Metabolic Engineering of Synechocystis sp. PCC 6803 for Production of the Plant Diterpenoid Manoyl Oxide

Abstract

Forskolin is a high value diterpenoid with a broad range of pharmaceutical applications, naturally found in root bark of the plant Coleus forskohlii. Because of its complex molecular structure, chemical synthesis of forskolin is not commercially attractive. Hence, the labor and resource intensive extraction and purification from C. forskohlii plants remains the current source of the compound. We have engineered the unicellular cyanobacterium Synechocystis sp. PCC 6803 to produce the forskolin precursor 13R-manoyl oxide (13R-MO), paving the way for light driven biotechnological production of this high value compound. In the course of this work, a new series of integrative vectors for use in Synechocystis was developed and used to create stable lines expressing chromosomally integrated CfTPS2 and CfTPS3, the enzymes responsible for the formation of 13R-MO in C. forskohlii. The engineered strains yielded production titers of up to 0.24 mg g(-1) DCW 13R-MO. To increase the yield, 13R-MO producing strains were further engineered by introduction of selected enzymes from C. forskohlii, improving the titer to 0.45 mg g(-1) DCW. This work forms a basis for further development of production of complex plant diterpenoids in cyanobacteria.

Keywords: MEP-pathway; Synechocystis; diterpenoid; forskolin; genetic tools; manoyl oxide.

Figure 1

Terpenoid biosynthesis via the MEP pathway in engineered Synechocystis. The native enzymes relevant for this paper are marked in blue, and the introduced C. forskohlii enzymes in green. Hemi-, mono- and sesquiterpenoids have not been identified in Synechocystis and are marked with dashed boxes. Abbreviations used: G3P = glyceraldehyde 3-phosphate, DXP = 1-deoxy-d-xylulose 5-phosphate, MEP = methylerythritol-4-phosphate, CDP-ME = diphosphocytidylyl methylerythritol, CDP-MEP = methylerythritol-2-phosphate, ME-cPP = methylerythritol-2,4-cyclodiphosphate, HMBPP = hydroxymethylbutenyl diphosphate, IPP = isopentenyl diphosphate, DMAPP = dimethylallyl diphosphate, GPP = geranyl diphosphate, FPP = farnesyl diphosphate, GGPP = geranylgeranyl diphosphate, NADP(H) = nicotinamide adenine dinucleotide phosphate, CTP/CMP = cytidine tri(mono)phosphate, PPi = diphosphate, ATP/ADP = adenosine tri(di)phosphate, Fd^red/^ox = ferredoxin reduced/oxidized, OPP = diphosphate group.

Figure 2

Schematic overview and utilization of the pEERM vectors. The base pEERM vector (A) and the open-reading frame (ORF) to be inserted (B) are cut with XbaI and PstI and ligated together (step 1). Additional genes can be cloned downstream of first gene by cutting the new vector (C) with SpeI and PstI and the next insert (D) with XbaI and PstI, and ligating them (step 2). A SpeI/XbaI scar will form between the two inserts in the resulting plasmid. When all genes have been inserted, the final construct (E) can be directly transformed into the Synechocystis genome through homologous recombination.

Figure 3

RT-PCR analysis of engineered strains. Total RNA was converted into cDNA and the presence and abundance of transcripts was verified with gene specific primers and specific amounts of PCR cycles. −RT = RNA samples before conversion to cDNA using reverse transcriptase amplified with 16S primers, gDNA = genomic DNA of TPS-PDG.

Figure 4

Detection of 13-MO by GC–MS analysis. 13R-MO detection in extracts of engineered Synechocystis strain TSP-N, grown with and without 2.5 μM Ni²⁺ induction, comparing with authentic standard and extracts of the wild type strain. Identification of 13R-MO was confirmed by the retention time and mass spectra of an authentic 13R-MO standard.

Figure 5

13R-MO production in engineered Synechocystis strains. 13R-MO was quantified from cell pellets of strains with CfTPS2 and CfTPS3 inserted in different sites on the genome and with combinations of CfDXS and CfGGPPS, in cells grown at low light (A) and high light (B). Results represent the mean of six biological replicates, error bars represent standard deviation. n.d. = no 13R-MO detected, DCW = dry cell weight.

Figure 6

Pigment analysis. Carotenoid content (left axis) and chlorophyll content (right axis) of 13R-MO producing strains of Synechocystis at low light (A) and high light (B). Asterisks represents significant differences between carotenoid samples, * = p < 0.05, ** = p < 0.01. Results represent the mean of two technical replicates per three biological replicates, error bars represent the standard deviation from the biological replicates. DCW = dry cell weight.