Synechocystis PCC 6803 overexpressing RuBisCO grow faster with increased photosynthesis

Abstract

The ribulose-1,5-bisphosphate (RuBP) oxygenation reaction catalyzed by Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is competing with carboxylation, being negative for both energy and carbon balances in photoautotrophic organisms. This makes RuBisCO one of the bottlenecks for oxygenic photosynthesis and carbon fixation. In this study, RuBisCO was overexpressed in the unicellular cyanobacterium Synechocystis PCC 6803. Relative RuBisCO levels in the engineered strains FL50 and FL52 increased 2.1 times and 1.4 times, respectively, and both strains showed increased growth, photosynthesis and in vitro RuBisCO activity. The oxygen evolution rate increased by 54% and 42% on per chlorophyll basis, while the in vitro RuBisCO activity increased by 52% and 8.6%, respectively. The overexpressed RuBisCO were tagged with a FLAG tag, in strain FL50 on the N terminus of the large subunit while in strain FL52 on the C terminus of the small subunit. The presence of a FLAG tag enhanced transcription of the genes encoding RuBisCO, and, with high possibility, also enhanced the initiation of translation or stability of the enzyme. However, when using a streptavidin-binding tag II (strep-tag II), we did not observe a similar effect. Tagged RuBisCO offers an opportunity for further studying RuBisCO expression and stability. Increased levels of RuBisCO can further improve photosynthesis and growth in the cyanobacterium Synechocystis PCC 6803 under certain growth conditions.

Keywords: FLAG tag; Increased growth; Photosynthesis; RuBisCO; Strep-tag II; Synechocystis PCC 6803.

Fig. 1

Genomic organization in the engineered Synechocystis strains FL75 and FL76 compared to in the wild type cells (A). Complete segregation of FL75 (B) and FL76 (C) were confirmed by PCRs. In FL75 and FL76, rbc on genome was tagged with a FLAG tag on the N terminus of the large subunit and on the C terminus of the small subunit, respectively separately. Numbers indicate the specific primers used in the PCRs to examine segregation. For further details, see Table 1.

Fig. 2

Growth of engineered Synechocystis PCC 6803 strains control (WT+Km^r-vector), FL50, and FL52. A, Chlorophyll a content. B, Optical density at 750 nm (OD₇₅₀). FL50 and FL52 had rbc gene, coding RuBisCO having FLAG tag fused to the large subunit N terminus or small subunit C terminus separately, overexpressed on pPMQAK1. Mean±SD is from six biological replicates, six technical replicates. Asterisks indicate that the differences observed between the respective engineered strain and the control strain are significant (One-way ANOVA, P<0.05). For strain information, see Table 1.

Fig. 3

Photosynthesis demonstrated by maximal oxygen evolution rate of engineered Synechocystis PCC 6803 strains control (WT+Km^r-vector), FL50, and FL52. A, Oxygen evolution rate per chlorophyll a content. B, Oxygen evolution rate per OD₇₅₀. FL50 and FL52 had rbc gene, coding RuBisCO having FLAG tag fused to the large subunit N terminus or small subunit C terminus, respectively, overexpressed on pPMQAK1. Mean±SD is from six biological replicates, six technical replicates. Asterisks indicate that the differences observed between the respective engineered strain and the control strain are significant (One-way ANOVA, P<0.05). For strain information, see Table 1.

Fig. 4

Relative RuBisCO transcript levels of engineered Synechocystis PCC 6803 strains control (WT+Km^r-vector), rbc, FL50, FL52, FL50strep, and FL52strep measured with reverse transcription poly chain reaction (RT-PCR). FL50 and FL52 had rbc gene, coding RuBisCO having FLAG tag fused to large subunit N terminus or small subunit C terminus separately, overexpressed on pPMQAK1. In FL50strep and FL52strep, strep-tag II was used instead of FLAG. 500 ng RNA was used for cDNA synthesizing and 2 µl cDNA was used to synthesize double strand DNA. For 16 s, 18 cycles were done and for rbc, 27 cycles were used. Wild type genomic DNA (gDNA) was used in PCR as a positive control. 1 µl and 4 µl indicated the volume loaded for DNA gel. Each strain has biological duplicates. For strain information, see to Table 1.

Fig. 5

Relative RuBisCO content of engineered Synechocystis PCC 6803 strains control (WT+Km^r-vector), rbc, FL50, FL52, FL50strep, and FL52strep. A, SDS-PAGE and Western immunoblot, primary antibody anti-rbcL IgG was used to detect RuBisCO large subunit. B, RuBisCO content normalized to control strain determined by Quantity One. Each strain had biological duplicate. FL50 and FL52 had rbc gene, coding RuBisCO having FLAG tag fused to the large subunit N terminus or small subunit C terminus separately, overexpressed on pPMQAK1. In FL50strep and FL52strep, strep-tag II was used instead of FLAG tag. Standard derivation is from two biological replicates and two technical replicates. Asterisks indicate the differences observed between the respective engineered strain and the control strain are significant (One-way ANOVA, P<0.05). For strain information, see Table 1.

Fig. 6

Relative total RuBisCO activity (A) and relative specific RuBisCO activity (B) of engineered Synechocystis PCC 6803 strains control (WT+Km^r-vector), FL50, FL52, control (WT+Km^r-genome), FL75, and FL76. Total RuBisCO activity was calculated as ¹⁴C incorporation per crude protein per time. Specific RuBisCO activity was calculated as ¹⁴C incorporation per RuBisCO protein per time. FL50 and FL52 had rbc gene, coding RuBisCO having FLAG tag fused to the large subunit N terminus or small subunit C terminus separately, overexpressed on pPMQAK1. In FL75 and FL76, rbc on genome was tagged with FLAG on large subunit N terminus or small subunit C terminus separately. Mean±SD is from four biological replicates, four technical replicates. Asterisks indicate the differences between the respective engineered strain and the control strain is significant (One-way ANOVA, P<0.05). FL50 and FL52 were normalized to control strain (WT+Km^r-vector) while FL75 and FL76 were normalized to control strain (WT+Km^r-genome). Even though the respective control strains, WT+Km^r-vector and WT+Km^r-genome, both were set to 1.0, it does not mean that the RuBisCO activity was the same in the two strains. For strain information, see Table 1.

Fig. 7

Relative RuBisCO content of engineered Synechocystis PCC 6803 strains, control (WT+Km^r-genome), FL75, FL76, FL50G, and FL52G. A, SDS-PAGE and Western immunoblot, primary antibody anti-rbcL IgG was used to detect RuBisCO large subunit. B, RuBisCO content normalized to control strain determined by Quantity One. In FL75 and FL76, rbc on genome was tagged with FLAG on large subunit N terminus and small subunit C terminus separately. FL50G and FL52G had rbc gene, coding RuBisCO having FLAG tag fused to the large subunit N terminus or small subunit C terminus separately, overexpressed on chromosome slr1608 site. Each strain had biological duplicate. Standard derivation is from two biological replicates and two technical replicates. Asterisks indicate the differences observed between the respective engineered strain and the control C strain are significant (One-way ANOVA, P<0.05). For strain information, see Table 1.