Additional file 1 of Improving productivity of citramalate from CO2 by Synechocystis sp. PCC 6803 through design of experiment
Creators: name: Faulkner, Matthew
name: Andrews, Fraser
name: Scrutton, Nigel
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Genetics
subject: FOS: Biological sciences
subject: Biotechnology
subject: Environmental Sciences not elsewhere classified
subject: Ecology
subject: Biological Sciences not elsewhere classified

description: Additional file 1: Supplementary Fig. 1: Plasmid maps for the two versions of pAM2991 used in this study created in SnapGene. Supplementary Fig. 2: DNA electrophoresis agarose gel images. Lanes that were loaded are numbered in red along the top, empty lanes are unnumbered. A) The sizes and masses of the characteristic bands of Quick-Load 1 kb DNA Ladder, 10 μL of which was loaded in lane 1 in all gel images. B) An agarose gel with samples from colony PCR to confirm the presence of cimA. Boil preps of whole Synechocystis cells were used as template with the cimA_screen_F &amp; cimA_screen_R primer pair giving an expected size of 1387 bp. Lanes 2–9 of the top gel have independent colonies from BG11 spectinomycin plates used as template, the bottom gel lanes 2–3 has a positive control of pure pAM2991 prior to transformation used as template, lanes 4–5 has a negative control of untransformed Synechocystis stock used for the natural transformation. C) An agarose gel with samples from colony PCR to confirm the presence of the chloramphenicol resistance marker, all amplified with the leuC_screen_F &amp; leuC_screen_R primer pair expected size of 2896 bp. Lane 2 holds a positive control of the pure pAM2991_ΔleuC plasmid, lanes 3–5 hold colony PCRs from independent colonies of Synechocystis transformed with pAM2991_ΔleuC on a BG11 chloramphenicol plate, lane 6 holds a negative control lanes of untransformed Synechocystis, lanes 7–9 hold colony PCR samples from the same 3 independent colonies after replica plating onto a BG11 chloramphenicol and spectinomycin plate. D) An agarose gel confirming the insertion of the chloramphenicol resistance marker into the Synechocystis genome. Isolated gDNA was used as template for amplification with the leuC_genome_F &amp; leuC_genome_R primer pair expected size for WT is 5328 bp and ΔleuC is 4581 bp. E) An agarose gel showing colony PCRs on whole cell boil prep samples, lane 2 holds a negative control of untransformed Synechocystis with both primer pairs, lanes 3–6 use the same colonies from lanes 6–7 in, lanes 3–4 used the primer pair leuC_screen_F &amp; cmR_internal_R expected size 1130 bp, lanes 5–6 used the primer pair leuC_screen_R &amp; cmR_internal_F expected size 1230 bp. Supplementary Fig. 3: Consensus genome sequences from Oxford Nanopore dataof Synechocystis_WTprior to any of the changes made and Synechocystis_ΔleuC_pAM2991_cimA as used for production of citramalate from this study mapped to NCBI GCF_000009725.1 as a reference genome, created in SnapGene. Both consensus genomes contain leuC 100% identity match to the reference when aligned using Blastn indicating the predominant population is WT. Neither genome contains any significant homology to pAM2991cimA when attempting to align with Blastn, indicating the cimA expression plasmid has not integrated into the genome of the production strain. There is a slight decrease in coverage and slight indication of contamination in the Synechocystis_ΔleuC_pAM2991_cimA perhaps indicating the mixed population between ΔleuC and WT facilitating the knockdown of leuC. The raw sequence read data are available in fastq format as additional files 2 &amp; 3 on OSF https://osf.io/ay52x/ and on Figshare https://doi.org/10.6084/m9.figshare.27170022 . Supplementary Fig. 4: Typical UV–vis Diode Array Detectorand Refractive Index Detectorplots and a standard curve for the quantification of citramalate by HPLC. A) An example standard curve 0.1–10 g/L from one citramalate quantification by HPLC, a similar standard curve was generated alongside each batch of samples quantified. B) A typical RID trace from an experimental sample of Synechocystis in BG11, the peak at 10.699 min is citramalate, the peak at 7.322 is also present in BG11 only control samples. C) A typical DAD trace from an experimental sample of Synechocystis in BG11. Citramalate has no UV absorption so gives signal, there are no peaks other than the one at 6.975 min which is also present in BG11 only control samples. D) A typical RID trace from a standard curve sample of 10 g/L in BG11, the peak at 10.487 min is citramalate. E) A typical DAD trace from a standard curve sample of 10 g/L in BG11. Citramalate has no UV absorption so gives signal, there are no peaks other than the one at 6.823 min which is also present in BG11 only control samples. Supplementary Fig. 5: A box plot of the OD 680 nm &amp; 720 nm, growth, and citramalate titre of the strains tested in this study. Endpoint OD and HPLC measurements after 96 h 10 ml culture in 25 ml Nunc EasY flasks at 34 °C agitating at 140 RPM with 40 µmol photons m−2 s−1 warm white light, in BG11 medium. 5 independent colonies of wild typeand ΔleuC, with and without pAM2991CimA, were assayed for citramalate production under standard photosynthetic growth conditions. We observed citramalate production by all of sub-strains with pAM2991CimA, Synechocystis_ΔleuC_pAM2991CimA accumulated the most citramalate, significantly more than Synechocystis_WT_ pAM2991CimA by two-tailed T-test assuming equal variance. We did not observe citramalate accumulation above the detection limit of our HPLC method in controls, strains with no CimA or strains with an inactive CimA variant [1] insert in pAM2991. Without any functional LeuC, Synechocystis should be a leucine auxotroph. We did not observe leucine auxotrophy in ΔleuC genotypes, nor did we observe fully segregated knock outs, our strain appears to be heterogenous for WT and knock out despite repeated subculturing on selective media. Synechocystis_ ΔleuC was not segregated with leucine supplementation thus full segregation would be fatal; Synechocystis is polyploid [2] thus the ΔleuC was knocked-down not knocked-out. This ΔleuC knock-down improved citramalate accumulation without impacting upon growth. Optimisation of the ratio of WT to ΔleuC and thus the strength of the knock down was outside the scope of this study. Supplementary Fig. 6: Endpoint growth measurementsafter 96 h with citramalate supplemented, left final OD 720 nmand right responseto citramalate supplementation EC50 or 50% decrease in growth is indicated by the red line. The tolerance of each of the sub-strains to exogenous citramalate was determined by measuring growth over 96 h in BG11 supplemented with citramalate 0 – 25 g/L. EC50 was ~ 11.93 g/L. There was no discernible difference in the growth of any sub-strain with citramalate supplements 0–5 g/L. Supplementation of citramalate &gt; 10 g/L began to markedly reduce growth of all sub-strains, and no growth was observed in any sub-strain with &gt; 20 g/L, with no significant difference between OD 720 nm at inoculation and after 96 h for any strainby two-tailed T-test assuming equal variance. Supplementary Fig. 7: The growth in blue OD 720 nm, and citramalate production in orange, curves for all 24 vessels in the DOE. Each plot is labelled top left with the run and vessel ID, for example run 1 vessel 1 is R1V1. The exact condition for each vessel can be found by cross-referencing with Table 1. In the literature where productivity was assessed over time it appears to start high and decrease over time. For example, Namakoshi et al. [3] reported 2010 mg L−1 day−1 was reported for the first 24 h that decreased over time, over 72 h the rate was 986.6 mg L−1 day−1. We have observed a similar trend with an initial maximal rate soon after changing conditions evident in most vessels of the DOE, that also tends to decrease over time. Highly productive cultures were also beginning to lose cell viability. Aliquots taken at 96 h from R3V2, R3V3, R3V5, and R3V6 showed little to no growth when spotted onto BG11 agar plates. Similar tests from all other vessels gave clear growth without signs of contamination. Supplementary Fig. 8: Contour plots showing citramalate titre for each possible iteration of 2-factor interaction between CO2, blue light, white light, phosphate, nitrate, and OD 720 nm. There are clear minima and maxima and clear interactions between some factors. There is a particularly strong correlation between growth and citramalate titre as indicated by the tight maxima around OD 720 nm ~ 3 in all contour plots. There are clear optima maxima for example when blue and/or white light is high around high CO2 ~ 8%, low phosphate ~ 0.2 or 1/5th the concentration of BG 11 medium, low nitrate ~ 0.5–1 or 8.8–17.6 mM. Some plots have no clear maxima show no correlation between the two variables and titre, such as the plot of phosphate, nitrate, and citramalate titre. From these contour plots we can conclude that there are complex multi-factor interactions determining citramalate titre and no single variable tested is determinant for the observed titres. Supplementary Fig. 9: A summary of the models tested in JMP and the weighting of the DOE factors in the equation of the support vector machines model given as ‘T#6’. We made a predictive model to better examine the complex nature of the multi-factor interactions determining citramalate titre, and to extrapolate from the conditions tested experimentally. The experimental data were fitted to all the models available in JMPusing default parameters, excluding any that fit only when excluding 1 or more of our 24 data points as outliers. The support vector machines had the highest R2, 0.4884, and the best fit for a predicted vs actual citramalate titre plot. Supplementary Table 1: Primers used in this study. Supplementary Table 2: Plasmid sequences, these sequences were confirmed on plasmids isolated from Synechocystis using the “standard high concentration plasmid” sequencing service performed by Plasmidsaurus using Oxford Nanopore Technology with custom analysis and annotation.