Exploration of Acinetobacter baylyi ADP1 sulfur metabolism

International audienc

Discovery of new levansucrase enzymes with interesting properties and improved catalytic activity to produce levan and fructooligosaccharides

Mining for new levansucrase enzymes with high levan production, transfructosylating activity, and thermal stability and studying their kinetics and acceptor specificity

Exploration of Acinetobacter baylyi ADP1 sulfur metabolism

International audienc

Discovery and Enzymatic Screening of Genome-Mined Microbial Levanases to Produce Second-Generation β-(2,6)-Fructooligosaccharides: Catalytic Properties

International audienc

Auto-sufficient enzymatic cascade combining a non selective and promiscuous ADH and BVMOS: Application for dihydrocoumarin synthesis

Dihydrocoumarin is a plant metabolite widely used as flavoring agent in agro-food industries and also as common fragrance in cosmetics. As a lot of flavor chemicals, dihydrocoumarin is found in minor quantities in its natural plant sources, the sweet clover (Melilotus officinalis) and tonka beans (Dipteryx odorata) so processes are needed to produce this natural flavor in a efficient and eco-friendly manner. Baeyer-Villiger MonoOxygenases (BVMOs) are well known flavoenzymes able to transform efficiently ketone into ester or lactone with high regio-and stereoselectivities. Nevertheless, BVMOs are strictly NADPH-dependent, and therefore require a stoichiometric amount of the expensive nicotinamide cofactor. To address this issue, the multi-enzyme syntheses provide the opportunity to generate efficient auto-sufficient systems and only a limited number of such systems involving BVMOs has been reported to date. We will present here a new efficient access to dihydrocoumarin via a two-enzyme mediated oxidation of Indanol involving Alcohol Dehydrogenase (ADH) and BVMO. The originality of our approach comes from the features of ADH, an enzyme discovered from the biodiversity via a dedicated High Throughput Screening. The ADH is a NADP-dependent and non-enantioselective enzyme which enables on the one hand cofactor recycling and on the other hand a complete transformation of the racemic alcohol. Moreover, it presents a quite large scope of substrates. Several plasmid constructions and combinations have been tested first for the formation of e-caprolactone and compared in order to elaborate a versatile platform using ADH and different BVMOs. Then to optimize the formation of Dihydrocoumarin and limit the hydrolysis, we investigated the composition of the biotransformation medium in whole-cell process as well as in purified enzyme system.Still exploring the diversity of BVMOs activities, we wish to expand the range of potentially valued compounds obtained using these systems. (Ménil et al.

Characterization of L­-Serine O-succinyltransferases involved in l-cysteine biosynthesis in prokaryotes

Until very recently, acetylation was the only known way for activating L-serine in the penultimate step of L-cysteine biosynthesis, in bacteria and fungi. This reaction is catalyzed by L-serine O-acetyl transferases (SAT), encoded by cysE and srpH, and belonging to the transferase hexapeptide repeat family. Nevertheless, we demonstrated that L-serine is succinylated in Schizosaccharomyces pombe by an enzyme unrelated to the classical SAT (Bastard and Perret et al., Nat. Chem. Biol., 2017). This unique class of L-serine succinyl-CoA transferases (SST), called Cys2, is encoded by metX homologs whereas these latter are known to only be involved in L-methionine biosynthesis. The conservation of Cys2 in fungi suggests that the biosynthesis of L-cysteine via the novel metabolite O-succinyl L-serine (OSS) is a common treat in this kingdom. In contrast, few Cys2 homologs have been identified in bacteria, particularly in Xanthomonadales species. The phylogenetic proximity of these enzymes, along with the structural similarity of their active site suggest they share the same function. To support the in vivo role for OSS in Xanthomonadales, we kinetically characterized Cys2 from both Xanthomonas campestris and Frateuria aurantia. Their catalytic efficiency (kcat/Km) is in favor of the formation of OSS. We also report that their L-cysteine synthase (the next enzyme in L-cysteine biosynthesis pathway) are actually O-succinyl-L-serine sulfhydrylases. This new metabolite was detected in the metabolome of X. campestris. Together, these results demonstrate the role for this novel metabolite in L-cysteine biosynthesis in bacteria, too

Auto-sufficient enzymatic cascade combining a non selective and promiscuous ADH and BVMOS: Application for dihydrocoumarin synthesis

International audienceDihydrocoumarin is a plant metabolite widely used as flavoring agent in agro-food industries and also as common fragrance in cosmetics. As a lot of flavor chemicals, dihydrocoumarin is found in minor quantities in its natural plant sources, the sweet clover (Melilotus officinalis) and tonka beans (Dipteryx odorata) so processes are needed to produce this natural flavor in a efficient and eco-friendly manner. Baeyer-Villiger MonoOxygenases (BVMOs) are well known flavoenzymes able to transform efficiently ketone into ester or lactone with high regio-and stereoselectivities. Nevertheless, BVMOs are strictly NADPH-dependent, and therefore require a stoichiometric amount of the expensive nicotinamide cofactor. To address this issue, the multi-enzyme syntheses provide the opportunity to generate efficient auto-sufficient systems and only a limited number of such systems involving BVMOs has been reported to date. We will present here a new efficient access to dihydrocoumarin via a two-enzyme mediated oxidation of Indanol involving Alcohol Dehydrogenase (ADH) and BVMO. The originality of our approach comes from the features of ADH, an enzyme discovered from the biodiversity via a dedicated High Throughput Screening. The ADH is a NADP-dependent and non-enantioselective enzyme which enables on the one hand cofactor recycling and on the other hand a complete transformation of the racemic alcohol. Moreover, it presents a quite large scope of substrates. Several plasmid constructions and combinations have been tested first for the formation of e-caprolactone and compared in order to elaborate a versatile platform using ADH and different BVMOs. Then to optimize the formation of Dihydrocoumarin and limit the hydrolysis, we investigated the composition of the biotransformation medium in whole-cell process as well as in purified enzyme system.Still exploring the diversity of BVMOs activities, we wish to expand the range of potentially valued compounds obtained using these systems. (Ménil et al.

Auto-sufficient enzymatic cascade combining a non selective and promiscuous ADH and BVMOS: Application for dihydrocoumarin synthesis

Dihydrocoumarin is a plant metabolite widely used as flavoring agent in agro-food industries and also as common fragrance in cosmetics. As a lot of flavor chemicals, dihydrocoumarin is found in minor quantities in its natural plant sources, the sweet clover (Melilotus officinalis) and tonka beans (Dipteryx odorata) so processes are needed to produce this natural flavor in a efficient and eco-friendly manner. Baeyer-Villiger MonoOxygenases (BVMOs) are well known flavoenzymes able to transform efficiently ketone into ester or lactone with high regio-and stereoselectivities. Nevertheless, BVMOs are strictly NADPH-dependent, and therefore require a stoichiometric amount of the expensive nicotinamide cofactor. To address this issue, the multi-enzyme syntheses provide the opportunity to generate efficient auto-sufficient systems and only a limited number of such systems involving BVMOs has been reported to date. We will present here a new efficient access to dihydrocoumarin via a two-enzyme mediated oxidation of Indanol involving Alcohol Dehydrogenase (ADH) and BVMO. The originality of our approach comes from the features of ADH, an enzyme discovered from the biodiversity via a dedicated High Throughput Screening. The ADH is a NADP-dependent and non-enantioselective enzyme which enables on the one hand cofactor recycling and on the other hand a complete transformation of the racemic alcohol. Moreover, it presents a quite large scope of substrates. Several plasmid constructions and combinations have been tested first for the formation of e-caprolactone and compared in order to elaborate a versatile platform using ADH and different BVMOs. Then to optimize the formation of Dihydrocoumarin and limit the hydrolysis, we investigated the composition of the biotransformation medium in whole-cell process as well as in purified enzyme system.Still exploring the diversity of BVMOs activities, we wish to expand the range of potentially valued compounds obtained using these systems. (Ménil et al.

Purification and Characterization of Nitphym, a Robust Thermostable Nitrilase From Paraburkholderia phymatum

International audienceDespite the success of some nitrilases in industrial applications, there is a constant demand to broaden the catalog of these hydrolases, especially robust ones with high operational stability. By using the criteria of thermoresistance to screen a collection of candidate enzymes heterologously expressed in Escherichia coli , the enzyme Nit phym from the mesophilic organism Paraburkholderia phymatum was selected and further characterized. Its quick and efficient purification by heat treatment is of major interest for large-scale applications. The purified nitrilase displayed a high thermostability with 90% of remaining activity after 2 days at 30°C and a half-life of 18 h at 60°C, together with a broad pH range of 5.5–8.5. Its high resistance to various miscible cosolvents and tolerance to high substrate loadings enabled the quantitative conversion of 65.5 g⋅L –1 of 3-phenylpropionitrile into 3-phenylpropionic acid at 50°C in 8 h at low enzyme loadings of 0.5 g⋅L –1 , with an isolated yield of 90%. This study highlights that thermophilic organisms are not the only source of industrially relevant thermostable enzymes and extends the scope of efficient nitrilases for the hydrolysis of a wide range of nitriles, especially trans -cinnamonitrile, terephthalonitrile, cyanopyridines, and 3-phenylpropionitrile

Exploring natural biodiversity to expand access to microbial terpene synthesis

Background: Terpenes are industrially relevant natural compounds the biosynthesis of which relies on two well-established-mevalonic acid (MVA) and methyl erythritol phosphate (MEP)-pathways. Both pathways are widely distributed in all domains of life, the former is predominantly found in eukaryotes and archaea and the latter in eubacteria and chloroplasts. These two pathways supply isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the universal building blocks of terpenes. Results: The potential to establish a semisynthetic third pathway to access these precursors has been investigated in the present work. We have tested the ability of a collection of 93 isopentenyl phosphate kinases (IPK) from the biodiversity to catalyse the double phosphorylation of isopentenol and dimethylallyl alcohol to give, respectively IPP and DMAPP. Five IPKs selected from a preliminary in vitro screening were evaluated in vivo in an engineered chassis E. coli strain producing carotenoids. The recombinant pathway leading to the synthesis of neurosporene and lyco-pene, allows a simple colorimetric assay to test the potential of IPKs for the synthesis of IPP and DMAPP starting from the corresponding alcohols. The best candidate identified was the IPK from Methanococcoides burtonii (UniProt ID: Q12TH9) which improved carotenoid and neurosporene yields ~ 18-fold and > 45-fold, respectively. In our lab scale conditions, titres of neurosporene reached up to 702.1 ± 44.7 µg/g DCW and 966.2 ± 61.6 µg/L. A scale up to 4 L in-batch cultures reached to 604.8 ± 68.3 µg/g DCW and 430.5 ± 48.6 µg/L without any optimisation shown its potential for future applications. Neurosporene was almost the only carotenoid produced under these conditions, reaching ~ 90% of total carotenoids both at lab and batch scales thus offering an easy access to this sophisticated molecule. Conclusion: IPK biodiversity was screened in order to identify IPKs that optimize the final carotenoid content of engineered E. coli cells expressing the lycopene biosynthesis pathway. By simply changing the IPK and without any other metabolic engineering we improved the neurosporene content by more than 45 fold offering a new biosynthetic access to this molecule of upmost importance