Bio-Isobutene production: When the key enzymes are nowhere to be found

As of today, most industrial bio production processes are based on naturally occurring metabolic pathways or biochemical reactions, preventing the access to many of the chemistry’s largest market. For example, the development of bio-processes for the efficient production of light olefins such as propylene, linear butylene, butadiene and isobutene, remains a technological challenge: since these molecules are not synthesized by natural microorganisms, the design of a complete metabolic pathway for their production is hampered by the lack of identified enzymes able to perform the final biochemical step. In order to bridge this gap, Global Bioenergies has engineered artificial biocatalysts, and combined them with natural enzymes into metabolic pathways leading to the production of isobutene. Thus, in contrast with most former approaches, the new metabolic routes leading to isobutene involve non-naturally occurring reactions. The same type of approach was also used for butadiene and other molecules. The scale-up of this innovative bio-based production process is currently ongoing. Whereas a pilot plant with a capacity of 10 tons/year of oxidation-grade isobutene is running since 2014 in Pomacle (France) a demo plant with a capacity of 100 tons/year of polymer-grade isobutene has recently started operations on the refinery site of Leuna (Germany). It will cover the whole of isobutene’s wide product tree, including rubber applications. The company prepares now the first full-scale plant through a Joint-Venture with Cristal Union, named IBN-One. In the same time, Global Bioenergies is developing a strategy of diversification, in order to propose a pipeline of processes covering all possible feedstocks, from first to third generation

Predicted Effects of Missense Mutations on Native-State Stability Account for Phenotypic Outcome in Phenylketonuria, a Paradigm of Misfolding Diseases

Phenylketonuria (PKU) is a genetic disease caused by mutations in human phenylalanine hydroxylase (PAH). Most missense mutations result in misfolding of PAH, increased protein turnover, and a loss of enzymatic function. We studied the prediction of the energetic impact on PAH native-state stability of 318 PKU-associated missense mutations, using the protein-design algorithm FoldX. For the 80 mutations for which expression analyses have been performed in eukaryote systems, in most cases we found substantial overall correlations between the mutational energetic impact and both in vitro residual activities and patient metabolic phenotype. This finding confirmed that the decrease in protein stability is the main molecular pathogenic mechanism in PKU and the determinant for phenotypic outcome. Metabolic phenotypes have been shown to be better predicted than in vitro residual activities, probably because of greater stringency in the phenotyping process. Finally, all the remaining 238 PKU missense mutations compiled at the PAH locus knowledgebase (PAHdb) were analyzed, and their phenotypic outcomes were predicted on the basis of the energetic impact provided by FoldX. Residues in exons 7–9 and in interdomain regions within the subunit appear to play an important structural role and constitute hotspots for destabilization. FoldX analysis will be useful for predicting the phenotype associated with rare or new mutations detected in patients with PKU. However, additional factors must be considered that may contribute to the patient phenotype, such as possible effects on catalysis and interindividual differences in physiological and metabolic processes

Développement et caractérisation mécanique de membranes silicone architecturées

International audienceDes applications médicales nécessitent l'élaboration de membranes à anisotropie de comportement mécanique. La présente étude vise à proposer une solution à partir d'un seul matériau constitutif. Le principe repose sur la création de membranes architecturées en créant localement au niveau du Volume Elémentaire Représentatif des hétérogénéités de réticulation aux motifs contrôlés. Un matériau silicone est choisi pour la réalisation de ces membranes, à la fois pour sa facilité à le modifier chimiquement et ses propriétés élastomériques intrinsèques. Le degré de réticulation du silicone est maitrisé localement par irradiation UV d'un photo-inhibiteur avant vulcanisation : les zones irradiées réagissent moins en hydrosilylation, générant une phase plus élastique. Cette manipulation permet la création de membranes aux propriétés architecturées de par le contrôle local du degré de réticulation du réseau polymère

Directed evolution of prenylated FMN-dependent Fdc supports efficient in vivo isobutene production

From Springer Nature via Jisc Publications RouterHistory: received 2021-03-02, accepted 2021-07-29, registration 2021-08-20, pub-electronic 2021-09-06, online 2021-09-06, collection 2021-12Publication status: PublishedFunder: EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council); doi: https://doi.org/10.13039/100010663; Grant(s): pre-FAB 695013Abstract: Isobutene is a high value gaseous alkene used as fuel additive and a chemical building block. As an alternative to fossil fuel derived isobutene, we here develop a modified mevalonate pathway for the production of isobutene from glucose in vivo. The final step in the pathway consists of the decarboxylation of 3-methylcrotonic acid, catalysed by an evolved ferulic acid decarboxylase (Fdc) enzyme. Fdc belongs to the prFMN-dependent UbiD enzyme family that catalyses reversible decarboxylation of (hetero)aromatic acids or acrylic acids with extended conjugation. Following a screen of an Fdc library for inherent 3-methylcrotonic acid decarboxylase activity, directed evolution yields variants with up to an 80-fold increase in activity. Crystal structures of the evolved variants reveal that changes in the substrate binding pocket are responsible for increased selectivity. Solution and computational studies suggest that isobutene cycloelimination is rate limiting and strictly dependent on presence of the 3-methyl group

Computer design of obligate heterodimer meganucleases allows efficient cutting of custom DNA sequences

Meganucleases cut long (>12 bp) unique sequences in genomes and can be used to induce targeted genome engineering by homologous recombination in the vicinity of their cleavage site. However, the use of natural meganucleases is limited by the repertoire of their target sequences, and considerable efforts have been made to engineer redesigned meganucleases cleaving chosen targets. Homodimeric meganucleases such as I-CreI have provided a scaffold, but can only be modified to recognize new quasi-palindromic DNA sequences, limiting their general applicability. Other groups have used dimer-interface redesign and peptide linkage to control heterodimerization between related meganucleases such as I-DmoI and I-CreI, but until now there has been no application of this aimed specifically at the scaffolds from existing combinatorial libraries of I-CreI. Here, we show that engineering meganucleases to form obligate heterodimers results in functional endonucleases that cut non-palindromic sequences. The protein design algorithm (FoldX v2.7) was used to design specific heterodimer interfaces between two meganuclease monomers, which were themselves engineered to recognize different DNA sequences. The new monomers favour functional heterodimer formation and prevent homodimer site recognition. This design massively increases the potential repertoire of DNA sequences that can be specifically targeted by designed I-CreI meganucleases and opens the way to safer targeted genome engineering

Using protein design algorithms to understand the molecular basis of disease caused by protein–DNA interactions: the Pax6 example

Quite often a single or a combination of protein mutations is linked to specific diseases. However, distinguishing from sequence information which mutations have real effects in the protein’s function is not trivial. Protein design tools are commonly used to explain mutations that affect protein stability, or protein–protein interaction, but not for mutations that could affect protein–DNA binding. Here, we used the protein design algorithm FoldX to model all known missense mutations in the paired box domain of Pax6, a highly conserved transcription factor involved in eye development and in several diseases such as aniridia. The validity of FoldX to deal with protein–DNA interactions was demonstrated by showing that high levels of accuracy can be achieved for mutations affecting these interactions. Also we showed that protein-design algorithms can accurately reproduce experimental DNA-binding logos. We conclude that 88% of the Pax6 mutations can be linked to changes in intrinsic stability (77%) and/or to its capabilities to bind DNA (30%). Our study emphasizes the importance of structure-based analysis to understand the molecular basis of diseases and shows that protein–DNA interactions can be analyzed to the same level of accuracy as protein stability, or protein–protein interactions

Molecular basis of engineered meganuclease targeting of the endogenous human RAG1 locus

Homing endonucleases recognize long target DNA sequences generating an accurate double-strand break that promotes gene targeting through homologous recombination. We have modified the homodimeric I-CreI endonuclease through protein engineering to target a specific DNA sequence within the human RAG1 gene. Mutations in RAG1 produce severe combined immunodeficiency (SCID), a monogenic disease leading to defective immune response in the individuals, leaving them vulnerable to infectious diseases. The structures of two engineered heterodimeric variants and one single-chain variant of I-CreI, in complex with a 24-bp oligonucleotide of the human RAG1 gene sequence, show how the DNA binding is achieved through interactions in the major groove. In addition, the introduction of the G19S mutation in the neighborhood of the catalytic site lowers the reaction energy barrier for DNA cleavage without compromising DNA recognition. Gene-targeting experiments in human cell lines show that the designed single-chain molecule preserves its in vivo activity with higher specificity, further enhanced by the G19S mutation. This is the first time that an engineered meganuclease variant targets the human RAG1 locus by stimulating homologous recombination in human cell lines up to 265 bp away from the cleavage site. Our analysis illustrates the key features for à la carte procedure in protein–DNA recognition design, opening new possibilities for SCID patients whose illness can be treated ex vivo

E-Cadherin Destabilization Accounts for the Pathogenicity of Missense Mutations in Hereditary Diffuse Gastric Cancer

E-cadherin is critical for the maintenance of tissue architecture due to its role in cell-cell adhesion. E-cadherin mutations are the genetic cause of Hereditary Diffuse Gastric Cancer (HDGC) and missense mutations represent a clinical burden, due to the uncertainty of their pathogenic role. In vitro and in vivo, most mutations lead to loss-of-function, although the causal factor is unknown for the majority. We hypothesized that destabilization could account for the pathogenicity of E-cadherin missense mutations in HDGC, and tested our hypothesis using in silico and in vitro tools. FoldX algorithm was used to calculate the impact of each mutation in E-cadherin native-state stability, and the analysis was complemented with evolutionary conservation, by SIFT. Interestingly, HDGC patients harbouring germline E-cadherin destabilizing mutants present a younger age at diagnosis or death, suggesting that the loss of native-state stability of E-cadherin accounts for the disease phenotype. To elucidate the biological relevance of E-cadherin destabilization in HDGC, we investigated a group of newly identified HDGC-associated mutations (E185V, S232C and L583R), of which L583R is predicted to be destabilizing. We show that this mutation is not functional in vitro, exhibits shorter half-life and is unable to mature, due to premature proteasome-dependent degradation, a phenotype reverted by stabilization with the artificial mutation L583I (structurally tolerated). Herein we report E-cadherin structural models suitable to predict the impact of the majority of cancer-associated missense mutations and we show that E-cadherin destabilization leads to loss-of-function in vitro and increased pathogenicity in vivo

Design of Miniproteins by the Transfer of Active Sites Onto Small-Size Scaffolds

International audienceNatural miniproteins (e.g., animal toxins, protease inhibitors, defensins) can express specific and powerful biological activities by using a stable and minimal (<80 amino acids) structural motif. Artificial activities have been designed on these miniscaffolds by transferring previously identified protein active sites into regions structurally compatible with the site and permissive for sequence mutations. These newly designed miniproteins, presenting a specific and high activity within a small size and well-defined three-dimensional structure, represent novel tools in biology, biotechnology, and medical sciences, and are also useful intermediates to develop new therapeutic agents. The different steps used to design and characterize new bioactive miniproteins are here described in detail. Two successful examples are here reported. The first one is a metal-binding miniprotein (MBP, 37 residues), which possesses a metal specificity resembling that of natural carbonic anhydrase; the second is a CD4 mimic (CD4M33, 27 residues), which is a powerful inhibitor of HIV-1 entry but also a fully functional substitute of the human receptor CD4 and, hence, a potential component of an AIDS vaccine

Membranes élastomères en silicone à architecturation de module pour applications biomédicales

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