## 2 Results
### Limonene Production Optimization
Statistical significance (one-way ANOVA) compared to 0 mM isoprenol is represented by asterisks (** indicates a difference at the \(p\leq\) 0.01 level and * indicates a difference at the \(p\leq\) 0.05 level). To investigate the influence of the dodecane overlay extraction time on the limonene extraction we performed a scaled-up cultivation in a TAP medium of the three best limonene-producing strains, and collected the dodecane fractions after 1, 2, 3, 4, 5, 6 and 7 d growth (Figure 5c). We also investigated whether the volume of dodecane used has an effect, where a 300 mL TAP medium was supplemented with 5, 10, 15 or 20 mL dodecane as an extraction solvent (Figure 5d). We observed the highest limonene accumulation after 7 d, with titers of up to 23, 30 and 43 \(\mathrm{\SIUnitSymbolMicro g}\)/L from strains V12-118, V12-203 and V12-209, respectively, while a 15-mL overlay of dodecane gave the highest concentration of limonene (approximately 7 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) in all three strains (Figure 5d). [PERSON] et al. [32] investigated the dynamics of the secreted recombinant proteins from _C. reinhardtii_ and showed that it was essential to optimize the culture time for the best product yields. [PERSON] et al. [9] observed the dynamics of the accumulation of the sesquiterpenoid patchoulol in dodecane and found that the accumulation seemed to continue until the catabolic processes ceased after 144 h. We also found that the limonene accumulation increased in the dodecane fraction even after the algal cells had reached a stationary phase (Figure 5c and Supplementary Figure S2). [PERSON] et al. [26] showed that the dodecane overlay was appropriate for the limonene product capture and did not greatly affect the chlorophyll content or biomass yield of Synechococcus sp. PCC 7002, which was confirmed in our study. The dodecane overlay has also been used in two-phase cultures to capture the heterologous patchoulol [9], triterpenoids [10] and the sesquiterpene biodiesel precursor (E)-\(\alpha\)-bisabolene [11] from the cell wall-deficient _C. reinhardtii_ UVM4 strain. A 5% dodecane overlay was used in their studies, which is in accordance with our results. The dodecane overlay extraction method probably helps to avoid possible product-induced feedback inhibition, which is often observed in metabolic pathways. Moreover, limonene can kill _C. reinhardtii_ cells at a concentration of 0.2 mM [33]; therefore, the dodecane extraction will likely reduce the toxic effect of limonene. The IUP involves a double phosphorylation (pyrophosphorylation) of the isopentenol substrates, isoprenol or phenol, in two sequential steps in order to produce IPP and DMAPP, the main precursors in the terpenoid synthesis. As mentioned above, the specific concentration of feedstock used is critical for increasing the production of IPP. Thus, we grew the best limonene-producing strains, V12-118, V12-203 and V12-209 in a TAP medium supplemented with 5, 10, 15, 20 or 25 mM isoprenol or phenol to investigate the impact of the different substrates and the substrate concentrations on the limonene production. The results showed that phenol has a lethal effect on the strains, which did not survive at any phenol concentration tested (data not shown); in contrast, isoprenol has a low toxicity and is thus compatible with the _C. reinhardtii_ growth. The transformant strains showed the highest limonene production at a 10 mM isoprenol feed concentration, giving limonene productivities in V12-118, V12-203 and V12-209 of approximately 20, 37 and 39 \(\mathrm{\SIUnitSymbolMicro g}\)/L, respectively (Figure 5e).