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.

Evaluation of promoters and ribosome binding sites for biotechnological applications in the unicellular cyanobacterium Synechocystis sp. PCC 6803

“…In a previous study where we used P nrsB for the production of the high value compound manoyl oxide, we observed a decrease in production at high light, something we speculated could be due to reduced function of P nrsB at higher light12. In order to investigate this effect, we performed experiments to determine the influence of light intensity on EYFP expression as well as ethanol production.…”

“…An alternative set of regulated promoters are metal ion inducible promoters that have already been used for several applications8910111213. In Synechocystis , a set of genes responsible for Ni 2+ , Co 2+ , and Zn 2+ tolerance are all grouped together in a gene cluster14.…”

“…In cyanobacterial strains which have a functional induction of P trc and P lac such as Synechococcus elongatus PCC 7942, those are commonly used24. For Synechocystis production, P nirA have been used for production of carotenoids25, P nrsB for manoyl oxide production12 and P coaT , P petE , and a version of the Lac promoter were used for ethylene production13.…”

Evaluating Light‐Induced Promoters for the Control of Heterologous Gene Expression in Synechocystis sp. PCC 6803

“…Over the last fifteen years, P psbAII has been leveraged as a gene control element to drive gene expression within engineered systems for the production of various products within Synechocystis . A majority of work using P psbAII replaces the open reading frame of the psbAII gene with the target gene of interest, thus utilizing P psbAII in its native location within the genome . Lagarde and colleagues replaced the psbAII ORF with carotenoid genes which resulted in increased production under the control of P psbAII .…”

Oxygen‐responsive genetic circuits constructed in Synechocystis sp. PCC 6803

“…Up to this point, all genetic parts used in this work were integrated into Synechocystis ’ chromosome, replacing psbA1 and its promoter (Varman et al, ). Other neutral sites in the chromosome have been used for heterologous expression, including slr0168 encoding a hypothetical protein (Angermayr et al, ; Kunert et al, ; Viola et al, ), slr2031 encoding a phosphatase expressed in instances of nitrogen, or sulfate starvation (Ejima et al, ; Yu et al, ), and several other regions (Wang et al, ), including NSC1 (personal communication with Dr. Himadri Pakrasi, Supplementary Table VI) identified from a transcription start site map (Mitschke et al, ), psbA2 , and sqs (Englund et al, ). Only one replicon, RSF1010, has been shown to function in Synechocystis .…”