C15 FPP is present in the chloroplast
Green algae such as C. reinhardtii are eukaryotic photosynthetic organisms and must turn over all five prenyl diphosphate precursors (IPP, DMAPP, GPP, FPP, GGPP) needed for the synthesis of the most prominent isoprenoid classes. It is largely unknown in which abundance each product could be found in the algal cell depending on the cellular compartment and their metabolic role, and how these molecular species are transported across lipid bilayers . The comparison of specific terpene synthase yields does not offer much insight here, as each synthase enzyme has its own rate of reaction, promiscuity of products, and different terpenoid products can exhibit toxicity. Metabolic architectures in similar systems such as land plants, and genomic as well as biochemical evidence for C. reinhardtii  suggest that the C15 FPP precursor pool would be available in the cytosol/ER of the algal cell as a precursor for sterol and dolichol metabolism as well as farnesylation of proteins, whereas C20 GGPP acts as a precursor for the synthesis of pigments and prenyl plastoquinones in the chloroplast (Fig. 1).
By targeting respective synthases to specific subcellular compartments of the algal cell, the generation of specific heterologous terpenoid products provided a facile confirmation of these metabolite localizations [6–8]. It was previously unknown if, and to which extent, FPP would be freely available in the chloroplast. In one previous investigation , it was attempted with no success to use FPP as a substrate in the chloroplast of C. reinhardtii. In our previous work , a patchoulol synthase was targeted to the algal chloroplast and mitochondria without generating the sesquiterpenoid patchoulol. However, this was not systematically investigated and yields of single enzyme expression may have been too low to detect the product. In our work here, we find strong evidence that this C15 hydrocarbon precursor is indeed present in the chloroplast (Figs. 2 and 3). FPP-derived C15 bisabolene was detected when targeting the respective terpene synthase (AgBS) to the chloroplast, and titers were similar to strains with cytosolic targeting of this enzyme (Fig. 3, compare constructs v and i). It could be argued that the observed bisabolene production is from a small fraction of AgBS remaining in the cytosol during import to the chloroplast. However, significantly lower sesquiterpenoid yields would be expected and no residual fluorescence signals could be detected in this compartment, implying this is likely not the case (Fig. 3). Similar results were obtained with higher plants: in moss, targeting of a santalene synthase and a patchoulol synthase to the chloroplast also resulted in the expected sesquiterpenoid products . Although farnesyl pyrophosphate is likely present in the chloroplast, its function in this compartment is not clear. It may be the precursor to solanesyl diphosphate, used in the prenylation of plastoquinone, and is also an intermediate in the synthesis of C20 GGPP. A single predicted type II GGPPS catalyzes the formation of this C20 precursor from IPP and DMAPP in the plastid by sequentially adding C5 units to the growing prenyl chain . C10 GPP and C15 FPP are generated as intermediates, which have not been reported to be released from the enzyme as side products. However, a type II GGPP synthase from another green microalga, Haematococcus lacustris, exhibited weak product promiscuity for FPP in vitro , and the same was reported for a cyanobacterial GGPPS . Therefore, plastidic FPP may be rapidly displaced and re-bound from the GGPPS active site as a reaction species, and FPP leakage from the GGPPS may represent a remnant of a prokaryotic evolutionary ancestor. FPP import from the cytosol to the chloroplast may also explain the presence of this precursor, but import of prenyl diphosphates into plant chloroplasts has been excluded previously .
C5 IPP and DMAPP are more readily available in the algal chloroplast than in the cytosol
It was expected that isoprenoid turnover and flux towards prenyl precursors is higher in the chloroplast due to operation of the native MEP pathway in this compartment. Co-expression of ScERG20 and AgBS in the chloroplast increased bisabolene production ~ 16-fold over the parental strain (Fig. 2), whereas co-expression of the native CrFPPS and AgBS in the cytosol did not alter the yield (Additional file 11: Fig. S1). Co-overexpression of an FPPS and the patchoulol synthase in tobacco plastids also improved patchoulol production several-fold . This indicates a large plastidic pool of IPP and DMAPP, which is freely available in this compartment. Fusion of a terpene synthase to ScERG20(F96C) was previously used to boost diterpenoid product formation from the algal chloroplast due to channeling from C5 prenyl precursors via higher prenyl intermediates to the final product . We therefore hypothesized that the AgBS alone and in fusion with ScERG20 could serve as a reporter to estimate the abundances of FPP, IPP, and DMAPP in the cytosol and the chloroplast. Targeting of the AgBS-mVenus-ScERG20 fusion (construct x) to the chloroplast indeed resulted in 25-fold improved bisabolene yields compared to the reference construct (vector i) (Fig. 3). Exchange of the ScERG20 fusion partner with the native CrFPPS led to a less pronounced enhancement (ninefold), which may result from a lower activity of this enzyme compared to the yeast ERG20. There is strong evidence that the C5 precursor pool in the chloroplast is much larger than C5 in the cytosol. Although FPP was observed in the chloroplast by the capacity to produce bisabolene, it was not much greater than that natively available in the cytosol (Fig. 3). Nevertheless, the biosynthetic potential for sesquiterpenoid production from the plastid is markedly improved when FPPS is co-expressed to convert C5 precursors to FPP. A large supply of plastidic C5 prenyl precursors may reflect the natural ability of green algae to adapt to highly variable light conditions .
The unique nature of green algal FPP metabolism
A direct comparison of sesquiterpene production by the same enzyme targeted to cytoplasm or chloroplast may be hindered by several factors like differing chemical environments (pH, Mg2+ concentrations), differing substrate concentrations, and substrate channeling effects of multi-enzyme complexes. However, we observed heterologous bisabolene production at comparable low levels from AgBS targeted to either cellular compartment. The question, therefore, is where does the cytoplasmic FPP come from if overexpression of an FPPS in the cytoplasm does not increase sesquiterpene yields (Fig. 3)? This becomes more intriguing when one considers the specific localization of the CrFPPS, in a halo around the algal nucleus (Fig. 3), a region where the ER-nuclear association is expected to be found. This localization could enable efficient channeling of FPP to the squalene synthase (CrSQS) anchored in the ER membrane in proximity (Fig. 3, ), and would prioritize FPP for sterol synthesis. Indeed, knockdown of CrSQS results in increased sesquiterpene yields in the algal cytoplasm . Nevertheless, the origin of DMAPP and IPP in this situation seems enigmatic. It was hypothesized that targeting of the AgBS to the vicinity of these enzymes would also channel FPP to the active site of this sesquiterpene synthase and enhance bisabolene yields. Therefore, the AgBS-mVenus construct was fused to CrFPPS and the transmembrane domain of CrSQS. Although the desired targeting effect was achieved (Fig. 3), bisabolene yields did not improve and were actually lower than the AgBS alone targeted to the cytosol. Considering these observations, two metabolic modes of C5 and C15 prenyl unit generation and transport (or a mixture of both) may be possible:
(i) IPP and DMAPP are exported from the chloroplast at low rates in specific connection to the site of FPP synthesis. The generation of FPP by the native, nucleus envelope-associated CrFPPS could be highly efficient, hence cytosolic IPP and DMAPP concentrations are low enough to not yield improved bisabolene yields with FPPS fusion proteins. The transfer of FPP to the CrSQS could be inefficient, and FPP may leak into the cytosol, resulting in a freely available metabolic pool. This may enable uptake into mitochondria and supply protein prenylation and dolichol production. There are two IPP isomerases (Cre08.g381800, Cre11.g467544, Phytozome v5.6) in the genome of C. reinhardtii, one of which may be required to maintain stoichiometries of IPP and DMAPP that match the differing substrate demands for plastidic GGPP and cytosolic FPP synthesis, and suggests a significant role of the two C5 precursors in both compartments.
On the contrary (ii), FPP could be exported from the chloroplast, potentially by an ancient IPP/DMAPP transporter. In this case, the FPPS would play a minimal role in FPP synthesis, which would take place in the chloroplast. FPP exported from the chloroplast could assist cytosolic formation of various C15-derived terpenoids, e.g. sterols. In both situations, whether exported or not, the small amount of plastidic FPP could be a promiscuous by-product of GGPP synthesis, and possibly also assist plastidic SPP formation. In this case, FPP leakage from the GGPPS may represent a remnant of a prokaryotic evolutionary ancestor . However, it is questionable if the plastidic GGPPS is an FPP-leaking enzyme like in H. lacustris  and simultaneously the major source of (exported) FPP. Evidence against this was the finding that CrGGPPS knock-down resulted in increased FPP supply to yield improved bisabolene titers in the cytosol , indicating a more dominant role of IPP/DMAPP export. Indeed, here CrFPPS was active in conversion of IPP and DMAPP into C15 substrate in the algal chloroplast (Fig. 3). However, directing AgBS to the site of CrFPPS in cytoplasmic fusions had no improvement over the AgBS alone, suggesting a lack of free C5 precursor in this location. It is likely that C. reinhardtii, like higher plants, regulates cytosolic IPP/DMAPP abundance using an isopentenyl phosphate kinase (IPK) and a Nudix superfamily hydrolase as metabolic valve [9, 24]. A putative IPK is encoded in the genome of C. reinhardtii (Cre13.g579800, Uniprot A8HTR6), and could function together with a Nudix superfamily hydrolase (yet to be identified) which may explain low cytosolic C5 precursor abundances in this organism.
A mixture of both situations (i & ii) is possible, and in line with this, plant chloroplasts were found to be capable of FPP export by a yet unknown mechanism, which was determined to be slower than IPP export . Higher plants and green algae share many metabolic features, but algae differ as they have lost the cytoplasmic/peroxisomal mevalonate pathway and the respective ability of this pathway in higher plants to provide FPP in various compartments: while Chlorophycean algae possess a single annotated FPPS as described here, higher plants have multiple isoforms to generate FPP pools in mitochondria, peroxisomes, and likely in the cytosol relatively independently .
Candidates for a prenyl transporter
Members of all eukaryotic kingdoms require prenyl unit exchange across lipid bilayer membranes. Although export of C5, C10, and C15 isoprenoid precursors from higher plant chloroplasts has been reported [21, 26], its exact mechanism is unclear. The uniqueness and apparent controlled specificity of algal metabolic architecture may require a transporter to facilitate the observed C15 FPP availability in the cytoplasm of the alga. We sought to use bioinformatics to assist in finding previously uncharacterized transporters that may play a role in this. By narrowing putative transporters down to plastid membrane-localized candidates without known substrates and uptake function, we were able to find one protein sequence (UniProt A0A2K3DRV6, Phytozome Cre05.g232751) annotated as a multidrug efflux transporter. A CLiP mutant with an insertion in the coding sequence is available , indicating that this gene could be non-essential. It was surprising to us that only one candidate could be found, which has no significant similarity to transporters in other organisms except uncharacterized/hypothetical proteins in other algae and bacteria. Perhaps such a protein enabled the transport of amphipathic compounds like FPP and the loss of the MVA pathway in many algae species? However, this finding is preliminary and will need to be followed up by in vivo localization, substrate testing and possibly its expression in plants concomitant with MVA pathway knockout. Such investigations are, however, beyond the scope of this report.
AgBS catalytic efficiency limits abundant FPP conversion to bisabolene
Only marginal yield improvements were achieved through gene loading strategies to accumulate AgBS copies in the chloroplast in an ERG20 co-overexpression background (Fig. 4A) or to obtain more AgBS-reporter-ERG20 fusion protein (Fig. 4B). Bisabolene yields previously obtained from the cytosol after CrSQS knockdown  could also not be matched or out-competed with our strategy of tapping plastidic prenyl pools for sesquiterpene synthesis (Figs. 4 and 5). We expected that the chloroplast bears higher potential for this challenge, but this capacity can likely not be exhausted at this juncture. Green algal GGPP synthases were shown to convert not only C5 prenyl pyrophosphate but also C15 FPP to its C20 product , and it is possible that excess FPP in the chloroplast is rapidly scavenged by this enzyme in C. reinhardtii while being converted to sesquiterpene products at comparably low flux by catalytically inefficient sesquiterpene synthases like the AgBS. Perhaps a higher residence time of FPP due to SQS knockdown in the cytoplasm led to the increased titers observed in our previous work . A homologous algal GGPPS from H. pluvialis exhibited higher affinity (KM = 19 μM) towards FPP in vitro  than the AgBS with a KM of 49.5 ± 6.3 μM . An imbalance between rapid FPP generation by the ScERG20 FPPS and subsequent slow conversion to bisabolene by the AgBS may be responsible for this observation. Extra FPP produced by the ScERG20 is likely processed to GGPP and pigments rapidly by the native cellular machinery.
It could also be possible that the freely accessible IPP and DMAPP pools of the chloroplast were totally depleted by robust ScERG20 activity, leading to a limited increase in bisabolene titers in cell lines transformed with multiple AgBS-reporter-ScERG20 fusion constructs. However, no apparent effect on cellular pigment levels was observed, suggesting that the IPP and DMAPP pool was not yet depleted. Gene loading strategies like the one applied here may be obsolete in the near future due to improved transgene expression with optimized genetic tools such as synthetic promoters which can generate more recombinant protein products from single transformation events .
Light–dark cycling encourages heterologous isoprenoid production from the chloroplast
We have previously demonstrated that L:D illumination cycles and photoautotrophic conditions promote high production rates for heterologous diterpenoids (C20) from the chloroplast . In this work, elevated specific productivities of the sesquiterpene bisabolene could also be observed in these conditions (Fig. 5C), although diterpenoid production rates  could not be matched likely due to the low AgBS catalytic efficiency . Prenyl precursors might be more readily available for heterologous terpene synthases in the dark as the pull to their roles in light-processes, synthesis of pigments and electron transport chain compounds, is stopped. Green algae like C. reinhardtii are adapted to a broad range of fluctuating light intensities and therefore can rapidly react to sudden illumination upon dark phases. A large pool of prenyl substrates held in the dark may support this ability. Simultaneous provision of CO2 in excess could further stimulate flux towards isoprenoids due to NAB1-mediated de-repression of LHCII protein accumulation, which affects both bound chlorophylls as well as carotenoids representing a metabolic pull in these conditions .