Combinatorial deletions of glgC and phaCE enhance ethanol production in Synechocystis sp. PCC 6803

Abstract

Synechocystis sp. PCC 6803 is an attractive host for bio-ethanol production. In the present study, a nitrogen starvation approach was applied on an ethanol producing strain for inhibiting the growth, since ethanol production competes with the cell growth. The effect of gene deletions in the glycogen and polyhydroxybutyrate (PHB) synthesis pathways was investigated. Measurements of intracellular glycogen and PHB revealed that the glycogen was accumulated under the nitrogen starvation condition and the gene deletion of glycogen synthesis pathway caused the accumulation of PHB. The ethanol producing strain harboring deletions for both the glycogen and the PHB synthesis pathways (ΔglgCΔphaCE/EtOH) produced ethanol at the specific rate of 240mgg (dry cell weight) day under the nitrogen starvation condition. In a high cell density culture (OD=50) using this ΔglgCΔphaCE/EtOH strain, the ethanol production rates were 1.08 and 2.01gL day under light conditions of 40 and 80μmolms, respectively.

Effects of inactivation of the cyAbrB2 transcription factor together with glycogen synthesis on cellular metabolism and free fatty acid production in the cyanobacterium Synechocystis sp. PCC 6803

“…Then, the cell pellet was resuspended with the medium without copper and nitrogen and further cultivated for 4 days (Supporting Information Figure S7A). Consistent with previous reports showing the nonbleaching phenotype of ΔglgC upon nitrogen depletion (Carrieri et al, 2015(Carrieri et al, , 2012Gründel et al, 2012;Namakoshi et al, 2016;van der Woude et al, 2014), the ΔglgCΔaas::UcTE and PpetE-cyabrB2ΔglgCΔaas::UcTE strains maintained blue-green color under copper and nitrogen depleted conditions (Supporting Information Figure S7B). The fatty acid productivity during 10 days of copper depleted conditions showed a similar tendency to the results shown in Figure 7 (Figure 8).…”

“…The Δ glgC strain has been reported to excrete organic acids such as pyruvate and 2‐OG under nitrogen depleted conditions (Carrieri et al, ; Carrieri, Paddock, Maness, Seibert, & Yu, ; Gründel, Scheunemann, Lockau, & Zilliges, ; Namakoshi et al, ; van der Woude et al, ). Thus, we quantified pyruvate excreted into the medium after 14 days of incubation under copper depleted conditions (Supporting Information Figure S5A).…”

“…It has been reported that knockout of glgC is effective for increased production of desired products especially under nitrogen depleted conditions (Namakoshi et al, ; van der Woude et al, ). On the basis of the information, we modified our free fatty acid producing conditions from “14 days without copper” to “10 days without copper plus 4 days without copper and nitrogen.” After cultivation for 10 days without copper, cells were harvested by centrifugation and the supernatant was used for the quantification of free fatty acids.…”

“…Several groups have been reported that cultivation of the ΔglgC mutants under nitrogen depleted conditions caused growth arrest, nonbleaching phenotype and excretion of organic acids (Carrieri et al, 2015(Carrieri et al, , 2012Gründel et al, 2012;Namakoshi et al, 2016;van der Woude et al, 2014). Under the conditions, increased production of target chemicals was achieved in some cases (Namakoshi et al, 2016;van der Woude et al, 2014). Consistently, we observed nonbleaching phenotype (Supporting Information Figure S7) and increased free fatty acid production (Figure 8) in the ΔglgCΔaas::UcTE and PpetE-cyabrB2ΔglgCΔaas::UcTE strains after 4 days of incubation under nitrogen depleted conditions.…”

“…If we successfully redirect excess carbon through a desired biosynthetic pathway by genetic engineering, the ΔcyabrB2 mutant can be a promising platform for the photosynthetic production of fuels and chemicals. To date, advantages of glycogen-mutants of cyanobacteria on chemical production have been reported, for instance, production of lactic acid (van der Woude, Angermayr, Veetil, Osnato, & Hellingwerf, 2014) and ethanol (Namakoshi, Nakajima, Yoshikawa, Toya, & Shimizu, 2016) in Synechocystis sp. PCC 6803 and isobutanol in Synechococcus elongatus PCC 7942 (Li, Shen, & Liao, 2014).…”