Prerequisite for highly efficient isoprenoid production by cyanobacteria discovered through the over-expression of 1-deoxy-d-xylulose 5-phosphate synthase and carbon allocation analysis

Abstract

Cyanobacteria have recently been receiving considerable attention owing to their potential as photosynthetic producers of biofuels and biomaterials. Here, we focused on the production of isoprenoids by cyanobacteria, and aimed to provide insight into metabolic engineering design. To this end, we examined the over-expression of a key enzyme in 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, 1-deoxy-d-xylulose 5-phosphate synthase (DXS) in the cyanobacterium Synechocystis sp. PCC6803. In the DXS-over-expression strain (Dxs_ox), the mRNA and protein levels of DXS were 4-times and 1.5-times the levels in the wild-type (WT) strain, respectively. The carotenoid content of the Dxs_ox strain (8.4 mg/g dry cell weight [DCW]) was also up to 1.5-times higher than that in the WT strain (5.6 mg/g DCW), whereas the glycogen content dramatically decreased to an undetectable level. These observations suggested that the carotenoid content in the Dxs_ox strain was increased by consuming glycogen, which is a C-storage compound in cyanobacteria. We also quantified the total sugar (145 and 104 mg/g DCW), total fatty acids (31 and 24 mg/g DCW) and total protein (200 and 240 mg/g DCW) content in the WT and Dxs_ox strains, respectively, which were much higher than the carotenoid content. In particular, approximately 54% of the proteins were phycobiliproteins. This study demonstrated the major destinations of carbon flux in cyanobacteria, and provided important insights into metabolic engineering. Target yield can be improved through optimization of gene expression, the DXS protein stabilization, cell propagation depression and restriction of storage compound synthesis.

Culturing Synechocystis sp. Strain PCC 6803 with N 2 and CO 2 in a Diel Regime Reveals Multiphase Glycogen Dynamics with Low Maintenance Costs

“…An enrichment analysis based on the categories of the biological functions showed that phycobilisome proteins (P ϭ 0.006) were among these (see Table S7). The phycobilisome proteins are the most abundant proteins found in the cells (38), which is corroborated by our analysis, and represent a major investment of resources for the cell, explaining their stable levels. It is interesting to note that the phycobilisome proteins showed no change, whereas the apparent chlorophyll content changed over the course of the light period in a fashion similar to content in a culture exposed to a much lower light intensity (see Fig.…”

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

“…Expression of an additional, transgenic DXS increased production by 4.2 times to 0.45 mg g –1 DCW in TPS-PD while TPS-PG increased production 2.6 times and TPS-PDG 3.5 times compared to TPS-P ( Figure 5 A). The 4.2 times increase upon CfDXS overexpression is larger than previously reported for heterologous production of other terpenoids such as carotenes and the monoterpene limonene in Synechocystis with overexpression of the endogenous DXS, 30 , 31 indicating that the native DXS may be subjected to regulation, which may not apply to the enzyme from the higher plant. The titer reached in the highest producing strain (TPS-PD) is similar to those achieved for other terpenoids in cyanobacteria (see Pattanaik and Lindberg, 2015 9 and references therein).…”

Terpenoids and Their Biosynthesis in Cyanobacteria

“…DXS, the enzyme catalyzing the first step of the MEP pathway, has been identified as a bottleneck of the pathway [ 143 ]. To direct the carbon flux towards product formation, Kudoh et al [ 144 ] developed a DXS overexpression strain in Synechocystis , by expressing an extra copy of the native Synechocystis dxs gene under control of the highly active psbA2 promoter. The extra expression of DXS in the modified strain lead to an increase in carotenoid levels, by a factor of about 1.5, and a decrease in glycogen levels, which lead the authors to suggest that the increase in carotenoid content was due to consumption of glycogen.…”