We found a match
Your institution may have access to this item. Find your institution then sign in to continue.
- Title
Engineering styrene biosynthesis: designing a functional trans-cinnamic acid decarboxylase in Pseudomonas.
- Authors
García-Franco, Ana; Godoy, Patricia; Duque, Estrella; Ramos, Juan L.
- 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. Highlights: This study focuses on the conversion of sugars into styrene, a valuable chemical compound, using as a host the solvent-tolerant Pseudomonas putida DOT-T1E as a chassis. The biosynthesis of styrene involves a two-step process, starting with the conversion of L-phenylalanine into trans-cinnamic acid (tCA) through PAL enzymes, followed by decarboxylation to yield styrene. A synthetic trans-cinnamic acid decarboxylase was designed using a novel 'wholesale' approach that involved the derivation of consensus sequences from homologous yeast FDC1 genes with > 60% identity, leading to the design of a functional Pseudomonas codon-optimized protein named PSC1. Assays demonstrated successful decarboxylation of tCA into styrene by the PSC1 enzyme. Optimal conditions for PSC1 enzyme activity in vivo were determined, including pH and temperature. Highest styrene biosynthesis efficiency was achieved by co-expressing the pal and psc1 genes as an operon in P. putida CM12-5, a P. putida DOT-T1E derivative that produces L-phenylalanine, showcasing the significance of coordinated expression for improved chemical production. The utilization of the solvent-tolerant Pseudomonas putida chassis as a biofactory for styrene production highlights the potential of microbial engineering for sustainable and environmentally-friendly chemical synthesis.
- Subjects
STYRENE; BIOSYNTHESIS; PSEUDOMONAS putida; PSEUDOMONAS; AROMATIC compounds
- Publication
Microbial Cell Factories, 2024, Vol 23, Issue 1, p1
- ISSN
1475-2859
- Publication type
Article
- DOI
10.1186/s12934-024-02341-0