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. 2024 Feb 28;23(1):69.
doi: 10.1186/s12934-024-02341-0.

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

Ana García-Franco  1   2 , Patricia Godoy  1 , Estrella Duque  1 , Juan L Ramos  3
Affiliations

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

Ana García-Franco et al. Microb Cell Fact. .

Abstract

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.

Keywords: Pseudomonas; Aromatic hydrocarbons; Decarboxylases; PAL enzymes; Styrene; Synthetic genes.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Enzymatic pathway to produce styrene from glucose, via the intermediates L-phenylalanine and trans-cinnamic acid. The two-step pathway from L-phenylalanine to styrene is achieved by co-expressing palN, encoding a phenylalanine ammonia lyase, and psc1, encoding a trans-cinnamic acid decarboxylase
Fig. 2
Fig. 2
Trans-cinnamic acid consumption by P. putida CM12-5 expressing different trans-cinnamic acid decarboxylases. P. putida CM12-5 without plasmid or bearing the plasmid pPSC1 or pPSD1 were grown on M9 minimal medium with glucose supplemented with 0.25, 0.5 or 1 mM trans-cinnamic acid. tCA concentrations were determined at the beginning of the assay (blue bars) and after 24-h cultivation (yellow bars). ANOVA analysis was performed for three-group comparisons and Tukey test was carried out between paired groups to determine the statistically significance (p-value < 0.05). The results shown are the averages and standard deviations of three independent assays
Fig. 3
Fig. 3
Trans-cinnamic acid and L-phenylananine consumption by P. putida CM12-5 (pPSC1) (A and B) and P. putida CM12-5 (pPALN) (C and D), respectively, at different pH and temperatures. For pH assays (A and C), cultures were grown at pH 5.8, pink open circles; pH 6.6, orange solid circles; pH 7.0, green open triangles; and pH 7.6, purple solid triangles. For temperature assays (B and D), cultures were grown in M9 minimal medium at pH 7.0 at 18 °C, pink open circles; 25 °C, orange solid circles; 30 °C, green open triangles; and 37 °C, purple solid triangles. Initial consumption rates correspond to the slope of a trendline during the first two hours of cultivation. The results shown are the averages and standard deviations of three independent assays
Fig. 4
Fig. 4
Biosynthesis of styrene by P. putida derivatives from glucose. The strains used were P. putida CM12-5 (pPALN, pPSC1), P. putida CM12-5 (pPALN_C1) and P. putida CM12-5 as control. Cells were grown on M9 minimal medium with 0,5% (w/v) glucose as the sole C-source. Production of L-phenylalanine (blue bars), trans-cinnamic acid (yellow bars) and styrene (pink bars) was determined. Two-tailed Student’s t-tests were performed to determine the statistical significance for two-group comparisons (p < 0.05). The results shown are the averages and standard deviations of three independent assays
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