally, for the integrated strain harboring the entire benzoic acid-independent pathway, the following reactions were added: ‘phenylpyruvic acid + O2 → S-mandelic acid + CO2’, ‘S-mandelic acid + FMN → phenylglyoxylic acid + FMNH2’, ‘phenylglyoxylic acid + H+ → benzaldehyde + CO2’, ‘benzaldehyde + NADPH + H+ → benzyl alcohol + NADP+’, ‘acetyl-CoA + benzyl alcohol → CoA + benzyl acetate’, transportation of benzyl acetate to extracellular space and a benzyl acetate exchange reaction.

To calculate the theoretical maximum flux of benzyl acetate biosynthesis in the integrated strains, flux balance analysis was conducted by maximizing the flux of benzyl acetate production reaction. To calculate the theoretical maximum flux of benzyl acetate biosynthesis in the co-culture systems, we constructed a combined model of strain 1 and strain 2 (that is, upstream and downstream strains, respectively) whose components are separated by their own compartments. We solved the following optimization problem for the combined model:

$$max {v}_{{{mathrm{benzyl}}}{{mathrm{acetate}}}{{mathrm{production}}}}$$

$${mathrm{s.t.}}sum _{jin M}{S}_{i,,j}^{k}, {v}_{j}^{k}=0,forall iin {N}^{k},$$

$${v}_{j,{{mathrm{lb}}}}^{k}le {v}_{j}^{k}le {v}_{j,{{mathrm{ub}}}}^{k},forall jin {M}^{k},$$

$${X}^{1}{v}_{{{mathrm{inter}}}}^{1}+{X}^{2} {v}_{{{mathrm{inter}}}}^{2}ge 0$$

In the above optimization problem, the variables are defined as follows: ({v}_{j}^{k}), a flux of a metabolic reaction j in strain k; ({S}_{i,,j}^{k}), a coefficient of metabolite i which participates in metabolic reaction j in strain k; ({v}_{j,{{mathrm{ub}}}}^{k}), the upper bound of a metabolic reaction j in strain k; ({v}_{j,{{mathrm{lb}}}}^{k}), the lower bound of a metabolic reaction j in strain k; ({v}_{{{mathrm{inter}}}}^{k}), a flux of the intermediate chemical (that is, benzoic acid for benzoic acid-dependent pathway and benzyl alcohol for benzoic acid-independent pathway) production of strain k; Xk, the cell density of strain k; Nk and Mk represent the sets of metabolites and reactions of strain k, respectively. s.t., such that. We assumed no biomass production or ATP requirement for non-growth associated maintenance when calculating the theoretical maximum flux of benzyl acetate biosynthesis. Thus, the specific cell growth rate was set to zero, leading to constant cell densities. Therefore, the above optimization problem can be formulated as follows, where n = X2/X1.

$$max {v}_{{{mathrm{benzyl}}}{{mathrm{acetate}}}{{mathrm{production}}}}$$

$${mathrm{s.t.}}sum _{jin M}{S}_{i,,j}^{k}, {v}_{j}^{k}=0,forall iin {N}^{k},$$

$${v}_{j,{{mathrm{lb}}}}^{k}le {v}_{j}^{k}le {v}_{j,{{mathrm{ub}}}}^{k},forall jin {M}^{k},$$

$${v}_{{{mathrm{inter}}}}^{1}+n, {v}_{{{mathrm{inter}}}}^{2}ge 0$$

To analyze the theoretical maximum flux of benzyl acetate production of the co-culture system based on the ratio of cell density between strain 1 and strain 2 (that is, the upstream and downstream strains), the optimization problem was solved with varying n. Throughout the simulations, the upper bound of glucose uptake flux was set at 10 mmol g−1 dry cell weight (DCW) h−1 and the ATP maintenance requirement was set at 0 mmol g−1 DCW h−1.

Techno-economic analysis and sensitivity analysis

A microbial fermentation-based configuration for production of benzyl acetate from d-glucose was developed in BioSTEAM (Fig. 6a and Supplementary Discussion 13). Techno-economic analysis of the microbial-based benzyl acetate production process based on the delayed co-culture of the Bn1 and Bn-BnAc3 strains and sensitivity analysis on the internal rate of return at the break-even point were conducted based on the configuration constructed in BioSTEAM and a set of parameters surveyed from diverse sources (Supplementary Tables 24). Supplementary Discussion 13 provides a detailed information on the techno-economic analysis and the sensitivity analysis conducted in this study.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.