An HMM approach expands the landscape of sesquiterpene cyclases across the kingdom Fungi.

Sesquiterpene cyclases (STC) catalyse the cyclization of the C15 molecule farnesyl diphosphate into a vast variety of mono- or polycyclic hydrocarbons and, for a few enzymes, oxygenated structures, with diverse stereogenic centres. The huge diversity in sesquiterpene skeleton structures in nature is primarily the result of the type of cyclization driven by the STC. Despite the phenomenal impact of fungal sesquiterpenes on the ecology of fungi and their potentials for applications, the fungal sesquiterpenome is largely untapped. The identification of fungal STC is generally based on protein sequence similarity with characterized enzymes. This approach has improved our knowledge on STC in a few fungal species, but it has limited success for the discovery of distant sequences. Besides, the tools based on secondary metabolite biosynthesis gene clusters have shown poor performance for terpene cyclases. Here, we used four sets of sequences of fungal STC that catalyse four types of cyclization, and specific amino acid motives to identify phylogenetically related sequences in the genomes of basidiomycetes fungi from the order Polyporales. We validated that four STC genes newly identified from the genome sequence of Leiotrametes menziesii, each classified in a different phylogenetic clade, catalysed a predicted cyclization of farnesyl diphosphate. We built HMM models and searched STC genes in 656 fungal genomes genomes. We identified 5605 STC genes, which were classified in one of the four clades and had a predicted cyclization mechanism. We noticed that the HMM models were more accurate for the prediction of the type of cyclization catalysed by basidiomycete STC than for ascomycete STC.

In vitro Applications of the Terpene Mini-Path 2.0.

In 2019 four groups reported independently the development of a simplified enzymatic access to the diphosphates (IPP and DMAPP) of isopentenol and dimethylallyl alcohol (IOH and DMAOH). The former are the two universal precursors of all terpenes. We report here on an improved version of what we call the terpene mini-path as well as its use in enzymatic cascades in combination with various transferases. The goal of this study is to demonstrate the in vitro utility of the TMP in, i) synthesizing various natural terpenes, ii) revealing the product selectivity of an unknown terpene synthase, or iii) generating unnatural cyclobutylated terpenes.

Exploring the Temperature Effect on Enantioselectivity of a Baeyer-Villiger Biooxidation by the 2,5-DKCMO Module: The SLM Approach.

Temperature is a crucial parameter for biological and chemical processes. Its effect on enzymatically catalysed reactions has been known for decades, and stereo- and enantiopreference are often temperature-dependent. For the first time, we present the temperature effect on the Baeyer-Villiger oxidation of rac-bicyclo[3.2.0]hept-2-en-6-one by the type II Bayer-Villiger monooxygenase, 2,5-DKCMO. In the absence of a reductase and driven by the hydride-donation of a synthetic nicotinamide analogue, the clear trend for a decreasing enantioselectivity at higher temperatures was observed. "Traditional" approaches such as the determination of the enantiomeric ratio (E) appeared unsuitable due to the complexity of the system. To quantify the trend, we chose to use the 'Shape Language Modelling' (SLM), a tool that allows the reaction to be described at all points in a shape prescriptive manner. Thus, without knowing the equation of the reaction, the substrate ee can be estimated that at any conversion.

Photobiocatalytic Oxyfunctionalization with High Reaction Rate using a Baeyer-Villiger Monooxygenase from Burkholderia xenovorans in Metabolically Engineered Cyanobacteria.

Baeyer-Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to lactones under very mild reaction conditions. This enzymatic route is hindered by the requirement of a stoichiometric supply of auxiliary substrates for cofactor recycling and difficulties with supplying the necessary oxygen. The recombinant production of BVMO in cyanobacteria allows the substitution of auxiliary organic cosubstrates with water as an electron donor and the utilization of oxygen generated by photosynthetic water splitting. Herein, we report the identification of a BVMO from Burkholderia xenovorans (BVMO Xeno ) that exhibits higher reaction rates in comparison to currently identified BVMOs. We report a 10-fold increase in specific activity in comparison to cyclohexanone monooxygenase (CHMO Acineto ) in Synechocystis sp. PCC 6803 (25 vs 2.3 U gDCW -1 at an optical density of OD750 = 10) and an initial rate of 3.7 ± 0.2 mM h-1. While the cells containing CHMO Acineto showed a considerable reduction of cyclohexanone to cyclohexanol, this unwanted side reaction was almost completely suppressed for BVMO Xeno , which was attributed to the much faster lactone formation and a 10-fold lower K M value of BVMO Xeno toward cyclohexanone. Furthermore, the whole-cell catalyst showed outstanding stereoselectivity. These results show that, despite the self-shading of the cells, high specific activities can be obtained at elevated cell densities and even further increased through manipulation of the photosynthetic electron transport chain (PETC). The obtained rates of up to 3.7 mM h-1 underline the usefulness of oxygenic cyanobacteria as a chassis for enzymatic oxidation reactions. The photosynthetic oxygen evolution can contribute to alleviating the highly problematic oxygen mass-transfer limitation of oxygen-dependent enzymatic processes.

Extension of the Terpene Chemical Space: the Very First Biosynthetic Steps.

The structural diversity of terpenes is particularly notable and many studies are carried out to increase it further. In the terpene biosynthetic pathway this diversity is accessible from only two common precursors, i. e. isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Methods recently developed (e. g. the Terpene Mini Path) have allowed DMAPP and IPP to be obtained from a two-step enzymatic conversion of industrially available isopentenol (IOH) and dimethylallyl alcohol (DMAOH) into their corresponding diphosphates. Easily available IOH and DMAOH analogues then offer quick access to modified terpenoids thus avoiding the tedious chemical synthesis of unnatural diphosphates. The aim of this minireview is to cover the literature devoted to the use of these analogues for widening the accessible terpene chemical space.

The Terpene Mini-Path, a New Promising Alternative for Terpenoids Bio-Production.

Terpenoids constitute the largest class of natural compounds and are extremely valuable from an economic point of view due to their extended physicochemical properties and biological activities. Due to recent environmental concerns, terpene extraction from natural sources is no longer considered as a viable option, and neither is the chemical synthesis to access such chemicals due to their sophisticated structural characteristics. An alternative to produce terpenoids is the use of biotechnological tools involving, for example, the construction of enzymatic cascades (cell-free synthesis) or a microbial bio-production thanks to metabolic engineering techniques. Despite outstanding successes, these approaches have been hampered by the length of the two natural biosynthetic routes (the mevalonate and the methyl erythritol phosphate pathways), leading to dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP), the two common universal precursors of all terpenoids. Recently, we, and others, developed what we called the terpene mini-path, a robust two enzyme access to DMAPP and IPP starting from their corresponding two alcohols, dimethylallyl alcohol and isopentenol. The aim here is to present the potential of this artificial bio-access to terpenoids, either in vitro or in vivo, through a review of the publications appearing since 2016 on this very new and fascinating field of investigation.

Divorce in the two-component BVMO family: the single oxygenase for enantioselective chemo-enzymatic Baeyer-Villiger oxidations.

Two-component flavoprotein monooxygenases consist of a reductase and an oxygenase enzyme. The proof of functionality of the latter without its counterpart as well as the mechanism of flavin transfer remains unanswered beyond doubt. To tackle this question, we utilized a reductase-free reaction system applying purified 2,5-diketocamphane-monooxygenase I (2,5-DKCMO), a FMN-dependent type II Baeyer-Villiger monooxygenase, and synthetic nicotinamide analogues (NCBs) as dihydropyridine derivatives for FMN reduction. This system demonstrated the stand-alone quality of the oxygenase, as well as the mechanism of FMNH2 transport by free diffusion. The efficiency of this reductase-free system strongly relies on the balance of FMN reduction and enzymatic (re)oxidation, since reduced FMN in solution causes undesired side reactions, such as hydrogen peroxide formation. Design of experiments allowed us to (i) investigate the effect of various reaction parameters, underlining the importance to balance the FMN/FMNH2 cycle, (ii) optimize the reaction system for the enzymatic Baeyer-Villiger oxidation of rac-bicyclo[3.2.0]hept-2-en-6-one, rac-camphor, and rac-norcamphor. Finally, this study not only demonstrates the reductase-independence of 2,5-DKCMO, but also revisits the terminology of two-component flavoprotein monooxygenases for this specific case.

Tuning of the enzyme ratio in a neutral redox convergent cascade: A key approach for an efficient one-pot/two-step biocatalytic whole-cell system.

The efficiency of a versatile in vivo cascade involving a promiscuous alcohol dehydrogenase, obtained from a biodiversity search, and a Baeyer-Villiger monooxygenase was enhanced by the independent control of the production level of each enzyme to produce ε-caprolactone and 3,4-dihydrocoumarin. This goal was achieved by adjusting the copy number per cell of Escherichia coli plasmids. We started from the observation that this number generally correlates with the amount of produced enzyme and demonstrated that an in vivo multi-enzymatic system can be improved by the judicious choice of plasmid, the lower activity of the enzyme that drives the limiting step being counter-balanced by a higher concentration. Using a preconception-free approach to the choice of the plasmid type, we observed positive and negative synergetic effects, sometimes unexpected and depending on the enzyme and plasmid combinations. Experimental optimization of the culture conditions allowed us to obtain the complete conversion of cyclohexanol (16 mM) and 1-indanol (7.5 mM) at a 0.5-L scale. The yield for the conversion of cyclohexanol was 80% (0.7 g ε-caprolactone, for the productivity of 244 mg·L -1 ·h -1 ) and that for 1-indanol 60% (0.3 g 3,4-dihydrocoumarin, for the productivity of 140 mg·L -1 ·h -1 ).

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 lycopene, 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.

Membrane Protein Solubilization and Composition of Protein Detergent Complexes.

Membrane proteins are typically expressed in heterologous systems with a view to in vitro characterization. A critical step in the preparation of membrane proteins after expression in any system is the solubilization of the protein in aqueous solution, typically using detergents and lipids, to obtain the protein in a form suitable for purification, structural or functional analysis. This process is particularly difficult as the objective is to prepare the protein in an unnatural environment, a protein detergent complex, separating it from its natural lipid partners while causing the minimum destabilization or modification of the structure. Although the process is difficult, and relatively hard to master, an increasing number of membrane proteins have been successfully isolated after expression in a wide variety of systems. In this chapter we give a general protocol for preparing protein detergent complexes that is aimed at guiding the reader through the different critical steps. In the second part of the chapter we illustrate how to analyze the composition of protein detergent complexes; this analysis is important as it has been found that compositional variation often causes irreproducible results.

Prevalence and specificity of Baeyer-Villiger monooxygenases in fungi.

Out of 107 fungal strains belonging to three phyla (Ascomycota, Basidiomycota and Zygomycota) and 46 genera, 86 exhibited Baeyer-Villiger monooxygenase (BVMO) activity against racemic bicyclo[3.2.0]heptenone. The strains were classified into three "profiles" based on regio- and enantioselectivity. Statistical analyses of our results, extended by literature data, showed that these profiles could be related to the taxonomic classification of the strains, and suggest that the BVMOs from the Zygomycota phylum may be different in their primary structures from established ones.

Ultrafast excited state processes in Roseobacter denitrificans antennae: comparison of isolated complexes and native membranes.

Roseobacter (Rsb.) denitrificans is a marine aerobic anoxygenic photosynthetic purple bacterium with an unusually high-800 nm absorption band. Ultrafast excited state processes have been intensively studied in the past in order to understand why the energy transfer efficiency between photosynthetic antennae approaches unity and recently it has been proved that the organization of the antennae proteins within the membranes plays an important role. Thanks to the development of genetic manipulation and to the capability of Rsb. denitrificans to grow anaerobically as well, it is possible to construct several mutants in order to compare the ultrafast dynamics between isolated complexes and complexes embedded in membrane environments. Time resolved fluorescence and transient absorption have been applied to isolate LH2, genetically modified membranes with LH2-only and wild type membranes with both LH2 and LH1 antennae of Rsb. denitrificans, in order to understand the effect of the membrane environment on the energy transfer efficiency. A global analysis is applied to calculate the lifetime of the excited states of LH2 and LH1, and although there is shortening of the relaxation lifetime of the LH2-only membranes with respect to the isolated LH2, we find an energy transfer efficiency from LH2 to LH1 of 95%, which still approaches unity.

Molecular origins and consequences of High-800 LH2 in Roseobacter denitrificans.

Roseobacter denitrificans is a marine bacterium capable of using a wide variety of different metabolic schemes and in particular is an anoxygenic aerobic photosynthetic bacterium. In the work reported here we use a deletion mutant that we have constructed to investigate the structural origin of the unusual High-800 light-harvesting complex absorption in this bacterium. We suggest that the structure is essentially unaltered when compared to the usual nonameric complexes but that a change in the environment of the C(13:1) carbonyl group is responsible for the change in spectrum. We tentatively relate this change to the presence of a serine residue in the α-polypeptide. Surprisingly, the low spectral overlap between the peripheral and core light-harvesting systems appears not to compromise energy collection efficiency too severely. We suggest that this may be at the expense of maintaining a low antenna size.

Forces guiding assembly of light-harvesting complex 2 in native membranes.

Interaction forces of membrane protein subunits are of importance in their structure, assembly, membrane insertion, and function. In biological membranes, and in the photosynthetic apparatus as a paradigm, membrane proteins fulfill their function by ensemble actions integrating a tight assembly of several proteins. In the bacterial photosynthetic apparatus light-harvesting complexes 2 (LH2) transfer light energy to neighboring tightly associated core complexes, constituted of light-harvesting complexes 1 (LH1) and reaction centers (RC). While the architecture of the photosynthetic unit has been described, the forces and energies assuring the structural and functional integrity of LH2, the assembly of LH2 complexes, and how LH2 interact with the other proteins in the supramolecular architecture are still unknown. Here we investigate the molecular forces of the bacterial LH2 within the native photosynthetic membrane using atomic force microscopy single-molecule imaging and force measurement in combination. The binding between LH2 subunits is fairly weak, of the order of k(B)T, indicating the importance of LH2 ring architecture. In contrast LH2 subunits are solid with a free energy difference of 90 k(B)T between folded and unfolded states. Subunit α-helices unfold either in one-step, α- and ß-polypeptides unfold together, or sequentially. The unfolding force of transmembrane helices is approximately 150 pN. In the two-step unfolding process, the ß-polypeptide is stabilized by the molecular environment in the membrane. Hence, intermolecular forces influence the structural and functional integrity of LH2.

Antenna mixing in photosynthetic membranes from Phaeospirillum molischianum.

We have investigated the adaptation of the light-harvesting system of the photosynthetic bacterium Phaeospirillum molischianum (DSM120) to very low light conditions. This strain is able to respond to changing light conditions by differentially modulating the expression of a family of puc operons that encode for peripheral light-harvesting complex (LH2) polypeptides. This modulation can result in a complete shift between the production of LH2 complexes absorbing maximally near 850 nm to those absorbing near 820 nm. In contradiction to prevailing wisdom, analysis of the LH2 rings found in the photosynthetic membranes during light adaptation are shown to have intermediate spectral and electrostatic properties. By chemical cross-linking and mass-spectrometry we show that individual LH2 rings and subunits can contain a mixture of polypeptides derived from the different operons. These observations show that polypeptide synthesis and insertion into the membrane are not strongly coupled to LH2 assembly. We show that the light-harvesting complexes resulting from this mixing could be important in maintaining photosynthetic efficiency during adaptation.

Membrane protein solubilization.

A critical step in any in vitro analysis of membrane proteins is the solubilization of the membrane to extract the protein of interest in an active form to obtain an aqueous solution containing the membrane protein complexed with detergents and lipids in a form suitable for purification and further analysis. This process is particularly delicate as the aim is to maximally disrupt the lipid components of the membrane while putting the protein components in an un-natural detergent environment without perturbing them. Looked at this way, it is remarkable that it ever works. Although the process is difficult and hard to master, an increasing number of membrane proteins have been successfully solubilized in active forms, allowing some general principles to be established that we illustrate in the method developed in this chapter.

High-resolution architecture of the outer membrane of the Gram-negative bacteria Roseobacter denitrificans.

The outer membrane of Gram-negative bacteria protects the cell against bactericidal substances. Passage of nutrients and waste is assured by outer membrane porins, beta-barrel transmembrane channels. While atomic structures of several porins have been solved, so far little is known on the supramolecular structure of the outer membrane. Here we present the first high-resolution view of a bacterial outer membrane gently purified maintaining remnants of peptidoglycan on the perisplasmic surface. Atomic force microscope images of outer membrane fragments of the size of approximately 50% of the bacterial envelope revealed that outer membrane porins are by far more densely packed than previously assumed. Indeed the outer membrane is a molecular sieve rather than a membrane. Porins cover approximately 70% of the membrane surface and form locally regular lattices. The potential role of exposed aromatic residues in the formation of the supramolecular assembly is discussed. Finally, we present first structural data of the outer membrane porin from the marine Gram-negative bacteria Roseobacter denitrificans, and we perform a sequence alignment with porins of known structure.

Quinone pathways in entire photosynthetic chromatophores of Rhodospirillum photometricum.

In photosynthetic organisms, membrane pigment-protein complexes [light-harvesting complex 1 (LH1) and light-harvesting complex 2 (LH2)] harvest solar energy and convert sunlight into an electrical and redox potential gradient (reaction center) with high efficiency. Recent atomic force microscopy studies have described their organization in native membranes. However, the cytochrome (cyt) bc(1) complex remains unseen, and the important question of how reduction energy can efficiently pass from core complexes (reaction center and LH1) to distant cyt bc(1) via membrane-soluble quinones needs to be addressed. Here, we report atomic force microscopy images of entire chromatophores of Rhodospirillum photometricum. We found that core complexes influence their molecular environment within a critical radius of approximately 250 A. Due to the size mismatch with LH2, lipid membrane spaces favorable for quinone diffusion are found within this critical radius around cores. We show that core complexes form a network throughout entire chromatophores, providing potential quinone diffusion pathways that will considerably speed the redox energy transfer to distant cyt bc(1). These long-range quinone pathway networks result from cooperative short-range interactions of cores with their immediate environment.

Arsenite oxidation by a chemoautotrophic moderately acidophilic Thiomonas sp.: from the strain isolation to the gene study.

An autotrophic bacterium able to gain energy from the oxidation of arsenite was isolated from arsenite-containing acid mine drainage waters. It belongs to the genus Thiomonas as shown by DNA-DNA hybridization experiments, 16S rRNA gene sequence, quinone and fatty acid content analyses. Carboxysomes were observed and the cbbSL genes encoding the ribulose 1,5-bisphosphate carboxylase/oxygenase were detected, confirming that this bacterium is able to fix CO(2). Arsenite oxidation was catalysed by a membrane-bound enzyme, and this activity was detected essentially in cells grown in the presence of arsenite. The genes encoding the two subunits of the arsenite oxidase of the Thiomonas isolate have been sequenced. The small subunit has a characteristic Tat signal sequence and contains the residues binding the [2Fe-2S] Rieske-type cluster. The large subunit has the [3Fe-4S] cluster-binding motif as well as the residues proposed to bind arsenite. In addition, most of the residues interacting with the molybdenum cofactor are conserved. The genes encoding both subunits belong to an operon, likely with a gene encoding a cytochrome c. The expression of this operon is greater in cells grown in the presence than in the absence of arsenite, in agreement with a transcriptional regulation in the presence of this metalloid.

Bacterial immobilization and oxidation of arsenic in acid mine drainage (Carnoulès creek, France).

The acid waters (pH=2.73-3.37) originating from the Carnoulès mine tailings contain high dissolved concentrations of arsenic (1-3.5 mmol l(-1)) and iron (20-40 mmol l(-1)). At the outlet, arsenite predominates. During the first 30 m of downflow, 20-60% is removed by coprecipitation with Fe(III). This process results from bacterially mediated As- and Fe-oxidation. The precipitation rates in the creek depend on the oxygen concentration in spring water and are lower during the dry summer period when the anoxic character of the spring water inhibits the activity of oxidizing bacteria. Ex situ experiments show that the presence of bacteria-rich precipitates increases the As- and Fe-removal rates. Three strains of bacteria promoting the oxidation of As have been isolated, and two of them have the characteristics of Thiomonas ynys1. The third strain, which is not identified yet, also catalyzes the oxidation of Fe.