Development of SyneBrick Vectors As a Synthetic Biology Platform for Gene Expression in Synechococcus elongatus PCC 7942

Abstract

Cyanobacteria are oxygenic photosynthetic prokaryotes that are able to assimilate CO₂ using solar energy and water. Metabolic engineering of cyanobacteria has suggested the possibility of direct CO₂ conversion to value-added chemicals. However, engineering of cyanobacteria has been limited due to the lack of various genetic tools for expression and control of multiple genes to reconstruct metabolic pathways for biochemicals from CO₂. Thus, we developed SyneBrick vectors as a synthetic biology platform for gene expression in Synechococcus elongatus PCC 7942 as a model cyanobacterium. The SyneBrick chromosomal integration vectors provide three inducible expression systems to control gene expression and three neutral sites for chromosomal integrations. Using a SyneBrick vector, LacI-regulated gene expression led to 24-fold induction of the eYFP reporter gene with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) inducer in S. elongatus PCC 7942 under 5% (v/v) CO₂. TetR-regulated gene expression led to 19-fold induction of the GFP gene when 100 nM anhydrotetracycline (aTc) inducer was used. Gene expression decreased after 48 h due to degradation of aTc under light. T7 RNA polymerase-based gene expression resulted in efficient expression with a lower IPTG concentration than a previously developed pTrc promoter. A library of T7 promoters can be used for tunable gene expression. In summary, SyneBrick vectors were developed as a synthetic biology platform for gene expression in S. elongatus PCC 7942. These results will accelerate metabolic engineering of biosolar cell factories through expressing and controlling multiple genes of interest.

Keywords: SyneBrick vectors; Synechococcus elongatus PCC 7942; T7 promoters; gene expression; synthetic biology.

Figure 1

Design features of SyneBrick vectors for gene expression in S. elongatus PCC 7942. (A) SyneBrick vector sections: promoters, antibiotic markers, chromosomal integrational sites and gene of interests (GOI). Standard SyneBrick vector (pSe1Bb1s-GFP) features are two homologous regions of neutral site I (NSIa and NSIb) for chromosomal integration, gene expression of a target under control of the inducible trc promoter and LacI repressor, a transcriptional terminator, spectinomycin-resistance selection, and replication of origin (pUC19) for cloning in E. coli. Sections are selected to construct a SyneBrick vector. For expansion of SyneBrick vectors, two promoters (TetA and T7 promoters), two antibiotic makers (kanamycin or chloramphenicol) and neutral sites for chromosomal integration (site II or site III) are available as interchangeable parts for the standard SyneBrick vector. (B) The nomenclature for the library of SyneBrick vector library is described. SyneBrick vectors constructed in this work are in Table 1. (C) Gel images of recombinant cyanobacterial strains. Strains were verified by PCR using oligonucleotides (arrows). Sequences are in Table S1. DNA fragments from wild-type and strains 1, 2, 3, and 4 are shown in gel images. DNA sequences were verified.

Figure 2

Gene expression analysis of engineered S. elongatus PCC 7942 using SyneBrick vectors. (A) Growth of S. elongatus PCC 7942 wild type under 5% (v/v) CO₂ bubbling in BG-11 supplemented with indicated concentrations of IPTG measured at OD₇₃₀ (black square, 0 mM; red circle, 0.01 mM; blue triangle, 0.1 mM; inverted pink triangle, 1 mM; green diamond, 10 mM). (B) Growth of engineered S. elongatus PCC 7942 (Se1Bb1s-eYFP and Se1Bb1s-GFP) under 5% (v/v) CO₂ bubbling in BG-11 supplemented with indicated concentrations of IPTG, measured at OD₇₃₀ (upper panel). Specific fluorescence (intensity per OD₇₃₀) for cyanobacterial cultures (lower panel). Se1Bb1s-None was the control strain (open black square). Symbols for IPTG used are as in (A). (C) Strain Se1Bb2s-GFP was used to measure growth and specific fluorescence under aTC induction (black square, 0 mM; red circle, 10 nM; blue triangle, 100 nM; inverted pink triangle, 200 nM; green diamond, 1000 nM). Experiments were performed in triplicate cultures. All data are mean ± standard deviation (SD) from triplicate cultures.

Figure 3

Design features of SyneBrick vectors 2.0 for gene expression using the T7 promoter in S. elongatus PCC 7942. (A) Scheme for genetic control of target gene (e.g. eYFP). With IPTG, T7 RNA polymerase (T7 TNAP) was expressed and its cognate T7 promoter was used to express target genes. Sequences of original T7 promoter and sequence variants (T7.3 and T7.4) with binding and strength regions (Temme et al., 2012). (B) Construction of cyanobacterial strains using pSe2Bb7k-eYFP, pSe2Bb7.3k-eYFP, pSe2Bb7.4k-eYFP, and pSe3Bb1c-T7RNAP vectors for expression of eYFP protein under control of T7 variant promoters. Sequencing primers are in Table S1. (C) Gel images of recombinant cyanobacterial strains. Strains were verified by PCR using oligonucleotides (arrows). Sequences are in Table S1. DNA fragments from wild-type and strains 1, 2, and 3 are shown in gel images. DNA sequences were verified.

Figure 4

Gene expression analysis of engineered S. elongatus PCC 7942 using SyneBrick 2.0 vectors. (A) Growth of engineered S. elongatus PCC (pSe23Bb7kc-eYFP/P, pSe23Bb7.3kc-eYFP/P, and pSe23Bb7.4kc-eYFP/P) under 5% (v/v) CO₂ bubbling in BG-11 supplemented with indicated concentrations of IPTG, measured at OD₇₃₀ (black square, 0 mM; red circle, 0.01 mM; blue triangle, 0.1 mM; inverted pink triangle, 1 mM; green diamond, 10 mM). (B) Specific fluorescence (intensities per OD₇₃₀) were calculated for strains. Experiments were performed in triplicate cultures. All data are presented as mean ± standard deviation (SD) from triplicate cultures.