High density cultivation for efficient sesquiterpenoid biosynthesis inSynechocystissp. PCC 6803

Abstract

Cyanobacteria and microalgae are attractive phototrophic host systems for climate-friendly production of fuels and other high-value chemicals. The biosynthesis of an increasing diversity of industrially relevant compounds such as terpenoids has been demonstrated in recent years. To develop economically feasible and sustainable process designs, major challenges still remain regarding intracellular carbon partitioning, specific metabolic pathway activities and efficient cultivation strategies. Here, we present a technical study on comparative characteristics of sesquiterpene and sesquiterpene alcohol accumulation in engineered strains of Synechocystis sp. PCC 6803 (substrain GT-U) under different growth conditions and cell densities. This study particularly focuses on the basic applicability of a commercial High Density Cultivation platform in the presence of a dodecane overlay, which serves as a standard in-situ extractant and sink for various hydrophobic biochemicals. Significantly, the presented data demonstrate high volumetric productivities of (E)-α-bisabolene under high-density conditions that are more than two orders of magnitude higher than previously reported for cyanobacteria. Operating in a two-step semi-batch mode over a period of eight days, average final volumetric titers of 179.4 ± 20.7 mg * L -1 were detected. Likewise, the sesquiterpene alcohols (-)patchoulol and (-)-α-bisabolol accumulated to many times higher levels in high density cultivation than under standard batch conditions, with final titers of 17.3 ± 1.85 mg * L -1 and 96.3 ± 2.2 mg * L -1 , respectively. In contrast, specific product accumulation (mg * L -1 * OD750 -1 ) was compromised particularly for bisabolene in the high density system during phases of high biomass accumulation rates. Volumetric productivities were high during linear growth at high densities, distinctly outperforming standard batch systems. While the presented data highlight the benefits of high-density strategies for highly efficient phototrophic terpenoid production, they further point at the presence of major metabolic bottlenecks for engineered terpenoid biosynthesis and the requirement for systematic and/or targeted strategies to sustainably redirect inherent carbon fluxes in cyanobacteria. Together, our data provide additional insights into growth-and density-related effects on the efficiency of product accumulation, introducing low-scale High Density Cultivation as a rapid and efficient platform for screening of heterologous terpenoid production in cyanobacteria.

Impact of irradiance and inorganic carbon availability on heterologous sucrose production in Synechococcus elongatus PCC 7942

“…Curiously, under these conditions of high inorganic carbon availability, we observed little, but significant, difference in growth rate in the sucrose-exporting strains despite a significant rate of carbon export in the form of sucrose (Figures 2A, B, E). Since nutrients in BG-11 medium can become limiting at higher density, we next evaluated an enriched medium (CD) designed to support high-density cultures, such as those achievable in the HDC system (Dienst et al, 2020). When cultures were grown in CD media, non-exporting strains reached 11.1 OD 750 over the course of 5 days (Figure 2C), while also secreting a disproportionally high level of sucrose for an uninduced culture (Figure 2D).…”

Engineered Terpenoid Production inSynechocystissp. PCC 6803 Under Different Growth Conditions

“…48 A recent study reported a yield of 17.3 mg L -1 patchoulol from Synechocystis using a High-Density Cultivation (HDC) system. 40 Though an impressive yield, it is based on smallscale (8 mL) cultures grown to an OD750 of 32 under 750 µE m -2 s -1 light, and the authors noted that specific productivity did not increase compared to batch cultures. For small-scale cultures that did not receive increasing light or media replenishment during growth and were supplied 100 µE m -2 s -1 light (growth conditions comparable to this work), yields of patchoulol were reduced to 590 µg L -1 , around two-fold greater than the highest yield achieved in this work.…”

“…In Synechocystis, expression of the patchoulol synthase gene under a strong copper inducible promoter yielded 17.3 mg L -1 patchoulol. 40 That study utilized a highly optimized small-scale High-Density Cultivation (HDC) system with nutrient rich media, bicarbonate, and increasing light intensity. The impressive patchoulol yield achieved reflects the high cell densities that can be reached using HDC; however, when grown under typical batch conditions, the yields were similar to those achieved for other terpenoids.…”