Construction of an artificial consortium of Escherichia coli and cyanobacteria for clean indirect production of volatile platform hydrocarbons from CO2

Abstract

Ethylene and isoprene are essential platform chemicals necessary to produce polymers and materials. However, their current production methods based on fossil fuels are not very efficient and result in significant environmental pollution. For a successful transition more sustainable economic model, producing these key polymeric building blocks from renewable and sustainable resources such as biomass or CO₂ is essential. Here, inspired by the symbiotic relationship of natural microbial communities, artificial consortia composed of E. coli strains producing volatile platform chemicals: ethylene and isoprene and two strains of cyanobacteria phototrophically synthesizing and exporting sucrose to feed these heterotrophs were developed. Disaccharide produced by transgenic cyanobacteria was used as a carbon and electron shuttle between the two community components. The E. coli cscB gene responsible for sucrose transport was inserted into two cyanobacterial strains, Thermosynechococcus elongatus PKUAC-SCTE542 and Synechococcus elongatus PCC7942, resulting in a maximal sucrose yield of 0.14 and 0.07 g/L, respectively. These organisms were co-cultured with E. coli BL21 expressing ethylene-forming enzyme or isoprene synthase and successfully synthesized volatile hydrocarbons. Productivity parameters of these co-cultures were higher than respective transgenic cultures of E. coli grown individually at similar sucrose concentrations, highlighting the positive impact of the artificial consortia on the production of these platform chemicals.

Keywords: co-culture; cyanobacteria; ethylene; isoprene; microbial community; sucrose.

Figure 1

Schematic diagram of the artificial consortium system used in this study. Thermosynechococcus E542 and Synechococcus PCC7942 engineered to secrete sucrose under osmotic stress to support the growth of transgenic Escherichia coli strains capable of the synthesis of ethylene or isoprene. CO₂, carbon dioxide; HCO₃⁻, bicarbonate; G3P, glyceraldehyde 3-phosphate; GLC, glucose; FRU, fructose; PYR, pyruvate; UDP-Glc, uridine diphosphate glucose; F6P, fructose 6-phosphate; SUC, sucrose; Ac-CoA, acetyl CoA; CIT, citrate; AKG, alpha ketoglutarate; SUC, succinate; OAA, oxaloacetate; L-Glu, glutamate; L-Arg, L-Arginine; ETH, ethylene; DMAPP, dimethylallyl pyrophosphate; ISP, isoprene; ATP, adenosine triphosphate; ADP, adenosine diphosphate; NADP⁺, oxidized nicotinamide adenine dinucleotide phosphate; NADPH, reduced nicotinamide adenine dinucleotide phosphate; CscB, sucrose permease; CscA, sucrose hydrolase; IspS, isoprene synthase; and efe, ethylene forming enzyme.

Figure 2

The comparison of sucrose yield of the PCC7942_cscB⁺ (A), E542_cscB⁺ (D), accumulated dry weight (B,E), and sucrose to biomass ratio (C,F) grown under different light and osmotic pressure in BG11 growth medium. Height of the bars shows the mean of three independent experiments with error bars representing SD from measurements.

Figure 3

The comparison of sucrose yield (A), and growth curve (B), of engineered E542_cscB⁺ and 7,942_cscB⁺ strains at different temperatures grown in BG-11 medium under salt stress. Distribution of the data points shows the mean of three independent experiments with error bars representing SD from measurements.

Figure 4

Changes of gene expression of BL21_efe-PS and BL21_IspS_EG⁺ in the cultures with sucrose as the sole carbon source calculated using the relevant ratio of the mRNA value in the M9 medium to the mRNA value in the LB with 2^−ΔΔCT method (Livak and Schmittgen, 2001).

Figure 5

The comparison of sucrose yield of the engineered strains of E542_cscB⁺ and PCC7942_cscB⁺ in different co-culture media during 48-h long cultivation. Height of the bars shows the mean of three independent experiments with error bars representing SD from measurements.