Photosynthetic Conversion of CO2 Into Pinene Using Engineered Synechococcus sp. PCC 7002

Abstract

Metabolic engineering of cyanobacteria has received much attention as a sustainable strategy to convert CO2 to various longer carbon chain fuels. Pinene has become increasingly attractive since pinene dimers contain high volumetric energy and have been proposed to act as potential aircraft fuels. However, cyanobacteria cannot directly convert geranyl pyrophosphate into pinene due to the lack of endogenous pinene synthase. Herein, we integrated the gene encoding Abies grandis pinene synthase into the model cyanobacterium Synechococcus sp. PCC 7002 through homologous recombination. The genetically modified cyanobacteria achieved a pinene titer of 1.525 ± 0.l45 mg L−1 in the lab-scale tube photobioreactor with CO2 aeration. Specifically, the results showed a mixture of α- and β-pinene (∼33:67 ratio). The ratio of β-pinene in the product was significantly increased compared with that previously reported in the engineered Escherichia coli. Furthermore, we investigated the photoautotrophic growth performances of Synechococcus overlaid with different concentrations of dodecane. The work demonstrates that the engineered Synechococcus is a suitable potential platform for β-pinene production.

Exploring the Potential of the Model Cyanobacteria Synecho-Coccus PCC 7002 and PCC 7942 for the Photoproduction of High-Value Terpenes: Comparison With Synechocystis PCC 6803

“…As terpenes are volatile chemicals, cyanobacteria engineered for terpene production are routinely grown under a dodecane overlay to trap terpene, which are subsequently quantified by gaz chromatography coupled to mass spectrometry [1]. The growth of S.7002 was not affected by a dodecane overlay (Figure 3), in agreement with previous studies on S.7002 [8,9]. In contrast, the growth of S.7942 was strongly delayed by a dodecane overlay (Figure 3), but S.7942 strains precultivated under dodecane grew subsequently well under dodecane (Figure 3), a finding not discussed by previous workers [10][11][12].…”

“…The presently observed production of pinene by S.7002 (∼60 μg.L -1 after 21 days) is lower than the previously reported level (1.5 mg.L -1 after 6 days, [9]) observed after cloning into the endogenous pAQ1 plasmid (we used the non-cyanobacterial pC vector derived from the broad-host-range plasmid RSF1010 [20]) a Abies grandis gene encoding a 6xhistidine-tagged pinene synthase (we used the native Pinus taeda enzyme) expressed from the cpc promoter (we used the lambda-phage pR promoter). As both the pAQ1 and pC vectors on one hand, and the cpc and pR promoters on the other hand, function well in cyanobacteria [3] we assume that the 6xhistidine-tagged pinene synthase from Abies grandis is more efficient than the native Pinus taeda enzyme we have used in this study (Figure 4D), and our previous analysis of pinene production in S.6803 [20], Figure S5D).…”

Synthetic biology in marine cyanobacteria: Advances and challenges

“…Prochlorococcus isolates, the most abundant photosynthetic organisms, have primarily been isolated from tropical and Flexibility "Model" marine cyanobacterium Fast-growing Genome integration sites (Vogel et al, 2017) Counterselection method (Begemann et al, 2013) Characterised genetic elements (Markley et al, 2015;Pérez et al, 2017;Jones et al, 2021a) Marker excision with recombinases (Jones et al, 2021b) Target downregulation (Zess et al, 2016;Gordon et al, 2016) Nozzi et al, 2017Hasunuma et al, 2019;Yang et al, 2021 Synechococcus sp.…”

“…In fact, several genomic integration sites have been characterized for minimal metabolic disruption, allowing insertion of genetic constructs at different loci (Ruffing et al, 2016;Vogel et al, 2017). Integration in the native plasmid pAQ1 also offers an interesting option due to the higher copy number compared to genome integration (Yang et al, 2021). Natural transformation tends to be the most common DNA transfer for genomic integration in Synechococcus 7002 by selecting transformants through antibiotic resistance.…”

“…In fact, a significant number of non-native target compounds have now been produced by Synechococcus 7002 at a laboratory scale. To name a few, bioproduction of the hydrocarbons limonene and bisabolene (Davies et al, 2014), the pigment astaxanthin (Hasunuma et al, 2019), the amino acid L-lysine 10.3389/fmicb.2022.994365 (Korosh et al, 2018), the building block 2,3-butanediol (Nozzi et al, 2017), the potential aircraft fuel pinene (Yang et al, 2021), the fatty acid lauric acid (Work et al, 2015) or the biomaterial precursor polyhydroxyalkanoate (Zhang et al, 2015) has been achieved. The chemical diversity of these compounds further highlights the industrial importance of this chassis organism for a greener future.…”