M Nocente

Ye O Kazakov

J Garcia

V G G Kiptily

J Ongena

M Dreval

M Fitzgerald

S E E Sharapov

Z Stancar

H Weisen

Y Baranov

A Bierwage

T Craciunescu

A Dal

E De La Luna

R Dumont

P Dumortier

J Eriksson

L Giacomelli

C Giroud

V Goloborodko

G Gorini

E Khilkevitch

K K Kirov

M Iliasova

P Jacquet

P Lauber

E Lerche

M J Mantsinen

A Mariani

S Mazzi

F Nabais

M F F Nave

J Oliver

E Panontin

D Rigamonti

A Sahlberg

M Salewski

A Shevelev

K Shinohara

P Siren

S Sumida

M Tardocchi

D Van Eester

J Varje

A Zohar

Generation and observation of fast deuterium ions and fusion-born alpha particles in JET D -3 He plasmas with the 3-ion radio-frequency heating scenario

Keywords: Fast ion generation, radio-frequency heating, 3-ion scenario, D -3 He plasmas, MeV range ions

published or not. The documents may come

Generation and observation of fast deuterium ions and fusion-born alpha particles in JET D -3 He plasmas

with the 3-ion radio-frequency heating scenario

Introduction

Understanding the transport and confinement of fast ions and, in particular, fusion born alpha particles in burning plasmas is one of the key missions to demonstrate the feasibility of fusion as an energy source. Although a number of aspects of alpha particle physics can only be studied in burning plasmas, some can be anticipated in present machines. To this end, the Joint European Torus (JET) has established an ambitious scientific program in preparation of its future deuterium-tritium (DTE2) campaign, aiming, among others, at developing dedicated scenarios where the effects of alpha particles from the fusion reactions can be studied [START_REF] Joffrin | Overview of the jet preparation for deuterium-tritium operation with the iter like-wall[END_REF][START_REF] Dumont | Scenario development for the observation of alpha-driven instabilities in jet dt plasmas[END_REF]. One possibility is offered by the 3-ion radio-frequency (RF) heating scenario [START_REF] Ye O Kazakov | On resonant icrf absorption in three-ion component plasmas: a new promising tool for fast ion generation[END_REF][START_REF] Ye O Kazakov | Efficient generation of energetic ions in multi-ion plasmas by radio-frequency heating[END_REF] in D-T plasmas, by accelerating tritons or deuterons from neutral beam injection (NBI) to high enough energies that maximize the D-T reactivity [START_REF] Kazakov | Plasma heating and generation of energetic d ions with the 3-ion icrf+ nbi scheme in mixed hd plasmas at jet-ilw[END_REF] at moderate input auxiliary heating power. This fast-ion scenario relies on the combined use of NBI and ICRF where NBI provides the source of resonant absorbers at the ion-ion hybrid (IIH) layer in mixed plasmas, where the left-hand RF polarization is strongly enhanced. The fast-ion distribution function of the RF-accelerated beam ions can be controlled by varying the ratio P ICRF /P NBI . In this way rather high neutron rates can be obtained with a moderate amount of total auxiliary heating power, as shown in proof-of-principle studies in H-D plasmas on JET [START_REF] Kazakov | Plasma heating and generation of energetic d ions with the 3-ion icrf+ nbi scheme in mixed hd plasmas at jet-ilw[END_REF][START_REF] Ye | Physics and applications of the 3-ion icrf scenarios for fusion research[END_REF]. In view of demonstrating the feasibility of this heating scenario for alpha physics studies in D-T, we have performed a series of dedicated experiments in mixed D - 3 He plasmas on JET (B 0 ≈ 3.7T, I p = 2.5MA, n e0 ≈ 6 × 10 19 m -3 , f = 32.2-33.0 MHz, dipole phasing; B 0 , I p , n e0 and f are here the core magnetic field, plasma current, core electron density and applied RF frequency, respectively). In these studies, large 3 He concentrations n( 3 He)/n e ≈ 20-25% were used to purposely position the IIH layer in the plasma core, where acceleration of D-NBI ions with ICRF waves was expected to take place (see figure 1). Note the dual function for 3 He ions in this fast-ion scenario. The concentration of 3 He ions not only defines the location of the IIH layer (similar to the effect of the isotopic H:D ratio in earlier experiments with the 3-ion D-(D NBI )-H scenario [START_REF] Kazakov | Plasma heating and generation of energetic d ions with the 3-ion icrf+ nbi scheme in mixed hd plasmas at jet-ilw[END_REF]), but the 3 He ions also act as the target in D -3 He fusion reactions [START_REF] Nocente | Cross section of the d+ 3he α+ p reaction of relevance for fusion plasma applications[END_REF] with deuterons accelerated by ICRF: D (fast) + 3 He (target) → 4 He(3.6MeV) + p (14.7MeV) [START_REF] Joffrin | Overview of the jet preparation for deuterium-tritium operation with the iter like-wall[END_REF] With the IIH layer placed on-axis, the cyclotron resonances for thermal D and 3 He ions are located at the high field side and low field side off-axis, as shown in Fig. 1. Note that neither thermal D nor 3 He ions can fulfil the resonance condition for the fundamental ion cyclotron interaction, ω = ω ci + k v ,i , at the IIH layer, where most of the power absorption takes place thanks to the left handed polarization of the RF wave electric field in this region. Here, k and v are the wavenumber and particle velocity parallel to the confining magnetic field. Similar to earlier experiments in mixed H-D plasmas, tangential NBI injectors with E NBI ≈ 100 keV and pitch λ = v /v ≈ 0.62 were used to provide resonant absorbers at the position of the IIH layer. Guided by former experiments with the 3-ion D -(D NBI ) -H scenario and associated PION analysis [START_REF] Kazakov | Plasma heating and generation of energetic d ions with the 3-ion icrf+ nbi scheme in mixed hd plasmas at jet-ilw[END_REF][START_REF] Mj Mantsinen | Modelling of three-ion icrf schemes with pion[END_REF], we varied the ICRF and NBI power (P ICRF ≈4-6MW, P NBI ≈ 3-20MW) and the ratio P ICRF /P NBI , as main actuators to change the fast-ion distribution of RF-accelerated D NBI ions. This resulted in D-D neutron rates that ranged between ≈ 2 × 10 15 s -1 and ≈ 1 × 10 16 s -1 in the series of discharges we have performed, at variable P ICRF /P NBI . The extended set of JET fast-ion measurements, in particular neutron [START_REF] Eriksson | Measuring fast ions in fusion plasmas with neutron diagnostics at jet[END_REF] and gammaray spectrometers [START_REF] Nocente | Mev range particle physics studies in tokamak plasmas using gamma-ray spectroscopy[END_REF][START_REF] Vg Kiptily | Gamma ray diagnostics of high temperature magnetically confined fusion plasmas[END_REF], have been used to provide information on the energy and spatial distribution of the accelerated deuterium and fusion-born alpha particles [START_REF] Moseev | Recent progress in fast-ion diagnostics for magnetically confined plasmas[END_REF][START_REF] Salewski | Mev-range velocity-space tomography from gamma-ray and neutron emission spectrometry measurements at jet[END_REF]. Recent upgrades of the gamma-ray diagnostic at JET [START_REF] Nocente | High resolution gamma ray spectroscopy at mhz counting rates with labr {3} scintillators for fusion plasma applications[END_REF][START_REF] Nocente | Gamma-ray spectroscopy at mhz counting rates with a compact labr3 detector and silicon photomultipliers for fusion plasma applications[END_REF][START_REF] Rigamonti | Performance of the prototype labr3 spectrometer developed for the jet gamma-ray camera upgrade[END_REF][START_REF] Rigamonti | The upgraded jet gamma-ray cameras based on high resolution/high count rate compact spectrometers[END_REF] made it possible to unambiguously identify the production of MeV-range deuterons and fusion-born alpha particles and to study some of their properties.

In this paper we describe the main experimental results obtained in recent fast-ion experiments on JET with the 3-ion D-(D NBI )-3 He scenario, including the unambiguous confirmation of the generation of co-passing energetic D ions and fusion-born alpha particles in the plasma core. These plasmas show a number of additional, interesting phenomena linked to the presence of the energetic ions generated by the 3-ion scenario.

In particular, the rich variety of fast-ion driven Alfvén eigenmodes (AEs) observed raise a number of questions that deserve detailed further investigations. Rather surprisingly, the complex AE activity seems not to be detrimental for the thermal confinement as indicated, among others, by the systematic observation of T i ≈ T e in these plasmas with dominant core electron heating from the fast ions. Another important observation is the systematic appearance of reversed shear AEs (RSAEs) during monster sawteeth (>≈ 1 s), indicating that a non monotonic q-profile was developed and sustained in these plasmas by the large fast-ion populations generated by the 3-ion ICRF scenario.

Experimental results

Figure 2 shows the time evolution of the main physical parameters of two JET-ILW pulses #94698 and #95679, where the D -(D NBI ) - ) was observed in the phase with P ICRF ≈5.8MW. In pulse #95679 with higher NBI power and lower P ICRF /P NBI , the period of sawtooth oscillations was significantly shorter, varying between ≈200-300 ms and ≈700 ms.

There is a remarkable increase in the neutron rate when ICRF is applied in combination with NBI. In pulse #94698, the neutron rate increased from ≈ 3 × 10 14 s -1 in the NBI only phase to ≈ 2.3 × 10 15 s -1 in the first ICRF + NBI phase with ≈4.0MW of ICRF, and later to ≈ 8.2 × 10 15 s -1 when the ICRF power reached ≈6.0 MW. Note the continuous increase in neutron rate and plasma stored energy during the monster sawtooth phase in #94698. In pulse #95679, an even higher neutron rate of ≈ 1 × 10 16 was achieved. The high efficiency of the generation of energetic D ions in the plasma core has been confirmed by measurements of the neutron spectrum with the TOFOR instrument [START_REF] Gatu | The 2.5-mev neutron time-of-flight spectrometer tofor for experiments at jet[END_REF], which has a vertical line-of-sight passing through the plasma core. TOFOR measurements for #95679 imply that most of the deuterons had MeV range energies, up to about ≈2 MeV. The production of energetic D ions occurs together with the generation of fusion-born alpha particles from the D -3 He reaction. This is demonstrated in Figure 3, showing the gamma-ray spectra recorded during the combined ICRF + NBI phase in #95679 by two independent gamma-ray spectrometers. One is a high-purity Germanium detector [START_REF] Tardocchi | Spectral broadening of characteristic γ-ray emission peaks from c 12 (he 3, p γ) n 14 reactions in fusion plasmas[END_REF], that has the same vertical line-of-sight passing through the plasma core as TOFOR (Fig. 3 (a)), while the other is a LaBr 3 (Ce) detector [START_REF] Nocente | Energy resolution of gamma-ray spectroscopy of jet plasmas with a labr 3 scintillator detector and digital data acquisition[END_REF], that has a tangential line of sight passing about 30 cm below the magnetic axis (Fig. 3 (b)). Two regions in the recorded gamma-ray spectra are important to understand the fast-ion observations and reveal more information on the fast-ion populations in these D -3 He plasmas. First, we note several peaks in the interval between 2 MeV and 5 MeV. Those in the energy range E γ =2 MeV to 4 MeV originate from nuclear reactions between fast D ions and 9 Be, namely d + 9 Be → n + 10 B * and d + 9 Be → p + 10 Be * , and that result in the production of the excited heavy nuclei 10 B * and 10 Be * , which decay through the emission of a gamma-ray [START_REF] Eriksson | Dual sightline measurements of mev range deuterons with neutron and gamma-ray spectroscopy at jet[END_REF]. 9 Be is the main low-Z impurity in JET-ILW plasmas. The observation of these lines confirms the presence of fast D ions with energies of ≈0.5 MeV and higher [START_REF] Eriksson | Dual sightline measurements of mev range deuterons with neutron and gamma-ray spectroscopy at jet[END_REF][START_REF] Vg Kiptily | γ-ray diagnostics of energetic ions in jet[END_REF], at which the cross sections for D + 9 Be reactions start to become significant. Of special interest is, furthemore, the peak at E γ =4.44 MeV and that comes from the α + 9 Be → n + 12 C * reaction between 4 He ions in the MeV range and 9 Be impurities in the plasma. The fact that these 4 He ions are confined α particles from the D+ 3 He reaction (see equation 1), and not unwanted 4 He impurities that may unexpectedly absorb some RF power, comes from the observation of counts in the energy range E γ > 10 MeV, see Fig. 3 (b). Note that in addition to the main channel of the D + 3 He fusion reactions with an alpha particle as fusion product (see Eq. 1), there is a weak electromagnetic branch of this reaction, i.e. d + 3 He → γ + 5 Li , with 5 Li as the fusion product and a probability ≈10 -5 . This leads to the generation of high-energy gammas, E γ ≈ 17 MeV [START_REF] Se Sharapov | Fusion product studies via fast ion d-d and d-3he fusion on jet[END_REF][START_REF] Nocente | Conceptual design of the radial gamma ray spectrometers system for α particle and runaway electron measurements at iter[END_REF]. In other words, for each 10 5 α particles generated by d + 3 He → α + p , there must be approximately one 17 MeV gamma-ray born from the d + 3 He → γ + 5 Li reaction and that we detect as the broad structure in the spectrum of figure 3 (b) because of the peculiar detection mechanism of the 17 MeV gamma-rays, as well as due to their ≈1.2 MeV intrinsic broadening at the source [START_REF] Nocente | Gamma-ray emission spectrum from thermonuclear fusion reactions without intrinsic broadening[END_REF][START_REF] Nocente | Conceptual design of the radial gamma ray spectrometers system for α particle and runaway electron measurements at iter[END_REF]. In summary, the simultaneous observation of E γ > 10 MeV counts in the gamma-ray spectrum of figure 3 (b), together with the E γ = 4.44 MeV line of figure 3 (a), suggests that α particles are being produced by d + 3 He → α + p , and are confined long enough to be detected by the gamma-ray emission from α + 9 Be → n + 12 C * .

Strong core localization of energetic D ions generated with the 3-ion D -(D NBI ) -3 He scenario has been independently confirmed by the measurements with the JET neutron camera [START_REF] Adams | The jet neutron emission profile monitor[END_REF], consisting of 10 horizontal and 9 vertical channels. Figure 1 shows the spatial profile of the neutron emission and the tomographic inversion of the neutron camera data for pulse #95679, highlighting that neutrons are mostly born in a small zone of ≈20 cm around the magnetic axis. This result confirms the theoretical expectations for the 3-ion ICRF scenario that energetic D ions are effectively generated in the vicinity of the IIH layer in the core of D -3 He plasmas, see Fig. 1.

The large population of fast ions, deuterons and fusion-born alpha particles, is accompanied by a rich variety of Alfvénic eigenmodes (AEs). Figure 4 shows the MHD spectrograms measured by the magnetic coils in pulses #94698 and #95679, including toroidicity-(TAEs) and ellipticity-induced (EAEs), reversed shear (RSAEs) and global n = 0 AEs (GAEs; for simplicity, these are still marked as EAEs in the figure as they appear in that frequency range) [START_REF] Nikolai N Gorelenkov | Energetic particle physics in fusion research in preparation for burning plasma experiments[END_REF]. An assessment of the nature of these MHD modes identifies those in the frequency range between 500 kHz and 600 kHz as EAEs and GAEs, while modes in the range 200 kHz to 300 kHz are identified as TAEs. RSAEs start at a frequency of about 100 kHz and sweep up to the TAE frequencies, unless a sawtooth crash interrupts this evolution. All of these different AEs were regularly observed in this series of discharges in D -3 He plasmas, suggesting that they must be related to the particular experimental conditions created by the use of the 3-ion scenario.

Furthermore, the presence of a specific type of AE instability depends on the duration of the sawtooth period. The discharges can be grouped in two categories, those with sawtooth periods of some hundreds of ms and those with periods of some seconds. The MHD spectrogram for the first type of discharges (see Fig. 4(b)) shows that EAEs and GAEs are particularly strong in this case, while RSAEs are short lived. Vice versa, during long-period sawteeth the RSAE frequency fully evolves until it approaches that of the TAE gap, see Fig. 4(a). By using the set of Mirnov coils available at JET we can further determine the toroidal number n of the observed modes. Besides instabilities with n = 1 through n = 4, both in the TAE and EAE frequency range, we also systematically observe modes with n = 0 (identified as a GAE) and EAEs with negative mode numbers, in particular n = -1. The spatial localization of the AEs is provided by the correlation reflectometer, available for most of the discharges, indicating that the AEs are typically located close to the plasma core, within ≈20 cm from the magnetic axis. For discharges with long sawtooth periods, the AE dynamics are more complex. As for cases with short sawteeth, EAEs appear about 90 ms after the crash and persist for about 100 ms. An important observation in these discharges is the presence of RSAEs with n = 3, n = 2 and n = 1, with frequencies evolving in time from the Geodesic Acoustic Mode (GAM) up to the TAE frequencies. Interestingly, a continuous increase in neutron rate and plasma stored energy is regularly observed during the monster sawtooth phases with RSAEs. The very observation of RSAEs implies that the q-profile must be nonmonotonic in this phase. Since non-inductive current drive was not purposely applied by external means, an intrinsic mechanism must be present that drives the current necessary for the reversal in the q-profile. As discussed in the next section, the strong core localization of the RF power deposition characteristic of the 3-ion ICRF scenarios leads to a very effective generation of a large population of fast co-passing D ions in the central region of the plasma. These fast D-ions are the obvious candidate particles to drive non-inductive current, which could contribute to the modification of the q-profile [START_REF] Ye | Physics and applications of the 3-ion icrf scenarios for fusion research[END_REF]. Despite the rich variety of MHD activities driven by the energetic ions, we find that these modes do not have a detrimental effect on the global plasma parameters. On the contrary, the energy confinement time and the ion and electron temperature profiles of these plasmas suggest that there is an overall beneficial effect of the fast particles on thermal plasma transport. Figure 5 shows the electron and ion temperature profiles measured in JET pulse #94700 (P ICRF ≈6.0MW and P NBI ≈6.3MW), that illustrates the type of T e and T i profiles observed in this series of discharges. The ion temperature profile was obtained by charge exchange analysis of lines from Neon, which was purposely injected into the plasma for T i measurements. As the developed fast-ion scenario efficiently generates MeV-range energetic ions, collisional electron heating is a dominant heating source in the plasma core, i.e. for a normalized toroidal radius ρ < 0.2 according to TRANSP [START_REF] Budny | A standard dt supershot simulation[END_REF] simulations. In particular, TRANSP predicts that the electron heating is twice as large as ion heating at the location of the magnetic axis. Despite that, we systematically observe T i ≈T e for all the pulses where good-quality Ne charge-exchange data are available. The observation that T i /T e ≈1 in plasmas with dominant fastion electron heating is very promising in view of ITER. Despite the L-mode nature of the plasmas, a rather high plasma stored energy was systematically observed in the phases with the combined ICRF and NBI heating, compared to NBI only. A prominent example comes from the direct comparison between discharges #94701 and #94704, where the same total heating power of 14 MW was applied by combined NBI+ICRF (#94701) or NBI only (#94704) (see figure 12 of [START_REF] Kazakov | Plasma heating and generation of energetic d ions with the 3-ion icrf+ nbi scheme in mixed hd plasmas at jet-ilw[END_REF]). The plasma stored energy is twice as large for #94701 compared to #94704. This observation, in combination with the direct comparison of T e and T i profiles for D -3 He plasmas heated with NBI-only at the same total heating power, plasma density and other operational conditions (see Fig. 12 in [START_REF] Kazakov | Plasma heating and generation of energetic d ions with the 3-ion icrf+ nbi scheme in mixed hd plasmas at jet-ilw[END_REF]), implies that turbulent transport may have been reduced when the 3-ion D -(D NBI ) -3 He was applied for plasma heating.

The rich and partly unexpected variety of phenomena that are present in the discharges we have studied raise a number of questions that require a detailed further investigation. In what follows, we provide first hypotheses to explain the observations. A further detailed assessment is ongoing and will be reported in future papers.

Discussion

We first focus on the nature of the orbits of the fast deuterons generated with ICRF. As follows from quasi-linear theory [START_REF] Eriksson | On the orbit-averaged monte carlo operator describing ion cyclotron resonance frequency wave-particle interaction in a tokamak[END_REF][START_REF] Howard | Fast-wave heating of a two-component plasma[END_REF][START_REF] Allan N Kaufman | Quasilinear diffusion of an axisymmetric toroidal plasma[END_REF], the evolution of the fast-ion distribution function f 0 under ICRF is described by

< Q(f 0 ) >= N,n,ω L N,n,ω (D 0 L N,n,ω f 0 ) (2)
where D 0 is a diffusion coefficient and the operator L n,N,ω is given by

L n,N,ω = ω ∂ ∂E + ω c0 n -Λω E ∂ ∂Λ + N ∂ ∂P Φ (3)
Here, ω is the angular RF frequency, n is the cyclotron harmonic (n = 1 for the 3ion ICRF scenarios), ω c0 is the on-axis cyclotron resonance frequency, N is the toroidal mode number and φ is the toroidal angle. In this formalism, the phase space trajectories of resonant ions are described by changes of the energy E, normalized magnetic moment Λ (Λ = µB 0 /E, with µ the magnetic moment and B 0 the magnetic field at the magnetic axis) and toroidal component of the canonical angular momentum P φ . For a toroidally symmetric system ∂ ∂P φ = 0. When collisions are neglected, we find from Eqs. 2 and 3 that small changes in E and Λ during ICRF acceleration are given by (see also Equation [START_REF] Eriksson | Dual sightline measurements of mev range deuterons with neutron and gamma-ray spectroscopy at jet[END_REF] in [START_REF] Eriksson | On the orbit-averaged monte carlo operator describing ion cyclotron resonance frequency wave-particle interaction in a tokamak[END_REF])

∆Λ = (Λ ∞ -Λ) ∆E E (4)
where Λ ∞ = ω c0 /ω. We note that Λ ∞ acts as a phase space attractor, i.e. it is the asymptotic normalized magnetic moment that is approached by the particle as its energy increases under the application of ICRF. Equation 4 implies a detailed relationship between the particle pitch λ = v /v and its energy E. By noting that λ 2 = 1 -Λ 2 , one can show that

λ(E) = λ 2 ∞ + E 0 E (λ 2 0 -λ 2 ∞ ) (5)
which mathematically implies that λ → λ ∞ as E → ∞, and where λ 0 is the pitch of the fast particle at the beginning of the acceleration.

For the 3-ion D -(D NBI ) -3 He scenario with the IIH layer located on-axis (B 0 =3.7T, f = 32.2-33 MHz), λ ∞ ≈0.34-0.38. Furthermore, the trapped-passing boundary in the plasma region very close to the magnetic axis has a peculiar structure that facilitates the presence of co-passing fast ions with v > 0 at energies above NBI injection energy. In this way, the combination of the strong localization of the RF power deposition in the plasma core, the non-standard trapped/passing boundary in that region and the quasilinear evolution of the fast ions ensure that co-passing NBI ions stay on passing orbits and do not become trapped as they absorb RF power and increase their energy [START_REF] Ye | Physics and applications of the 3-ion icrf scenarios for fusion research[END_REF]. We note that this is possible only thanks to the peculiar properties of the 3-ion ICRF scenario, with very strong energy deposition and fast-ion generation in a small volume close to the magnetic axis (see also Figure 1). This is quite different from more common ICRF heating scenarios, e.g. minority ICRF heating, where the power absorption takes place in a larger (vertically stretched) plasma volume and at larger normalized radii ρ, such that ICRF-generated fast ions become predominantly trapped as they absorb RF power.

The result given by Eq. 5 is supported by TRANSP simulations for JET pulse #95679. Figure 6(a) shows the computed distribution function of the fast D ions in the core region of the plasma as a function of their pitch at E D = 0.8 MeV, 1.1 MeV and 1.4MeV. At the NBI injection energy, the population of fast D ions is peaked at λ 0 ≈ 0.62. As follows from Fig. 6(a), at high energies most of the ICRF-accelerated D-NBI ions have a pitch between 0.35 and 0.40. Note that the perpendicular energy of these fast ions represents ≈85% of the total energy, consistent with the fact that RF cyclotron interaction predominantly increases the perpendicular velocity of resonant ions. A direct confirmation of the co-passing nature of the fast deuterons comes from a detailed analysis of the shape of the 2.87 MeV gamma-ray peak born from D + 9 Be nuclear reactions and measured with a high purity Germanium (HpGe) detector. In Fig. 6(b) we show the sum of the experimental data from discharges #95677, #95679, #95680, #95683 where the HpGe was active, together with the expected shape of this line computed with the GENESIS code [START_REF] Tardocchi | Spectral broadening of characteristic γ-ray emission peaks from c 12 (he 3, p γ) n 14 reactions in fusion plasmas[END_REF][START_REF] Nocente | Neutron and gamma-ray emission spectroscopy as fast ion diagnostics in fusion plasmas[END_REF] and assuming that the fast deuterons have λ ≈ 0.4 (solid red line) and λ ≈ 0.0 (blue dotted line). The latter occurs in most of the commonly used ICRF scenarios. Besides the peak shape, with the code we can also compute the relative amount of gamma rays emitted in both cases. In this way, there is only one parameter to fit between simulations and data, i.e. the conversion factor between the output of the code for the λ ≈ 0.40 case (gammas/second/keV produced along the instrument line of sight) and the counts measured experimentally in the region of the 2.87 MeV peak. In agreement with theory, we find that the shape of the computed curves assuming λ ≈ 0.4 agrees rather well with the measured shape of the E γ = 2.87 MeV peak. For λ ≈ 0.0, instead, the model systematically predicts lower values at the centre of the line. This synthetic analysis confirms that the 3-ion ICRF scenario is effective in generating a large population of co-passing fast D ions with λ ≈ 0.3 -0.5 in the central region of the plasma. We note that the co-passing deuterons may provide a current contribution that favours the excitation of the RSAEs. As noted earlier, the very observation of RSAEs (which appear in between the sawtooth crashes) implies that the q-profile must be nonmonotonic and one candidate contribution to the reversal of q may come from the copassing deuteron population itself, which could be slightly off-axis due to the Shafranov shift or the spatial redistribution resulting from its interaction with the MHD activity.

While we are aware that the detailed relation between the presence of a co-passing deuteron population, the reversal of q and the excitation of RSAEs might be complex and therefore needs to be studied in detail, we also suggest that, if fully understood, it may transform the 3-ion ICRF scenario in a tool for tailoring the q profile in future applications. The topic will be the subject of future studies.

In addition to RSAEs, the fast ions excited various AE instabilities, particularly in the range between 500 kHz and 600 kHz with toroidal mode numbers n = 0 and n = -1. As the appearance of these modes occurs together with the generation of an unusually large alpha particle population, at the level of ≈ 10 16 particle/s according to TRANSP and thus comparable to the neutron emission from these plasmas as measured by the fission chambers (see figure 2), a possibility exists that some of the modes may be partly or entirely alpha driven. This applies in particular to the above mentioned n=0 and n=-1 instabilities, whose resonance conditions seem to be more easily satisfied by the MeV range, relatively isotropic alpha particle population according to an ongoing analysis [START_REF] Kiptily | Evidence for alfvén eigenmodes driven by alphaparticlesin d-3 he fusion experiments on jet[END_REF].

The rich variety of AE activities present in the plasma does not have a detrimental effect on the global plasma parameters but, on the contrary, comparable electron and ion temperature are observed in all our discharges with dominant fast-ion electron heating in the plasma core, as surmised for burning plasmas in ITER. Since this is accompanied by a factor 2 higher plasma stored energy compared with plasmas with same input heating power delivered by NBI only, we suggest that the presence of the large population of fast ions located in a small volume around the plasma core may play some key role in improving the energy confinement. A similar observation was made earlier in connection with experiments at ASDEX Upgrade and JET both in L-mode [START_REF] Mantica | Ion heat transport studies in jet[END_REF] and Hmode [START_REF] Challis | Improved confinement in jet high β plasmas with an iter-like wall[END_REF][START_REF] Mj Mantsinen | Bulk ion heating with icrf waves in tokamaks[END_REF] with different sources of fast ions such as 3 He ions generated with ICRF minority heating or NBI fast ions. An explanation of the turbulence mitigation observed in those experiments was advanced in a number of papers, where a key contribution of the energetic ions to the mitigation of Ion Temperature Gradient modes by different mechanisms was found through gyrokinetic simulations [START_REF] Citrin | Nonlinear stabilization of tokamak microturbulence by fast ions[END_REF][START_REF] Bonanomi | Turbulent transport stabilization by icrh minority fast ions in low rotating jet ilw l-mode plasmas[END_REF][START_REF] Di Siena | Electromagnetic turbulence suppression by energetic particle driven modes[END_REF][START_REF] Garcia | Key impact of finite-beta and fast ions in core and edge tokamak regions for the transition to advanced scenarios[END_REF][START_REF] Doerk | Gyrokinetic study of turbulence suppression in a jet-ilw power scan[END_REF]. Although the ICRF heating scheme adopted in our scenario is different with respect to that used in the former experiments, i.e. 3 ion versus 3 He minority ICRF, we share with the latter the presence of a significant, core localized population of fast ions in the MeV range, as well as the experimental observation of a larger than expected bulk ion temperature. For these reasons, we surmise that our scenario may be of interest also for studies on the effects that a large fast ion population in the MeV range can have on the mitigation of bulk plasma turbulence, in view of understanding the role that DT fusion born α particles may have in this respect [START_REF] Garcia | Isotope and fast ions turbulence suppression effects: Consequences for high-β iter plasmas[END_REF].

Conclusions

Dedicated experiments to generate alpha particles from the D -3 He fusion reaction were performed at JET. The experiments were conducted in D -3 He plasmas with the 3-ion D -(D NBI ) -3 He ICRF scenario, in which deuterium ions from NBI are accelerated up to MeV-range energies and 3 He acts as the target of the fusion reactions. The alpha particles (with a production rate in excess of 10 16 s -1 ) and co-passing fast deuterons are generated in a narrow zone around the plasma center as experimentally demonstrated by gamma-ray and neutron measurements. Most importantly, these plasmas show rather unique phenomena, such as a rich variety of AEs, in particular elliptical and reversed shear AEs, and ion and electron temperature profiles with very similar shape and central temperature values, despite the dominant fast-ion electron heating. We propose hypotheses that may explain our experimental findings such as, e.g., the possibility that fusion-born alpha particles contribute to drive some of the AEs, as well as effects from large populations of ICRF-generated co-passing deuterium ions on current drive and modifications of the q-profile. The results we have reported are highly relevant for the development of a scenario that maximizes the production of alpha particles and thus the observation of their effects on the plasma in forthcoming deuterium-tritium experiments at JET. The experimental conditions obtained in our discharges may therefore be of more general interest, as they seem to anticipate novel aspects of alpha particle physics in plasmas without tritium.

Figure 1 .

 1 Figure 1. The poloidal cross-section of the JET tokamak with off-axis location of the ion cyclotron resonance for thermal D and 3 He ions for 3-ion ICRF fast-ion experiments in D -3 He plasmas (3.7T/2.5MA, n e0 ≈ 6 × 10 19 m -3 , f = 32.2-33.0 MHz, dipole phasing, E NBI ≈ 100 keV). The coloured image is a tomographic reconstruction of the neutron emissivity for discharge #95679. The dashed lines are the lines of sight of the JET neutron camera [18].

Figure 3 .

 3 Figure 3. Gamma-ray spectra measured in JET pulse #95679. (a) Data obtained with the high-purity Germanium detector with a vertical line-of-sight passing through the plasma core. The most prominent gamma-ray lines are labelled by the reaction they come from. (b) Data from the LaBr 3 (Ce) detector with a horizontal line-of-sight that passes about 30 cm below the magnetic axis. The broad structure in the energy range E γ > 10 MeV comes from a weak branch of the D -3 He fusion reaction with 5 Li as the fusion product. For E γ < 5 MeV the same peaks of figure (a) are measured by this detector as well.

Figure 4 .

 4 Figure 4. A rich variety of fast-on driven Alfvén eigenmodes is observed in D -3 He plasmas using the 3-ion D -(D NBI ) -3 He scenario: (left) #94698 and (right) #95679.

Figure 5 .

 5 Figure 5. Illustration of the electron and ion temperature profiles in JET experiments in D -3 He plasmas, in which the 3-ion D -(D NBI ) -3 He ICRF scenario accelerated D-NBI ions to MeV range energies. The example shows T e and T i profiles measured in pulse #94700 at t = 10.6s (P ICRF ≈6.0MW, P NBI ≈6.3MW, n e0 ≈ 6×10 19 m -3 )

FastFigure 6 .

 6 Figure 6. (a) The distribution function of ICRF-accelerated fast D ions in #95679 at E D = 800keV, 1100 keV and 1400 keV as a function of the pitch parameter, computed by the TRANSP code. (b) The measured shape of the E γ = 2.87MeV gamma-ray peak, originating from the reactions between fast D and 9 Be, obtained by adding data from discharges #95677, #95679, #95680 and #95683. The solid and dashed lines show the calculated line shape, assuming the same energy distribution of the fast deuterons but for two different values of the pitch: λ ≈ 0 (blue, dashed) and λ ≈ 0.4 (red, solid).

to P aux. (MW) #94698 0 2 4 6 8 NBI ICRF Neutron rate × 10 15 n/s 0 2 4 6 8 10 T e0 (keV) 2 4 6 8 W p (MJ) Time, t (s) 1 2 3 4 6 7 8 9 10 11 P aux. (MW) #95679 0 2 4 6 8 NBI ICRF Neutron rate × 10 15 n/s 0 2 4 6 8 10 T e0 (keV) 2 4 6 8 W p (MJ) Time, t (s) 1 2 3 4 6 7 8 9 10 11 Figure

  2. Overview of two JET-ILW pulses in mixed D -3 He plasmas, where the 3-ion D -(D NBI ) -3 He scenario was applied (3.7T/2.5MA, n e0 ≈ 6 × 10 19 m -3 ,

	n3 He /n e ≈ 20-25%): (a) #94698 and (b) #95679. The panels from top to bottom
	show the NBI and ICRF power, neutron rate, the central electron temperature and
	the plasma stored energy.
	generate high-energy D ions in the core of the D -3 He plasma. In these experiments, a
	high central toroidal magnetic field of 3.7 T was applied and plasmas stayed in L-mode
	(3.7T/2.5MA, n e0 ≈ 6 × 10 19 m -3 , n( 3 He)/n e ≈ 20-25%). Pulses #94698 and #95679
	had a comparable maximum ICRF power (≈5.5-6.0MW), but different NBI power of
	≈4.6MW and ≈7.0 MW. The efficient generation of high-energy D ions with ICRF can
	be immediately seen by comparing the time traces for the measured neutron rate and
	central electron temperature in the phases with NBI-only and the combined NBI+ICRF
	phases. Note the larger ratio P ICRF /P NBI ≈1.3 in JET pulse #94698 as compared to
	P ICRF /P NBI ≈0.8 in #95679, resulting in quite different sawtooth dynamics in these two
	pulses. In pulse #94698, a monster sawtooth with ∆t sw ≈1.9s (t = 8.63-10.53s

3

He 3-ion scenario was applied

Acknowledgments

This work has been carried out within the framework of the EUROfusion Consortium and has received funding from the Euratom research and training programme 2014-2018 and 2019-2020 under grant agreement No 633053. The views and opinions expressed herein do not necessarily reflect those of the European Commission.


Pascale Cossart

Listeria monocytogenes is reported in all textbooks as an intracellular pathogen that replicates in the cytosol of human cells after its escape from the vacuole. However, recently this paradigm was challenged by observations that L. monocytogenes can also adopt a vacuolar lifestyle when persisting within cells [START_REF] Kortebi | Listeria monocytogenes switches from dissemination to persistence by adopting a vacuolar lifestyle in epithelial cells[END_REF]. As another example, Mycobacterium tuberculosis, which was thought for a long time to be a strictly intra-vacuolar pathogen, was recently shown to rupture the phagosome and to contact the cytosol of the phagocytes it infects [START_REF] Russell | The ins and outs of the Mycobacterium tuberculosis-containing vacuole[END_REF][START_REF] Simeone | ESX/type VII secretion systems and their role in host-pathogen interaction[END_REF]. This discovery was very important to better understand former unsolved steps of the disease process, as it is associated with altered intracellular signalling. In particular, this knowledge is important for the development of vaccines, a priority for tuberculosis research [START_REF] Groschel | Recombinant BCG Expressing ESX-1 of Mycobacterium marinum Combines Low Virulence with Cytosolic Immune Signaling and Improved TB Protection[END_REF]. Thus a strict distinction between vacuolar and cytosolic intracellular pathogens is not evident. Similarly, the strict distinction between extracellular and intracellular pathogens does not hold true anymore. In a very interesting opinion article Arturo Casadevall and Ferric Fang show that new findings blur the boundaries between extracellular and intracellular pathogens and they discuss the benefits and caveats arising from the intracellular pathogen concept.

Today, multidrug resistant bacteria are a worldwide public health threat with increasing mortality and tremendous costs. Thus multidisciplinary research efforts are of utmost importance to better understand resistance emergence, persistence, dissemination and to find new, effective drugs. In an illustration of how the knowledge of ancient civilisations might help to solve this modern problem, Julian Davis and Shekooh Behroozian provide insights on how to use native environmental resources for extraction of natural products, so called medicinals. In particular, they present a personal view on the antimicrobial properties of clay minerals and propose that these underexplored natural resources may provide an alternative means to fight pathogens resistant to commonly used drugs.

Another fascinating approach to fight antibiotic resistance is proposed by Jörg Vogel who provides new ideas on how species-specific programmable RNA antibiotics could be developed. Indeed, broad-spectrum antibiotics are killing many bacteria present in and on our body, including both beneficial as well as pathogenic ones. Thus, the possibility of selective killing of pathogenic and/or multidrug resistant bacteria is very appealing, and might lead to microbiome editing for health. While proposing different ways to use short antisense oligonucleotides as antimicrobials, he also highlights the caveats including specificity, delivery, resistance and persistence.

Antibiotics are mainly seen as drugs to treat bacterial infections by inhibiting bacterial growth or killing them. However, as most antibiotics are naturally produced by soil organisms, their primary ecological function in the environment may be different. Pishchany Gleb and Roberto Kolter consider possible ecological roles of antibiotics and discuss their alternative functions at sub-inhibitory concentrations, such as the modulation of gene expression or biofilm formation, their influence on the stability of RNA molecules, and effects on bacterial evolution by increasing mutagenesis rates and horizontal gene transfer through conjugation.

They argue that it is important to start studying the importance of these molecules in the context of multispecies communities and in environmental conditions to understand how antibiotics might affect microbial communities and to expand our knowledge on these other, understudied effects of antibiotics.

Fernando Baquero, Val Fernadez-Lanza, Melodie Duval and Teresa Coque consider another idea that puts the environment into the focus of antibiotic resistance, namely the impact of gene -environment interactions on the acquisition of antibiotic resistance determinants, the ecogenetics of antibiotic resistance. As a specific example of how the ecology of an organism may shape the acquisition of antibiotic resistance, they examine L. monocytogenes, as it is intriguing that these bacteria have retained susceptibility to the key antimicrobials used to treat infections. This is even more surprising as L. monocytogenes is found ubiquitous in the environment and in a large spectrum of animals and plants. The authors discuss the impacts of global ecology, population size, genome structure, environmental stresses, and suggest that the many biocide and heavy metal resistance genes that are present in the L. monocytogenes genome may also play a role.

Cameron Parson, Sangmi Lee and Sophia Kathariou discuss these heavy metal resistance genes in L. monocytogenes and other Gram-positive bacteria further. They give a comprehensive update on the plasmid-and chromosomal-borne cadmium and arsenic resistance genes, and discuss their importance for environmental persistence. They suggest that the food-processing environment exerts a major selection pressure for cadmium resistance in L. monocytogenes, however many natural Listeria isolates are also highly resistant to several toxic metals. Thus, further studies will help to understand the ecological and evolutionary factors that drive acquisition, spread and diversification of the genomic elements conferring heavy metal resistance.

Two important new concepts in RNA biology have been discovered by the group of Pascale Cossart that are highlighted in the two following articles. Pierre Mandin and Jörgen Johansson give a short overview on how bacteria sense temperature changes and the impact this might have on virulence. For many years it was known that bacteria can sense temperature changes, and that in pathogenic bacteria virulence gene expression is often correlated with the transition from the environment (low temperature) to the human host (37°C). An example of temperature-regulated virulence gene expression the PrfA thermosensor of L. monocytogenes is described and compared to other bacterial thermosensers. Alejandro Toledo-Arana and Iñigo Lasa discuss additional regulatory concepts by giving a masterly account of the advances made and new insights gained through the implementation of high-throughput methods for RNA profiling. Through genome-wide transcriptional profiling massive amount of antisense transcription, as well as conditional transcriptional termination and non-contiguous operons, have been discovered. Furthermore, the authors highlight riboswitch-dependent regulation of antisense RNA and overlapping transcription between neighbouring genes and present the related excludon concept, a new paradigm of regulation based on overlapping transcription.

Another rapidly expanding area of gene regulation studies was initiated by the discovery and characterization of small noncoding regulatory RNAs. Many of these control bacterial pathogenicity and the adaptation of bacteria to environmental stresses and to their hosts [START_REF] Oliva | Small RNAs, 5' UTR elements and RNA-binding proteins in intracellular bacteria: impact on metabolism and virulence[END_REF]. Jens Georg, David Lalaouna, Shengwei Hou, Steffen Lott, Isabelle Caldelari, Stefano Marzi, Wolfgang Hess and Pascale Romby present a comprehensive and exciting overview on how targets of small RNAs can be identified, a still very challenging task. They show how by combining powerful computational approaches with experimental approaches the full extent of small RNA-depended regulatory networks can be elucidated.

Peptidoglycan is an essential molecule for bacteria that maintains their shape, cell integrity and provides a protective function against changing environmental conditions. Furthermore, peptidoglycan can be altered within the host to impair responses triggered by pattern recognition receptors [START_REF] Pucciarelli | Within-Host Envelope Remodelling and its Impact in Bacterial Pathogen Recognition[END_REF]. Peptidoglycan is also studied as a target to control bacterial infections. However, these studies are rarely undertaken in natural environments but under laboratory conditions. Until now our knowledge on peptidoglycan stems mainly from few model bacteria, and intracellular bacteria have only been studied very recently. Francisco Garcia-del-Portillo provides an illuminating account of recent advances on probing peptidoglycan synthesis and assembly within eukaryotic host cells, with a particular focus on the enzymes involved in peptidoglycan metabolism in the context of symbiotic growth and persistent infection. He gives a detailed description of the enzymatic machinery identified mainly in Escherichia coli and of peptidoglycan recycling utilized by bacterial pathogens to evade immune defences. Furthermore, the current techniques that are used for analysis and isolation of cell wall muropeptides are described and examples of intracellular bacteria that have been analysed with these methods are summarised.

Peptidoglycan is also one of the three major constituents of the L. monocyogenes cell wall, along with anionic teichoic acid polymers and wall-associated and wall-anchored proteins.

Polymeric teichoic acid is divided into wall teichoic-acid, which is directly conjugated to the peptidoglycan, and lipoteichoic acid, which is tethered to the plasma membrane. The Listeria teichoic acid polymers have important functions such as regulating bacterial growth, ionhomeostasis, biofilm formation, interaction with bacteriophages, host cell invasion and virulence. The L. moncytogenes serotyping scheme classifies strains into different serotypes.

It recognizes structural and compositional changes on the cell wall surface and led to the discovery of strains belonging to specific serovars, which cause most human infections. In their MicroReview Eric Sumrall, Anja Keller, Yang Shen and Martin Loessner provide a comprehensive update on the synthesis, structural variability and function of teichoic acids in the genus Listeria. They discuss the impacts of teichoic acid decorations on serotype-specific designations, on phage resistance and virulence via wall-associated virulence proteins. They propose that the chemical synthesis of teichoic acids could be used for vaccine development. We are confident that these contributions and ideas will inspire young researchers to embrace the upcoming challenges of the New Microbiology [START_REF] Cossart | The New Microbiology: From Microbiomes to CRISPR[END_REF].

1

  EDITORIAL: CELEBRATING THE CAREER AND LEGACY OF PROFESSOR PASCALE COSSART Carmen Buchrieser Institut Pasteur, Biology of Intracellular Bacteria and CNRS UMR 3525, 28, Rue du Docteur Roux, 75724 Paris, France It is an enormous pleasure and honour for me to contribute this editorial to introduce the special issue that we have assembled to honour the numerous, unique and outstanding scientific achievements of Pascale Cossart and her group. Pascale Cossart, a distinguished Professor at the Institut Pasteur in Paris, France, has been a worldwide leader in the study of fundamental processes in molecular and cellular microbiology and host pathogen interactions for more than 30 years. Using mainly Listeria monocytogenes, but also Ricketssia conorii, as models she has made several seminal findings in the field of infection biology, cell biology and epigenetics. Her work on fundamental RNA biology and bacterial regulation, and more recently the discovery of a new antibiotic resistance mechanism that is called ribosome splitting, are examples of her elegant contributions to the field. In this Special issue of Molecular Microbiology, colleagues, former students, postdoctoral fellows and friends of Pascale Cossart have gathered together to contribute reviews and articles in the area of her major research interests. The special issue starts with a personal tribute to Pascale Cossart, contributed by Werner Goebel, a longstanding colleague and competitor who traces many of the ground-breaking findings made by Pascale Cossart and her group.

  These engineered, teichoic acid-based vaccines could fully exploit their immunogenic potential and might represent a new strategy to fight infections with Gram-positive bacteria. In the following article, Eric Sumrall, Christopher Schefer, Jeanine Rismonod, Angelika Gründling, Martin Loessner and Yang Shen present new research results on the function of teichoic acids in virulence of L. monocytogenes. They show that two different glycosyltransferases are required for the galactosylation of lipotechoic acid (LTA) and wall techioic acid (WTA), respectively. Thus L. monocytogenes encodes two surface-acting galactosyltransferases with distinct substrate specificities, which have an impact on the cell surface modification and InlB (an important virulence factor for invasion of eukaryotic cells) retention and function. The demonstration that modification of WTA alone is critical for InlB localization is interesting in that LTA and WTA are often referred to in the literature with minimal functional distinction. This study clearly indicates that these surface moieties have important functional differences, and leads one to wonder what other functions might be solely attributed to WTA versus LTA, or vice versa. Another important surface structure is pili. They have a pivotal role in the colonization of specific host tissues in many pathogenic bacteria. Frederico Iovino, Priyanka Nannapaneni, Brigitta Henriques-Normark and Staffan Normark provide a state-of-the art overview of the recent findings of type 1 pilus and its adhesive tip protein RrgA from Streptococcus pneumoniae. The type 1 pilus is an important yet less studied surface associated virulence determinant of pneumococcal surface proteins, and thus the presentation of biochemical, structural and host pathogen interaction studies showing the impact of type 1 pilus in colonization and pathogenicity are very timely. Pneumococcal type 1 pili seem to confer a competitive advantage for nasopharyngeal colonization of humans, and the RrgA tip protein might be a good addition to the group of proteins used for the pneumococcal vaccine. How did these different bacterial cell envelopes evolve? In particular, how did monoderm and diderm bacteria evolve? In the penultimate article of this issue Daniela Megrian, Najwa Taib, Jerzy Witwinowski, Christophe Beloin and Simonetta Gribaldo address the important question of cell envelope diversity across the bacterial domain. Based on the existence of diderm bacteria among the generally monoderm Firmicutes, the division of Gram-positive (monoderm) and Gram-negative (diderm bacteria) is discussed. Using an in depth and comprehensive phylogenomic analyses, together with results from a new experimental model, Veillonella parvula, a diderm Firmicute, the authors propose that the diderm envelope is ancestral within the Firmicutes and the monoderm envelope evolved multiple times through loss of the outer membrane. This provocative hypothesis will stimulate discussions and further studies to understand bacterial cell envelope evolution. Finally, Megan de Ste Croix, Jonathan Holmes, Joseph Wanford, Richard Moxon, Marco Oggioni, and Christopher Bayliss discuss the role of bottlenecks in the evolution of diversity-generating mechanisms of microbes during bacterial transmission and infection. They provide an authoritative summary of progress in the field and challenges for future studies. This collection of articles reflects many of the areas of research to which Pascale Cossart has contributed, highlighting and building upon her outstanding findings over the last decades.


Prof Mugilarasi Purushothaman

Prof Snigdha Sarkar

Maho Morita

Muriel Gugger

Dr Eric W Schmidt

Dr Brandon I Morinaka

Genome mining based discovery of the cyclic peptide tolypamide and TolF, a Ser/Thr forward O-prenyltransferase

Keywords: biosynthesis, genome mining, natural products, prenylation, ribosomally synthesized and post-translationally modified peptides, RiPPs

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Over the past decade, peptides have secured a stronghold in the pharmaceutical market due to numerous advantages over traditional small molecule drugs. [1] Bioactive peptides are effective in targeting protein-protein interactions, have low incidences of off-target effects and cytotoxicity. However, these properties are sometimes compromised by their poor bioavailability caused by proteolytic degradation, rapid renal clearance and failure to cross cellular membranes. The bare peptides can be chemically modified in multiple ways to boost their therapeutic efficacy. [2] One means to improve membrane permeability is lipidation or prenylation, but the current methods for selective enzymatic peptide alkylation are relatively limited. [START_REF] Kowalczyk | Peptides and Peptide-based Biomaterials and their Biomedical Applications[END_REF] Prenyltransferase enzymes catalyze the intermolecular transfer of prenyl groups from isoprene donors to a variety of acceptors. [START_REF] Winkelblech | [END_REF] On peptides and proteins, these acceptors include electron-rich positions on aromatic rings and heteroatoms. The reaction is initiated by the generation of the allylic carbocation from prenyl diphosphate precursor, followed by nucleophilic attack by an electron rich group to produce the final prenylated product. A prenylation reaction can have multiple outcomes depending on isomerization of the intermediate carbocation, regiospecificity, chemoselectivity, and rearrangement of the preliminary product. As a result, the prenyltransferase enzyme family is capable of generating structurally diverse products from a limited number of precursors.

Cyanobactins are a chemically diverse class of ribosomally synthesized and posttranslationally modified peptide natural products (RiPPs) [5] isolated from cyanobacteria and have demonstrated anticancer and antimalarial activities. [6] Characteristic posttranslational modifications of this class are macrocyclization, heterocyclization (to form thiazole, oxazole, thiazoline, and oxazoline), and prenylation. Cyanobactin pathways show promise in biotechnology because many of the tailoring enzymes exhibit exceptionally relaxed substrate selectivity. [6,7] A significant subset of cyanobactins are tailored by the Ftype prenyltransferases, which append an isoprene unit to Ser, Thr, Tyr and Trp residues or at the N-or C-terminus of linear peptides. [6b, 6c, 8] These prenyltransferases have the same secondary structural motifs as ABBA type enzymes with a barrel of 10 anti-parallel β-sheets surrounded by a ring of 10 solvent exposed α-helices. [9] The interior of the active site is similar to other prenyltransferases, exhibiting strict selectivity for the prenyl donor, amino acid acceptor, and the direction of prenylation at either C-1 (forward) or C-3 (reverse) position of the dimethylallyl cation. [8b, 10] Although the prenyl donor is usually derived from dimethylallylpyrophosphate, one enzyme using geranyl pyrophosphate has been characterized. [11] In comparison to other prenyltransferases with capped active sites that sequester small substrates, cyanobactin F enzymes have a large, open hydrophobic cavity adjacent to the active site, which allows them to accommodate larger peptide substrates. [8b] This peculiarity also COMMUNICATION 2 gives the cyanobactin F enzymes strict selectivity for the prenylation donor and acceptor amino acid, but often extremely relaxed selectivity for the remainder of the peptide. This has proven to be useful in the biotechnological generation of diverse, alkylated peptides. [6b, 6c] In addition to these enzymes, a major fraction of F enzyme family exemplified by PatF appear to be essential biosynthesis, but are inactive and may serve a non-prenylating role in the pathways. [12] Prenylation is usually the last step of the cyanobactin biosynthetic pathway and in presence of inactive prenyltransferase genes, non-prenylated cyclic peptides are observed as mature natural products. [13] TruF1 was the first cyanobactin prenyltransferase to be unambiguously characterized both in vivo and in vitro. [8f, 13] However, the poor solubility and low in vitro activity of TruF1 prevented extensive structural characterization. TruF1 is the only identified prenyltransferase that modifies Ser and Thr hydroxyl groups, prenylating them with DMAPP in the reverse orientation. As such, there remains a significant gap in the cyanobactin chemical space, where there is no well-behaved, promiscuous catalyst for prenylation of Ser and Thr residues. Here, we analyzed the prenyltransferase sequence-function space to identify an orphan F enzyme bearing sequence similarity to TruF1. The pathway was de-orphanized through the identification of a novel cyanobactin natural product, tolypamide (1). The cyclic peptide was forward-prenylated on Thr, representing a previously unknown post-translational modification. The TolF prenyltransferase was characterized in vitro, demonstrating its selectivity for Ser or Thr forward prenylation in the context of several peptide sequences. TolF represents a robustly expressed and stable enzyme useful in the modification of Ser and Thr residues.

To investigate the sequence-function and chemical space of cyanobactin prenylation, a sequence similarity network (SSN) was generated for cyanobactin prenyltransferase InterPro family (IPR031037, TIGR04445) (Figure 1A). [14] This resulted in two large clusters, one containing inactive enzymes (PatF, TruF2, and TenF) and the other with enzymes catalyzing forward Oprenylation on Tyr (PagF and PirF). [8b, 11, 13, 15] The SSN also contained smaller clusters representing other regiospecific transformations such as N-prenylation on tryptophan (AcyF), prenylation on the terminal amino acid (AgeMTPT, MusF1, MusF2), C-prenylation on tryptophan (KgpF) and reverse Oprenylation on Tyr (LynF) (Figure 1C). [5b, 8a, 8c-e] Often, clustering is diagnostic for the function of uncharacterized enzymes, and indeed all characterized enzymes fell into clusters predictive of their chemistry. However, prenyltransferase TolF from Tolypothrix sp. clustered with LynF but only shared ~53% amino acid sequence identity, suggesting a possibly divergent function. TolF was compared with other characterized cyanobactin prenyltransferases in a Bayesian phylogenetic tree (Figure 1B), revealing that TolF is evolutionarily more closely related to TruF1, despite the proteins sharing only ~48% identity. We therefore predicted that TolF might produce new chemistry. Because of this possibility, and because no cyanobactins or cyanobactin biosynthetic genes from Tolypothrix had previously been described, we chose to investigate this pathway further.

The gene tolF was identified in Tolypothrix sp. PCC 7601, a freshwater filamentous cyanobacterium isolated from a lake in the state of Connecticut, USA. We identified the 10.4 kb long tol gene cluster from a 9.96 Mbp draft genome (Figure 2A), containing the signature genes involved in cyanobactin biosynthesis. [16] tolE encodes the precursor peptide, which is the substrate for enzymatic modification. TolE contains recognition sequences (RSs) I, II, and III, which should be responsible for docking with TolD, TolA, and TolG, respectively. [17] Collectively, if similar to other cyanobactin pathways, the four proteins (TolE, TolD, TolA and TolG) would produce an N-C cyclic peptide in which all Cys/Thr residues would be heterocyclized to thiazoline/ oxazoline residues. [18] TolE contained three copies of the core peptide sequence, TAPCYC, encoding the final cyclic natural product. Each core peptide was flanked by RSII (GVDAS) and RSIII (SYD), so that one equivalent of TolE would produce three equivalents of the cyclic peptide product. The role of TolF was unknown, but its evolutionary relationship to TruF1 suggested that it might prenylate the nonheterocyclized Thr residue present in the core peptide sequence.

To test our predictions, we initiated efforts to detect the cyanobactin product from the tol cluster in Tolypothrix sp. PCC 7601. The LC/MS chromatogram of the extracted culture medium showed a peak with two major ions at m/z 671.2666 Da and m/z 603.2047 Da (Figure 2B). The neutral mass difference of m/z 68 Da between the two ions is typical of the loss of isoprene. [5b, 19] Moreover, the mass of the parent ion precisely matches the structure predicted from the biosynthetic gene cluster. Large scale fermentation was carried out to isolate the compound, tolypamide (1), for spectroscopic studies.

Extensive 1D and 2D NMR analysis, including COSY, HSQC, HMBC and NOESY, was carried out to elucidate the structure of 1 (Table S1 andFigures S2-S6). The 1 H NMR spectrum showed characteristic features of a peptide, such as αmethine multiplets (H  5.5-3.5 ppm) and clear NH proton signals (H 8.18-7.23 ppm). Examination of the 2D NMR spectra led to the assignment of seven spin systems consisting of Thr, Ala, Pro, Tyr, a prenyl group and two Cys, which collectively matched our initial predicted core sequence. The NH peak at H 7.23 ppm in the Thr spin system indicated that it was not modified by heterocyclization. Each of the Cys spin systems showed a methylene carbon (C 37.3 -38.8 ppm) correlated to a methine carbon (C 77.9 -78.4 ppm) both resonating further downfield than usual. In addition, the absence of NH peaks in the Cys spin systems along with 13 C chemical shifts that were consistent with literature data to thiazoline-containing peptides, supported heterocyclization of both Cys residues to thiazoline rings. [START_REF] Rudi | [END_REF] The two methyl singlets (H 1.47 and 1.26 ppm) in the 1 H NMR were assigned to a terminal gem-dimethyl of a prenyl spin system indicating forward prenylation in 1. Further support for this assignment was obtained by the strong HMBC correlation between the β-proton (H 3.95 ppm) of Thr and the methylene carbon (C 65.3 ppm) of the prenyl group. Both these observations suggested that the prenyl group was attached to the Thr through an ether linkage, thus revealing the identification of the structure of forward-prenylated Thr (PrThr) unit, which to the best of our knowledge has not been previously observed.

The absolute configuration of (1) was assigned using a Marfey's type method, confirming the presence of L-Ala, L-Thr, and L-Cys. [21] During the hydrolysis, both Pro and Tyr racemized because of the adjacent azoline double bond. [22a] Subjecting 1 to ozonolysis prior to derivatization revealed the presence of L-Pro. The L-Pro in 1 was assigned as the trans rotamer based on the difference in 13 C chemical shifts of  and  carbons of Pro ( = 4.5 ppm). [22b] Hydrolysis in deuterium chloride led to a selective deuterium labelling of the D-isomers, demonstrating that D-Pro and D-Tyr were hydrolysis artefacts and revealing that all amino acids in 1 are in the L-configuration. [22c] Tolypamide is structurally unique among characterized cyanobactins in that the cyclic hexapeptide contains two thiazoline rings and a forward O-prenylated threonine. The latter is unprecedented among this class of natural products. Under the growth conditions tested, tolypamide ( 1) is the only detectable product by LC/MS which can be connected with the tol pathway. Although a number of cyanobactins have shown cytotoxic activity, [19b] tolypamide (1) did not show significant cytotoxic or antimicrobial activity against a panel of six cancer cell lines (DU145, A549, HeLa CCL2, HepG2, and MDA-MB 231) or bacterial strains (E. coli ATCC 25922, S. aureus ATCC 29737 and P. aeruginosa ATCC 9027), respectively. For functional validation, TolF was cloned, overexpressed, and used in biochemical assays (Figure S7). Short precursor peptides containing Thr were incubated with the recombinant TolF and the putative prenyl donor, dimethylallyl pyrophosphate (DMAPP) (Table 1). After overnight incubation, the reaction mixtures were monitored using LC/MS for product ions with increased mass of m/z 68 Da relative to the substrate mass (Figure 3 andS14).

To access the putative native substrate 2, synthetic peptide TAPCYCSYD (core + RSIII) was treated sequentially with broadsubstrate enzymes catalyzing heterocyclization, proteolytic cleavage and macrocyclization (Figure S13). When the substrate was treated with heterocyclase (RSI-TruD) the Cys residues were converted to thiazolines (C*). [8e] The resultant product TAPC*YC*SYD was incubated with macrocyclase, PatG to produce cyclic peptide 2 along with the linear cleaved recognition sequence, SYD. [8f] After overnight incubation with TolF and DMAPP, the native substrate 2 was quantitatively converted to tolypamide (1). The enzymatic reaction product 1 was compared to the natural product 1 obtained from Tolypothrix sp. PCC 7601, confirming its structural identity (Figure 3A). Interestingly, when TAPCYC (3; the linear TolE core sequence) was used, it was also modified on Thr, but with only 6% conversion after 16 h incubation. We further compared cyclic-[TSIAPFP] ( 4), an analog of the trunkamide natural product, finding that it was prenylated only on Thr with a conversion of 80%. Linear analog 5 (TSIAPFPSYD) was again prenylated only on Thr, with only a conversion of 5%. Thus, in two examples with quite different sequences, the cyclic variant was more efficiently prenylated than the linear variant.

Further linear tru pathway analogs 6, 7 and pag pathway substrate 9 were also relatively efficiently modified on Thr, with overnight conversions from 15-70%. Shorter substrates containing Thr were only slightly modified (4% on substrate 10) or were not modified at all (substrates 11 and 12). Although Ser was present in substrates 4-7, only Thr was modified. Since TruF1 reverse-prenylates both Ser and Thr, we sought to determine whether TolF also prenylates Ser. We designed substrates 13 and 14, analogs of substrate 7 in which the reactive Thr is replaced with Ser. Tandem mass spectrometry provided unambiguous evidence that 7 was prenylated on the terminal Thr, while 13 and 14 were prenylated on the terminal Ser (Figure S15). However, only a very minor amount of prenylation was detected for substrates 13 and 14 (<10% conversion compared to 7). Thus, TolF appears to prefer Thr, unlike TruF1.

We further used this subset of related substrates to answer the major scientific question posed by this study: what governs the forward versus reverse prenylation of Ser and Thr residues? Extremely sequence-diverse natural and synthetic peptides are known to be reverse-prenylated on Ser/Thr residues by TruF1, which is unfortunately challenging to characterize biochemically. [15] Therefore, TolF offered us the opportunity to characterize the serine/threonine-prenylating class of enzymes for the first time and to obtain deeper insight into the regioselectivity of DMAPP addition. We hypothesized that TolF would be intrinsically forward-prenylating, regardless of the sequence of peptide substrate. To test this hypothesis, we used the most synthetically accessible robust substrate, the disulfide dimer 6. When 6 was treated with TolF and DMAPP at scale, both mono-prenylated and di-prenylated products were identified (Figure S14F). To obtain sufficient compound for purification, peptide dimer 6 was reduced using tris (2carboxyethyl)phosphine (TCEP) and treated with iodoacetamide (IAA) to produce 7, which when treated with TolF and DMAPP afforded mono-prenylated 8 in good yield (Figure 3B). The reduced form of 6 and compounds 7 and 8 were purified and used for NMR experiments (Figure S8).

The 1 H NMR and HSQC spectra were consistent with the proposed structures, and they were assigned using COSY and TOCSY experiments (Figure S9-S12 and Tables S3-S5). The peptide sequences were further supported by ROESY correlations and tandem mass spectrometry. Comparison of 1 H NMR spectra of 6 (reduced form), 7 and 8 shows the disappearance of the Thr hydroxyl group (H 5.51 ppm) and emergence of 1 H chemical shifts from forward prenyl group in compound 8, indicating that the prenyl group is installed on the Thr residue (Figure S9). The 1 H NMR spectrum of 8 showed the signature chemical shifts associated with a forward prenyl moiety, with diastereotopic α-methylene protons (H 4.01, 3.87 ppm), αmethine proton (H 5.24 ppm) and sharp gem-dimethyl singlets (H 1.60, 1.69 ppm). [23] The isoprene α-methylene protons (H 4.01, 3.87 ppm) showed ROESY correlations with Thr via the Thr βproton (δH 3.63 ppm) and γ-proton (H 1.13 ppm) (Figure S10). Taken together with the Thr, Ser and Tyr chemical shifts in 6, these data unambiguously demonstrate that TolF is forward prenylating on Thr.

Previous studies indicated that TruF1 is likely strictly Ser/Thr reverse prenylating, while here we demonstrate that TolF is rigorously forward prenylating, preferring Thr. Both TruF1 and TolF are more broadly accommodating of other residues flanking the essential Ser/Thr. Previous crystal structures of prenyltransferases complexed with substrates and prenyl donors allow some general conclusions about this class to be drawn. In the PagF (forward Tyr prenylating) structure, phenolic oxygen of the substrate is 1 Å closer to the C-1 carbon atom of dimethylallyl group than the C-3 carbon, resulting in favourable O-prenylation of Tyr. [8b] A recent structure of a non-cyanobactin enzyme, the Trp N-prenyltransferase, CymD showed that the C-3 carbon of the 10.1002/anie.202015975

Accepted Manuscript

Angewandte Chemie International Edition COMMUNICATION 5 dimethylallyl group is 2.5 Å closer to the substrate than the C-1 carbon, leading to facile reverse prenylation. [24] We expect similar structural nuances give rise to different regiospecificities of TolF and TruF1, although their relatively distant sequence similarity makes direct comparison challenging (Figure S16). % Conversion [d] Significance

Cyclic-[TAPC*YC*] (2) [b] NR [e] Single amino acid H-Thr-Thr-Thr-OH ( 12) NR [e] Linear tripeptide SVPALcTYD ( 13) [c] 6% N-terminal Ser SVPALcSYD ( 14) [c] 4% N-terminal Ser

[a] The site of prenylation is underlined in the substrate on the basis of NMR or MS/MS data. In some cases, it cannot be determined by MS. TolF was selective for DMAPP and could not accept isopentenyl pyrophosphate (IPP) or longer C10-C20 isoprenoids including geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). A closer examination of TolF sequence alignment with other prenyltransferases revealed that the residues involved in sequestering the pyrophosphate moiety of the isoprene donor are conserved in TolF, including Arg66, Lys135, His137, Tyr191 and Arg298 (Figure S16). [8b] The carbocation intermediate generated from isoprene pyrophosphate donor is also shielded from solvent at the active site cavity by several hydrophobic residues. In 2018, Estrada et al. showed that a single Phe to Gly mutation at the hydrophobic active site of PagF, shifts the donor preference to GPP from DMAPP. [11b] This is a direct consequence of active site expansion in presence of smaller glycine residue. In TolF, the equivalent residue is the larger Met232, which might result in a more constricted active site incapable of accommodating C10-C20 isoprenoid cofactors.

In conclusion, we show how focused genome mining leads to the discovery of a novel cyanobactin, tolypamide (1) and a new post-translational modification with applications in peptide and pharmaceutical chemistry. TolF catalyzes a new regio-and chemoselective isoprenylation reaction capable of modifying diverse peptides. To our knowledge, TolF is the first example of a cyanobactin prenyltransferase that catalyzes the forward prenylation of Ser/Thr in peptides. With the discovery of TolF, nearly every chemically possible combination of peptide Oprenylation has now been observed, including hydroxyl forward and reverse prenylation, phenolic OH forward and reverse prenylation, and carboxylate prenylation. Coupled with the known C-and N-modifying F proteins, there is now a broad toolset for alkylating diverse peptide sequences. Cyanobactin pathways remain a rich source novel natural products and biosynthetic machinery that is useful in biotechnology. It is notable that there are many further orphan F proteins and other unknown proteins revealed in SNN diagrams, implying an even broader diversity awaiting discovery. Finally, the discovery TolF as a robust and stable Ser/Thr O-prenyltransaferase creates opportunities to study the structural biology and evolution of this enzyme family.

Figure 1 .

 1 Figure 1. Sequence-function and chemical space of cyanobactin prenyltransferases. A) Sequence similarity network showing the clustering of cyanobactin prenyltransferases according to their regiospecificity using an alignment score threshold of 86. B) Bayesian phylogenetic tree of TolF with previously characterized prenyltransferases show TolF falls in the same clade as TruF1, showing similar substrate preference. C) Regioselective and chemoselective modes of prenylation carried out natively by characterized cyanobactin prenyltransferases. Reverse and forward prenylation patterns are represented within and outside the dashed box, respectively. The type of transformation is indicated by the atom (italicized) followed by the residue, Nterm or Cterm. The enzyme is shown in parenthesis. The R-group = Me and H for Thr and Ser, respectively.

Figure 2 .

 2 Figure 2. Isolation of the cyanobactin tolypamide (1) from Tolypothrix sp. A) The tol gene cluster and partial sequence of precursor peptide (TolE) with the core regions in bold. RSI, RSII and RSIII are annotated. B) Total ion chromatogram of the crude extract of Tolypothrix sp. PCC 7601. The compound of interest, tolypamide (1) eluted at tR = 13.80 mins. Inlay is the extracted mass spectrum of the peak at tR = 13.80 mins with m/z = 671.26 Da and m/z = 603.20 Da (loss of the prenyl group). The other peaks observed did not show m/z values which could be correlated to a tol pathway product. C) 2D NMR correlations establishing the planar structure of 1. D) Structure of tolypamide (1)

Figure 3 .

 3 Figure 3. In vitro prenyltransferase activity of TolF. A) Extracted ion chromatograms of overnight TolF reaction with substrate 2 to generate tolypamide 1 (top), no DMAPP negative control (middle) and comparison with authentic tolypamide 1 standard (bottom). B) Extracted ion chromatograms (EICs) for overnight TolF reaction with compound 7 to produce 8 (top) and no DMAPP negative control (bottom). The m/z values [M+H] + for the compounds are indicated above each peak in the EICs. The Cys (c) residue in 7 is alkylated with iodoacetamide.

  [b] C* indicates thiazoline generated using RSI-TruD,[c] c indicates Cys treated with iodoacetamide,[d] % conversion were calculated from MS traces and do not represent isolated yield. % Conversions were calculated using the peak area (A) from extracted ion chromatograms from reactions incubated for 16 h. % Conversion = (Aproduct/Aproduct + Astarting material) x 100. [e] NR = No reaction

Table 1 .

 1 Prenylation selectivity of TolF

	Substrate [a]

Acknowledgements

This work was funded by NIH GM122521 (E.W.S), MOE Tier 1 R148000258114 and R148000271114 (B.I.M) and Carnot-Pasteur Microbe Santé (M.G). S.S is supported by the Skaggs Graduate Research Fellowship. M.P is supported by the MOE Research Scholarship. We thank Dr. Bailey Miller for assistance with NMR spectroscopy, Dr. Wenjia Gu for providing RSI-TruD, Prof. Lu Yixin and Dr. Wang Nengzhong for assistance with ozonolysis, and the Drug Discovery and Optimization Platform (NUS) for cytotoxicity assays.

Entry for the Table of Contents

Insert graphic for Table of Contents here.

TolF is a novel O-prenyltransferase which catalyzes forward O-prenylation on threonine and serine residues. A combination of genome mining, natural products chemistry, and biochemical assays were used to identify the biosynthetic gene cluster, natural product, and functionally validate this enzyme. This study fills a gap in the sequence-function space of prenyltransferases and introduces TolF as a promising tool in synthetic biology.

Institute and/or researcher Twitter usernames: @NUSingapore, @nuspharmacydept 10.1002/anie.202015975

Accepted Manuscript