CYAO Project CYAO: Cyanobacterial Platform Optimised for Bioproduction
Excitation Emission Map Syn6803

Excitation and emission wavelength dependence of fluorescence spectra in whole cells of the cyanobacterium Synechocystis sp. PPC6803: Influence on the estimation of Photosystem II maximal quantum efficiency.
Remelli W, Santabarbara S.
Biochim Biophys Acta (2018) 1859:1207-1222.
The fluorescence emission spectrum of Synechocystis sp. PPC6803 cells, at room temperature, displays: i) significant bandshape variations when collected under open (F0) and closed (FM) Photosystem II reaction centre conditions; ii) a marked dependence on the excitation wavelength both under F0 and FM conditions, due to the enhancement of phycobilisomes (PBS) emission upon their direct excitation. As a consequence: iii) the ratio of the variable and maximal fluorescence (FV/FM), that is a commonly employed indicator of the maximal photochemical quantum efficiency of PSII (Fpc, PSII), displays a significant dependency on both the excitation and the emission (detection) wavelength; iv) the FV/FM excitation/emission wavelength dependency is due, primarily, to the overlap of PSII emission with that of supercomplexes showing negligible changes in quantum yield upon trap closure, i.e. PSI and a PBS fraction which is incapable to transfer the excitation energy efficiently to core complexes. v) The contribution to the cellular emission and the relative absorption-cross section of PSII, PSI and uncoupled PBS are extracted using a spectral decomposition strategy. It is concluded that vi) Fpc, PSII is generally underestimated from the FV/FM measurements in this organism and, the degree of the estimation bias, which can exceed 50%, depends on the measurement conditions. Spectral modelling based on the decomposed emission/cross-section profiles were extended to other processes typically monitored from steady-state fluorescence measurements, in the presence of an actinic illumination, in particular non-photochemical quenching. It is suggested that vii) the quenching extent is generally underestimated in analogy to FV/FM but that viii) the location of quenching sites can be discriminated based on the combined excitation/emission spectral analysis.

Inducible Carotenoid Production

Non-endogenous ketocarotenoid accumulation in engineered Synechocystis sp. PCC 6803.
Menin B, Santabarbara S, Lami A, Musazzi S, Villafiorita Monteleone F, Casazza AP.
Physiol Plant. (2018) doi: 10.1111/ppl.12900.
The cyanobacterium Synechocystis sp. PCC 6803 is a model species commonly employed for biotechnological applications. It is naturally able to accumulate zeaxanthin (Zea) and echinenone (Ech), but not astaxanthin (Asx), which is the highest value carotenoid produced by microalgae, with a wide range of applications in pharmaceutical, cosmetics, food and feed industries. With the aim of finding an alternative and sustainable biological source for the production of Asx and other valuable hydroxylated and ketolated intermediates, the carotenoid biosynthetic pathway of Synechocystis sp. PCC 6803 has been engineered by introducing the 4,4' -carotene oxygenase (CrtW) and 3,3' -carotene hydroxylase (CrtZ) genes from Brevundimonas sp. SD-212 under the control of a temperature-inducible promoter. The expression of exogenous CrtZ led to an increased accumulation of Zea at the expense of Ech, while the expression of exogenous CrtW promoted the production of non-endogenous canthaxanthin and an increase in the Ech content with a concomitant strong reduction of -carotene (-car). When both Brevundimonas sp. SD-212 genes were coexpressed, significant amounts of non-endogenous Asx were obtained accompanied by a strong decrease in -car content. Asx accumulation was higher (approximately 50% of total carotenoids) when CrtZ was cloned upstream of CrtW, but still significant (approximately 30%) when the position of genes was inverted. Therefore, the engineered strains constitute a useful tool for investigating the ketocarotenoid biosynthetic pathway in cyanobacteria and an excellent starting point for further optimisation and industrial exploitation of these organisms for the production of added-value compounds.

green week

Comparative excitation-emission dependence of the FV/FM ratio in model green algae and cyanobacterial strains.
Santabarbara S, Villafiorita Monteleone F, Remelli W, Rizzo F, Menin B, Casazza AP.
Physiol Plant. (2019) doi: 10.1111/ppl.12931.
The emission spectra collected under conditions of open (F0) and closed (FM) photosystem II (PSII) reaction centres are close-to-independent from the excitation wavelength in Chlamydomonas reinhardtii and Chlorella sorokiniana, whereas a pronounced dependence is observed in Synechocystis sp. PCC6803 and Synechococcus sp. PCC7942, instead. The differences in band-shape between the F0 and FM emission are limited in green algae, giving rise only to a minor trough in the FV/FMspectrum in the 705-720?nm range, irrespectively of the excitation. More substantial variations are observed in cyanobacteria, resulting in marked dependencies of the measured FV/FM ratios on both the excitation and the detection wavelengths. In cyanobacteria, the maximal FV /FM values (0.5-0.7), observed monitoring at approximately 684 nm and exciting Chl a preferentially, are comparable to those of green algae; however, FV /FM decreases sharply below approximately 660 nm. Furthermore, in the red emission tail, the trough in the FV /FM spectrum is more pronounced in cyanobacteria with respect to green algae, corresponding to FV /FM values of 0.25-0.4 in this spectral region. Upon direct phycobilisomes excitation (i.e. >520 nm), the FV /FM value detected at 684 nm decreases to 0.3-0.5 and is close-to-negligible (approximately 0.1) below 660 nm. At the same time, the FV spectra are, in all species investigated, almost independent on the excitation wavelength. It is concluded that the excitation/emission dependencies of the FV /FM ratio arise from overlapped contributions from the three independent emissions of PSI, PSII and a fraction of energetically uncoupled external antenna, excited in different proportions depending on the respective optical cross-section and fluorescence yield.

ET Modelling

Modelling electron transfer in photosystem I: Limits and perspectives
Santabarbara S, Casazza AP, Hastings G.
Physiol Plant. (2019) doi: 10.1111/ppl.12959.
Uncovering the parameters underlying the electron transfer (ET) in photosynthetic reaction centres is of importance for understanding the molecular mechanisms underpinning their functionality. The reductive nature of most cofactors involved in photosynthetic ET makes the direct estimation of their properties difficult. Photosystem I (PSI) operate in a highly reducing regime, making the assessment of cofactor properties even more difficult. Kinetic modelling coupled to a non-adiabatic description of ET is a useful approach in overcoming this hindrance. Here we review the theory and modelling approaches that have been used in assessing parameters associated with ET reactions in PSI, with particular attention to ET reactions involving the phylloquinones (PhQ) and the iron-sulphur clusters. In most modelling studies the goal is to estimate the driving force of ET, which usually is associated with the cofactor midpoint potentials. The driving force is sensitive to many factors, which define the ET rate, i.e. the reorganisation energy, the coupling with nuclear modes and the electronic matrix elements, which are explored and discussed here. The importance of an inclusive modelling of both forward and reverse ET processes is discussed and highlighted. It is shown that, although estimates are indeed sensitive to the exact parameter sets employed in the modelling, a general consensus is still attained, pointing to a scenario where Delta(G0A1A/FX)/Delta(G0A1B/FX) is weakly endergonic/exergonic, respectively. It is emphasised that to further refine those estimates, it will require a joint effort between computational modelling and more wide-ranging experimental studies.

La ricerca finanziata dalla Fondazione Cariplo nell'ambito del programma sulle Biotecnologie e sulla Bioeconomia. Fondazione Cariplo
CYAO-Web.V2. 18/09/2018