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Styrene Production in Genetically Engineered Escherichia coli in a Two-Phase Culture.

ABSTRACT: Styrene is an important industrial chemical. Although several studies have reported microbial styrene production, the amount of styrene produced in batch cultures can be increased. In this study, styrene was produced using genetically engineered Escherichia coli. First, we evaluated five types of phenylalanine ammonia lyases (PALs) from Arabidopsis thaliana (AtPAL) and Brachypodium distachyon (BdPAL) for their ability to produce trans-cinnamic acid (Cin), a styrene precursor. AtPAL2-expressing E. coli produced approximately 700 mg/L of Cin and we found that BdPALs could convert Cin into styrene. To assess styrene production, we constructed an E. coli strain that co-expressed AtPAL2 and ferulic acid decarboxylase from Saccharomyces cerevisiae. After a biphasic culture with oleyl alcohol, styrene production and yield from glucose were 3.1 g/L and 26.7% (mol/mol), respectively, which, to the best of our knowledge, are the highest values obtained in batch cultivation. Thus, this strain can be applied to the large-scale industrial production of styrene.

SUBMITTER: Noda S

PROVIDER: S-EPMC10801462 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature

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Styrene Production in Genetically Engineered Escherichia coli in a Two-Phase Culture.

Noda Shuhei S Fujiwara Ryosuke R Mori Yutaro Y Dainin Mayumi M Shirai Tomokazu T Kondo Akihiko A

Biotech (Basel (Switzerland)) 20240114 1

Styrene is an important industrial chemical. Although several studies have reported microbial styrene production, the amount of styrene produced in batch cultures can be increased. In this study, styrene was produced using genetically engineered Escherichia coli. First, we evaluated five types of phenylalanine ammonia lyases (PALs) from Arabidopsis thaliana (AtPAL) and Brachypodium distachyon (BdPAL) for their ability to produce trans-cinnamic acid (Cin), a styrene pr ...[more]

PMID: 38247732

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Project description:BackgroundRetinoids are lipophilic isoprenoids composed of a cyclic group and a linear chain with a hydrophilic end group. These compounds include retinol, retinal, retinoic acid, retinyl esters, and various derivatives of these structures. Retinoids are used as cosmetic agents and effective pharmaceuticals for skin diseases. Retinal, an immediate precursor of retinoids, is derived by β-carotene 15,15'-mono(di)oxygenase (BCM(D)O) from β-carotene, which is synthesized from the isoprenoid building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Retinoids are chemically unstable and biologically degraded via retinoic acid. Although extensive studies have been performed on the microbial production of carotenoids, retinoid production using microbial metabolic engineering has not been reported. Here, we report retinoid production using engineered Escherichia coli that express exogenous BCM(D)O and the mevalonate (MVA) pathway for the building blocks synthesis in combination with a two-phase culture system using a dodecane overlay.ResultsAmong the BCM(D)O tested in E. coli, the synthetic retinoid synthesis protein (SR), based on bacteriorhodopsin-related protein-like homolog (Blh) of the uncultured marine bacteria 66A03, showed the highest β-carotene cleavage activity with no residual intracellular β-carotene. By introducing the exogenous MVA pathway, 8.7 mg/L of retinal was produced, which is 4-fold higher production than that of augmenting the MEP pathway (dxs overexpression). There was a large gap between retinal production and β-carotene consumption using the exogenous MVA pathway; therefore, the retinal derivatives were analyzed. The derivatives, except for retinoic acid, that formed were identified, and the levels of retinal, retinol, and retinyl acetate were measured. Amounts as high as 95 mg/L retinoids were obtained from engineered E. coli DH5α harboring the synthetic SR gene and the exogenous MVA pathway in addition to dxs overexpression, which were cultured at 29°C for 72 hours with 2YT medium containing 2.0% (w/v) glycerol as the main carbon source. However, a significant level of intracellular degradation of the retinoids was also observed in the culture. To prevent degradation of the intracellular retinoids through in situ extraction from the cells, a two-phase culture system with dodecane was used. The highest level of retinoid production (136 mg/L) was obtained after 72 hours with 5 mL of dodecane overlaid on a 5 mL culture.ConclusionsIn this study, we successfully produced 136 mg/L retinoids, which were composed of 67 mg/L retinal, 54 mg/L retinol, and 15 mg/L retinyl acetate, using a two-phase culture system with dodecane, which produced 68-fold more retinoids than the initial level of production (2.2 mg/L). Our results demonstrate the potential use of E. coli as a promising microbial cell factory for retinoid production.

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Styrene Production in Genetically Engineered Escherichia coli in a Two-Phase Culture.

Publications

Styrene Production in Genetically Engineered <i>Escherichia coli</i> in a Two-Phase Culture.

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