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

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. This approach facilitated a ~ 23-fold increase in citramalate titre from initial unoptimised experiments. 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. We believe the decrease in productivity from 2-L to 5-L scale was likely due to the increased pathlength and shading for light delivery reducing incident light per cell. However, changes in productivity and growth characteristics are not uncommon when scaling up biotechnology processes and have numerous potential causes.

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. This is the first study using design of experiment to optimise overall carbon fixation rate and carbon partitioning to product, with the goal of improving the performance of a cyanobacterium as a host for biological carbon capture.

Keywords: Synechocystis; Carbon capture; Citramalate; Cyanobacteria; Methyl methacrylate; Photosynthesis.

Fig. 1

The reaction catalysed by citramalate synthase

Fig. 2

DOE endpoints, A a scatter plot and B a box plot, showing the final citramalate concentration, OD 720 and OD 680 nm of the 24, 2-L, reactors. C Principal component analysis displayed as a biplot. Blue light white light and CO₂ are the main factors correlating with citramalate titre whilst nitrate and phosphate correlate with growth. D A scatter plot of the OD 720 nm and titre of each individual vessel

Fig. 3

Reproducing the optimum citramalate titre from the DOE. These plots represent duplicates of the experimental maximum citramalate titre from the DOE (R3V5) in 5-L photobioreactors (5L1 and 5L2) both with 8% CO₂ (8% v (CO₂)/92% v (air)), 400 mmol (photon) m⁻² s⁻¹ blue and 400 mmol (photon) m⁻² s⁻¹ white light irradiation, 0.076 mM K₂HPO₄ and 52.8 mM NaNO₃. A A scatter plot of the of the OD 720 nm (A.U) and titre (g/L) from the most productive experimental DOE condition (R3V5) and repeats treated as a triplicate with the mean plotted and error bars showing 1 SD. B The same data split by individual photobioreactor experiment not as a triplicate, until ~ 80 h all 3 growth and production trends were similar, after ~ 80 h the repeats in BioFlo120 produced less citramalate than the DOE vessel. These three photobioreactors were started independently from independent seed cultures with OD 720 nm measurements as follows R3V5, seed = 3.572, starting = 0.261, 5L1, seed = 3.811, starting = 0.356, 5L2, seed = 2.164, starting = 0.174

Fig. 4

Predicted data from the DOE model vs DOE experimental citramalate titre data, A linear fit on all DOE data points (yellow), DOE data (yellow) including repeats of the maximum (blue) in 2 L (n = 3) and 5 L (n = 2) vessels to experimentally validate the prediction. A linear relationship between predicted and experimental citramalate titre indicates the model is in agreement with the experimental findings. There is a clear linear relationship between predicted and experimental citramalate titre between 0 and 3 g/L, there is a plateau at ~ 3–5 g/L therefore, a less clear linear relationship, and a decrease after ~ 5 g/L giving a non-linear relationship, suggesting the predicted citramalate titre and experimental titre are similar, in agreement, and thus the model is accurate at predicting titres up to ~ 3 g/L. The second linear fit (blue) on the repeat and DOE data points excluding the highest 2 points from the DOE with a much-improved fit vs panel A. B A repeat of the minimum citramalate titre prediction (similar to DOE vessel R2V3) citramalate titre (orange) and OD 720 nm (blue) displayed in a scatter plot with error bars representing 1 SD. C A repeat of the median citramalate titre prediction (similar to DOE vessel R4V7), and D) a repeat of the maximum citramalate titre prediction (similar to DOE vessel R3V5) both in a similar scatter plot

Fig. 5

Carbon partitioning across the 24 vessels of the DOE. A Rate of carbon fixed as biomass (blue) and citramalate (orange) per day and B relative carbon partitioning. R1-4 corresponds to the run and V1-8 corresponds to the vessel, on the far right 5 L corresponds to repeats of the DOE maxima in BioFlo120 (n = 2) and 0.5L corresponds to initial experiments in the FMT150 prior to the DOE (n = 5). Citramalate represents the major constituent peak in the HPLC data, it is possible other metabolites are released from the cell into the culture medium that were not detected by our HPLC method and were not accounted for in the carbon partitioning calculation