Pinene production using protein fusions
Purple non-sulfur photosynthetic bacteria use the endogenous MEP pathway to produce IPP and DMAPP, which are further catalyzed by CrtE to synthesize GPP. Then, GPP can be catalyzed to pinene by introducing an exogenous PS gene, as shown in Fig. 1a. However, CrtE have both GPP synthase and farnesyl diphosphate (FPP) synthase activity. Thus, CrtE can convert IPP and DMAPP to GPP, then to FPP. As an intermediate, GPP is more readily catalyzed to FPP, instead of generating monoterpenes, e.g. pinene[23].
To elevate the metabolic flux from GPP to pinene, a feasible strategy is to construct a fusion protein of GPPS and PS to make it easier for GPP to enter PS active site from GPPS active site [5, 8]. In this study, a pBBR-αGppsPs plasmid was constructed, in which a fusion gene of Abies grandis GPPS and PS was expressed under the strong trc promoter and with the ribosome binding site of RBSα. The pBBR-αGppsPs plasmid was transferred to R. sphaeroides by conjugation and the final strain (R. sphaeroides:pBBR-αGppsPs) was grown under light anaerobic conditions to produce pinene. Pinene titers were detected over time at various temperatures to obtain the optimal culturing temperature and duration. The results showed that maintaining the temperature at 30℃ was more suitable for pinene production as the rate of pinene production and the final pinene titers were higher at this condition (Fig. 2). It was observed the pinene titers gradually increasing to maximum and then reached plateau after culturing for 132 h at 30℃, so the optimal culturing duration was 132 h.
As the transcription of gpps-ps was induced by IPTG, we tested the effect of IPTG concentrations on pinene production. Figure 3 showed pinene production using GPPS-PS fusion protein at various IPTG concentrations, ranging from 0 to 1000 µM. At the IPTG concentration of 3 µM, the highest pinene titer was obtained, which was 97.51 µg/L in total (32.17 µg/L α-pinene and 65.34 µg/L β-pinene). When IPTG concentration was higher than 10 µM, the pinene titers were much lower than the value at the IPTG concentration of 3 µM. This suggests that the increase in IPTG concentration will inhibit the synthesis of pinene in R. sphaeroides. Thus, in the subsequent pinene production reaction, the concentration of IPTG was set to 3 µM.
Improving Pinene Production By Overexpression Of Key Genes
As most genes in isoprenoid pathway are expressed at low level under normal growth conditions, the common method is to increase the expression of key genes to improve the metabolic flux [24]. The genes coded for MEP pathway enzymes were studied and four genes (dxs, idi, ispD and ispF) were deemed to be the rate-limiting in E. coli [24, 25]. Lu et.al overexpressed dxs, dxr, ispD and idi simultaneously and increased Q10 yield by two folds in R. sphaeroides [21]. However, there is limited information about the MEP pathway in purple non-sulfur photosynthetic bacteria and the key genes were not confirmed by experiments.
In this study, the genes in MEP pathway were overexpressed and pinene production was tested, respectively, in order to determine the key genes. Then, the key genes were co-expressed to increase metabolic flux towards IPP and DMAPP.
Eight plasmids with the expression of dxs, dxr, ispD, ispE, ispF, ispG, ispH and idi were constructed based on pBBR-αGppsPs (Fig. 1b), and then transferred to R. sphaeroides, respectively. Figure 4a showed pinene production with overexpression of these genes. Pinene titers were improved to 105.34 and 109.73 µg/L with overexpression of dxs and idi, respectively, indicating that Dxs and Idi are rate-limiting enzymes of MEP pathway in R. sphaeroids, which is consistent with the reports that Dxs and Idi enhanced carbon flux of isoprenoid biosynthesis in E.coli [26, 27]. We therefore co-expressed idi and dxs together (pBBR-αGppsPs-IdiDxs) and further improved pinene production to 148.83 µg/L (Fig. 4b).
In contrast, overexpression of dxr, ispD, ispE, ispF, ispG and ispH, respectively, resulted in a decrease in pinene production. This suggests overexpression of these genes might cause metabolic burden or toxicity.
Considering that Dxr was reported to be a key enzyme in E.coli [28], dxr was overexpressed with idi and dxs together (pBBR-αGppsPs-IdiDxsDxr) to improve pinene production. As shown in Fig. 4b, the strain overexpressing idi, dxs and dxr produced a higher amount of pinene (174.08 µg/L) than the one overexpressing idi dxs combination. This result indicates that Dxr turns into a rate-limiting enzyme, when the primary rate-limiting steps are released, and overexpression of dxr may further improve metabolic flux. In summary, overexpressing idi, dxs and dxr simultaneously causes a great increase of pinene output.
Improving Pinene Production By Altering Rbs Of Gpps-ps Mrna
The expression level of GPPS-PS protein can be altered by changing ribosome binding site, as the ribosome binding site plays an important role in protein translation. Therefore, we considered increasing pinene production by optimizing ribosome binding site. Two standard RBS sequences (RBSβ and RBSγ) as well as RBSα, were all taken from iGEM toolbox. The strength of RBSα, RBSβ, and RBSγ were tested with GFP as the reporter protein. As shown in Fig. 5a, the fluorescent intensity of GFP is stronger with RBSγ compared to RBSα, but is weaker with RBSβ. Then, the ribosome binding site of GPPS-PS mRNA (RBSα) was replaced by a stronger RBS (RBSγ) and a weaker RBS (RBSβ), and two plasmids (pBBR-βGppsPs, pBBR-γGppsPs) were transferred to R.sphaeroides, respectively, then the resulting pinene titers were compared. Figure 5b shows that the pinene production can be improved by using a stronger RBS, but decreased with a weaker RBS. Thus, overexpression of GPPS-PS protein could increase pinene production, which sµggests the metabolic fluxes of downstream pathway might be inadequate in the strain harboring pBBR-αGppsPs.
Therefore, RBSα was replaced with RBSγ, and pBBR-γGppsPs-IdiDxs and pBBR-γGppsPs-IdiDxsDxr were constructed and transferred to R.sphaeroides, respectively. As shown in Fig. 6, overexpresssing idi and dxs increased the production of pinene to 401.43 µg/L and overexpressing idi, dxs and dxr together further improved the yield of pinene to 539.84 µg/L, which was 5.54 folds compared to R. sphaeroides:pBBR-αGppsPs strain. Thus, overexpression of key enzymes of the MEP pathway and the downstream pathways simultaneously can substantially increase pinene production.