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

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.

Insights into the susceptibility of Pseudomonas putida to industrially relevant aromatic hydrocarbons that it can synthesize from sugars.

Pseudomonas putida DOT-T1E is a highly solvent tolerant strain for which many genetic tools have been developed. The strain represents a promising candidate host for the synthesis of aromatic compounds-opening a path towards a green alternative to petrol-derived chemicals. We have engineered this strain to produce phenylalanine, which can then be used as a raw material for the synthesis of styrene via trans-cinnamic acid. To understand the response of this strain to the bioproducts of interest, we have analyzed the in-depth physiological and genetic response of the strain to these compounds. We found that in response to the exposure to the toxic compounds that the strain can produce, the cell launches a multifactorial response to enhance membrane impermeabilization. This process occurs via the activation of a cis to trans isomerase that converts cis unsaturated fatty acids to their corresponding trans isomers. In addition, the bacterial cells initiate a stress response program that involves the synthesis of a number of chaperones and ROS removing enzymes, such as peroxidases and superoxide dismutases. The strain also responds by enhancing the metabolism of glucose through the specific induction of the glucose phosphorylative pathway, Entner-Doudoroff enzymes, Krebs cycle enzymes and Nuo. In step with these changes, the cells induce two efflux pumps to extrude the toxic chemicals. Through analyzing a wide collection of efflux pump mutants, we found that the most relevant pump is TtgGHI, which is controlled by the TtgV regulator.

Addressing the energy crisis: using microbes to make biofuels.

Much of the energy being used to power our lives comes from fossil fuels such as coal, natural gas and petroleum. These energy sources are non-renewable, are being exhausted and also pollute the air, water and soil with toxic chemicals. Their mining, transportation, refining and use are associated with a large carbon footprint that contributes significantly to global warming. In addition, the geopolitical complexities surrounding the main fossil fuel producers create risks and uncertainties around the world. Replacing fossil fuels with clean, renewable forms of energy is paramount to creating a sustainable and healthy future, and for laying the foundations for global political stability and prosperity. Using biomass from plants, microbes can produce biofuels that are identical to or perform as well as fossil fuels. In addition of creating sustainable energy, advancing the biofuel industry will create new, high-quality rural jobs whilst improving energy security.

Synthesis of aromatic amino acids from 2G lignocellulosic substrates.

Pseudomonas putida is a highly solvent-resistant microorganism and useful chassis for the production of value-added compounds from lignocellulosic residues, in particular aromatic compounds that are made from phenylalanine. The use of these agricultural residues requires a two-step treatment to release the components of the polysaccharides of cellulose and hemicellulose as monomeric sugars, the most abundant monomers being glucose and xylose. Pan-genomic studies have shown that Pseudomonas putida metabolizes glucose through three convergent pathways to yield 6-phosphogluconate and subsequently metabolizes it through the Entner-Doudoroff pathway, but the strains do not degrade xylose. The valorization of both sugars is critical from the point of view of economic viability of the process. For this reason, a P. putida strain was endowed with the ability to metabolize xylose via the xylose isomerase pathway, by incorporating heterologous catabolic genes that convert this C5 sugar into intermediates of the pentose phosphate cycle. In addition, the open reading frame T1E_2822, encoding glucose dehydrogenase, was knocked-out to avoid the production of the dead-end product xylonate. We generated a set of DOT-T1E-derived strains that metabolized glucose and xylose simultaneously in culture medium and that reached high cell density with generation times of around 100 min with glucose and around 300 min with xylose. The strains grew in 2G hydrolysates from diluted acid and steam explosion pretreated corn stover and sugarcane straw. During growth, the strains metabolized > 98% of glucose, > 96% xylose and > 85% acetic acid. In 2G hydrolysates P. putida 5PL, a DOT-T1E derivative strain that carries up to five independent mutations to avoid phenylalanine metabolism, accumulated this amino acid in the medium. We constructed P. putida 5PLΔgcd (xylABE) that produced up to 250 mg l-1 of phenylalanine when grown in 2G pretreated corn stover or sugarcane straw. These results support as a proof of concept the potential of P. putida as a chassis for 2G processes.