Synechosytsis sp. PCC6803 citramalate production strains genome sequence data

Additional files containing genome sequence data for the strains constructed in this manuscript:

Improving production of citramalate from CO₂ by Synechocystis sp. PCC 6803 through design of experiment

Highlights

· Synechocystis sp. PCC 6803 produced 6.35 g/L of citramalate with a space-time yield of 1.59 g/L/day in 2 L photobioreactors, and 3.96 ± 0.23 g/L and 0.99 ± 0.06 g/L/day in 5 L photobioreactors, from carbon dioxide_.

· The application of design of experiment increased the rate of citramalate production from an initial 0.068 to optimised 1.59 g/L/day, a ~23-fold increase.

· The switch between growth and production can be controlled using process parameters in place of complex genetic circuits and chemical inducers.

· Optimised process parameters can increase overall carbon fixation rates of similar Synechocystis sp. PCC 6803 photobioreactor cultures from 0.12 g/L/day to 0.77 g/L/day.

Abstract

Background

Cyanobacteria have long been suggested as an industrial chassis for the conversion of carbon dioxide to products as part of a circular bioeconomy. The slow growth, carbon fixation rates, and limits of carbon partitioning between biomass and product in cyanobacteria must be overcome to fully realise this industrial potential. Typically, flux towards heterologous pathways is limited by the availability of core metabolites. Citramalate is produced in a single enzymatic step through the condensation of the central metabolites pyruvate and acetyl-CoA; improvements in citramalate productivity can, therefore, be used as a measure of overcoming this limitation. Furthermore, citramalate is a useful biomaterial precursor and provides a route to renewable methyl methacrylate and poly(methyl methacrylate), which is often traded as Perspex or Plexiglas.

Results

Here, we describe a phenomenon where the concerted optimisation of process parameters significantly increased citramalate production in Synechocystis sp. PCC 6803. Design of experiment principles were used to determine the optima for each parameter and the interplay between multiple parameters. The process of scale-up from batch cultures to 0.5, 2, and 5 L photobioreactors is described. At the 2 L scale, citramalate titres from carbon dioxide reached 6.35 g/L with space-time yields of 1.59 g/L/day whilst 5 L PBRs yielded 3.96 ± 0.23 g/L with a productivity of 0.99 ± 0.06 g/L/day. This approach facilitated a ~23-fold increase in citramalate titre from initial unoptimised experiments.

Conclusions

This work demonstrates that the use of a process parameter control regime can ameliorate precursor limitation and enhance citramalate production. Since pyruvate and/or acetyl-CoA give rise to numerous products of biotechnological interest, the workflow presented here could be employed to optimise flux towards other heterologous pathways. Understanding the factors controlling and thus increasing carbon partitioning to product will help progress cyanobacteria as part of a carbon-neutral circular bioeconomy.