Ethylene production with engineered Synechocystis sp PCC 6803 strains

Abstract

Background: Metabolic engineering and synthetic biology of cyanobacteria offer a promising sustainable alternative approach for fossil-based ethylene production, by using sunlight via oxygenic photosynthesis, to convert carbon dioxide directly into ethylene. Towards this, both well-studied cyanobacteria, i.e., Synechocystis sp PCC 6803 and Synechococcus elongatus PCC 7942, have been engineered to produce ethylene by introducing the ethylene-forming enzyme (Efe) from Pseudomonas syringae pv. phaseolicola PK2 (the Kudzu strain), which catalyzes the conversion of the ubiquitous tricarboxylic acid cycle intermediate 2-oxoglutarate into ethylene.

Results: This study focuses on Synechocystis sp PCC 6803 and shows stable ethylene production through the integration of a codon-optimized version of the efe gene under control of the Ptrc promoter and the core Shine-Dalgarno sequence (5'-AGGAGG-3') as the ribosome-binding site (RBS), at the slr0168 neutral site. We have increased ethylene production twofold by RBS screening and further investigated improving ethylene production from a single gene copy of efe, using multiple tandem promoters and by putting our best construct on an RSF1010-based broad-host-self-replicating plasmid, which has a higher copy number than the genome. Moreover, to raise the intracellular amounts of the key Efe substrate, 2-oxoglutarate, from which ethylene is formed, we constructed a glycogen-synthesis knockout mutant (ΔglgC) and introduced the ethylene biosynthetic pathway in it. Under nitrogen limiting conditions, the glycogen knockout strain has increased intracellular 2-oxoglutarate levels; however, surprisingly, ethylene production was lower in this strain than in the wild-type background.

Conclusion: Making use of different RBS sequences, production of ethylene ranging over a 20-fold difference has been achieved. However, a further increase of production through multiple tandem promoters and a broad-host plasmid was not achieved speculating that the transcription strength and the gene copy number are not the limiting factors in our system.

Keywords: Arginine; Cyanobacteria; Ethylene; Glycogen; Oxoglutarate; Sustainable; Synechocystis.

Fig. 1

Rate of ethylene production in Synechocystis strains expressing the efe gene under control of the Ptrc promoter and various RBSs. a Comparison of volumetric ethylene production rates; b comparison of biomass specific ethylene production rates; c typical growth curves of the wild-type and ethylene producing Synechocystis strains. The genetic makeup of the VPV strains is detailed in Table 1. Error bars indicate standard deviation for triplicate measurements

Fig. 2

Rates of ethylene production form Synechocystis strains expressing the efe gene under control of in-tandem promoters. a Comparison of the volumetric ethylene production rate; b comparison of the biomass-specific rate of ethylene production. Error bars indicate standard deviation for triplicate measurements

Fig. 3

Comparison of rate of ethylene production between wild-type Synechocystis, expressing efe from a plasmid (VPV55) and as chromosomal integration (VPV3), and comparison with the corresponding ∆glgC strains (VPV56 and VPV65). a Biomass specific production rates; b typical growth curves of ethylene producing Synechocystis strains mentioned above in comparison to their wild-type. Error bars in a indicate standard deviation for triplicate measurements

Fig. 4

Comparison of ethylene production in wild type and in a ∆glgC strain, with a plasmid- and a chromosome-incorporated efe gene. a Comparison of Biomass-specific ethylene production rates between wild-type Synechocystis strains expressing the efe gene from a plasmid (VPV55) or as a chromosomal integration construct (VPV3), in comparison to the glycogen-synthesis deficient Synechocystis strains expressing the efe gene on a plasmid (VPV56) or as a chromosomal integration construct (VPV65) under nitrogen limitation conditions, induced by growing the cells in nitrogen-restricted medium. b 2-Oxoglutarate measured in the supernatants of the ethylene-producing strains after measurement of the rate of ethylene-production. c Typical growth data of the mutant strains during the period in which the ethylene production experiments were conducted. Error bars indicate standard deviation for triplicate measurements for (a) and standard deviation for duplicate measurements for (b)

Fig. 5

Ethylene production and growth with aerated batch cultivation. Volumetric ethylene production (filled triangles, levels indicated on the left y-axis) and OD₇₃₀ (filled triangles, levels indicated on the right y-axis) plotted against time after inoculation. Error bars indicate standard deviation for triplicate measurements. Lines in the figure serve as a guide to the eye