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Quantitative proteomics analysis of an ethanol- and a lactate-producing mutant strain of Synechocystis sp. PCC6803.

Borirak O, de Koning LJ, van der Woude AD, Hoefsloot HC, Dekker HL, Roseboom W, de Koster CG, Hellingwerf KJ - Biotechnol Biofuels (2015)

Bottom Line: Also a general decrease in abundance of the protein synthesizing machinery of the cells and a specific induction of an oxidative stress response were observed in this mutant.In the lactic acid overproducing mutant, that expresses part of the heterologous l-lactate dehydrogenase from a self-replicating plasmid, specific activation of two CRISPR associated proteins, encoded on the endogenous pSYSA plasmid, was observed.For selected, limited, number of genes a striking correlation between the respective mRNA- and the corresponding protein expression level was observed, suggesting that for the expression of these genes regulation takes place primarily at the level of gene transcription.

View Article: PubMed Central - PubMed

Affiliation: Molecular Microbial Physiology, Swammerdam Institute for Life Sciences, and Netherlands Institute for System Biology, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands.

ABSTRACT

Background: This study aimed at exploring the molecular physiological consequences of a major redirection of carbon flow in so-called cyanobacterial cell factories: quantitative whole-cell proteomics analyses were carried out on two (14)N-labelled Synechocystis mutant strains, relative to their (15)N-labelled wild-type counterpart. Each mutant strain overproduced one specific commodity product, i.e. ethanol or lactic acid, to such an extent that the majority of the incoming CO2 in the organism was directly converted into the product.

Results: In total, 267 proteins have been identified with a significantly up- or down-regulated expression level. In the ethanol-producing mutant, which had the highest relative direct flux of carbon-to-product (>65%), significant up-regulation of several components involved in the initial stages of CO2 fixation for cellular metabolism was detected. Also a general decrease in abundance of the protein synthesizing machinery of the cells and a specific induction of an oxidative stress response were observed in this mutant. In the lactic acid overproducing mutant, that expresses part of the heterologous l-lactate dehydrogenase from a self-replicating plasmid, specific activation of two CRISPR associated proteins, encoded on the endogenous pSYSA plasmid, was observed. RT-qPCR was used to measure, of nine of the genes identified in the proteomics studies, also the adjustment of the corresponding mRNA level.

Conclusion: The most striking adjustments detected in the proteome of the engineered cells were dependent on the specific product formed, with, e.g. more stress caused by lactic acid- than by ethanol production. Up-regulation of the total capacity for CO2 fixation in the ethanol-producing strain was due to hierarchical- rather than metabolic regulation. Furthermore, plasmid-based expression of heterologous gene(s) may induce genetic instability. For selected, limited, number of genes a striking correlation between the respective mRNA- and the corresponding protein expression level was observed, suggesting that for the expression of these genes regulation takes place primarily at the level of gene transcription.

No MeSH data available.


Related in: MedlinePlus

Growth, product formation, and calculated total rates of carbon fixation of the two product-forming mutants, compared to the wild-type strain Synechocystis sp. PCC6803. a Growth and product formation of the wild-type (WT) and the two product-forming strains (SAA012 and SAW041). b Total carbon fixation rate of SAA012, SAW041, and the corresponding WT Synechocystis strain plotted against the cell density of the culture. Data are mean ± SD of three biological replicates.
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Fig1: Growth, product formation, and calculated total rates of carbon fixation of the two product-forming mutants, compared to the wild-type strain Synechocystis sp. PCC6803. a Growth and product formation of the wild-type (WT) and the two product-forming strains (SAA012 and SAW041). b Total carbon fixation rate of SAA012, SAW041, and the corresponding WT Synechocystis strain plotted against the cell density of the culture. Data are mean ± SD of three biological replicates.

Mentions: The two product-forming strains both grew considerably slower than the wild-type (WT) strain, which was to be expected in view of the large amount of carbon directly channeled into product. Maximum specific growth rates (µ) of SAA012 and SAW041 were reduced to 71 and 58% of the value of WT, respectively. The ethanol production observed in SAA012 was 9.88 ± 0.16 mM over 19 days, representing a maximum production rate of 1.33 ± 0.12 mmol l−1 day−1, while in the same period the lactic acid accumulated up to 6.19 ± 0.12 mM, with a rate of 0.36 ± 0.01 mmol l−1 day−1 (see Fig. 1a). From these physiological data the total carbon fixation rate (qCO2) was calculated (i.e. for conversion into biomass and into product). Due to the differences in growth rate between the strains (and the consequences this has on the degree of light saturation in the cultures), the qCO2 is here plotted as a function of biomass density in gDW l−1 (Fig. 1b). During exponential growth of the cells, when OD730 < 1 (i.e. up to approximately 0.2 gDW l−1), SAA012 exhibits a higher qCO2 than both WT and SAW041. The average increase in the qCO2 calculated was ~1.5-fold higher than the rate of CO2 fixation in the WT. In contrast, the lactic acid-forming strain SAW041 showed a small decrease in overall qCO2 at this cell density.Fig. 1


Quantitative proteomics analysis of an ethanol- and a lactate-producing mutant strain of Synechocystis sp. PCC6803.

Borirak O, de Koning LJ, van der Woude AD, Hoefsloot HC, Dekker HL, Roseboom W, de Koster CG, Hellingwerf KJ - Biotechnol Biofuels (2015)

Growth, product formation, and calculated total rates of carbon fixation of the two product-forming mutants, compared to the wild-type strain Synechocystis sp. PCC6803. a Growth and product formation of the wild-type (WT) and the two product-forming strains (SAA012 and SAW041). b Total carbon fixation rate of SAA012, SAW041, and the corresponding WT Synechocystis strain plotted against the cell density of the culture. Data are mean ± SD of three biological replicates.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4526308&req=5

Fig1: Growth, product formation, and calculated total rates of carbon fixation of the two product-forming mutants, compared to the wild-type strain Synechocystis sp. PCC6803. a Growth and product formation of the wild-type (WT) and the two product-forming strains (SAA012 and SAW041). b Total carbon fixation rate of SAA012, SAW041, and the corresponding WT Synechocystis strain plotted against the cell density of the culture. Data are mean ± SD of three biological replicates.
Mentions: The two product-forming strains both grew considerably slower than the wild-type (WT) strain, which was to be expected in view of the large amount of carbon directly channeled into product. Maximum specific growth rates (µ) of SAA012 and SAW041 were reduced to 71 and 58% of the value of WT, respectively. The ethanol production observed in SAA012 was 9.88 ± 0.16 mM over 19 days, representing a maximum production rate of 1.33 ± 0.12 mmol l−1 day−1, while in the same period the lactic acid accumulated up to 6.19 ± 0.12 mM, with a rate of 0.36 ± 0.01 mmol l−1 day−1 (see Fig. 1a). From these physiological data the total carbon fixation rate (qCO2) was calculated (i.e. for conversion into biomass and into product). Due to the differences in growth rate between the strains (and the consequences this has on the degree of light saturation in the cultures), the qCO2 is here plotted as a function of biomass density in gDW l−1 (Fig. 1b). During exponential growth of the cells, when OD730 < 1 (i.e. up to approximately 0.2 gDW l−1), SAA012 exhibits a higher qCO2 than both WT and SAW041. The average increase in the qCO2 calculated was ~1.5-fold higher than the rate of CO2 fixation in the WT. In contrast, the lactic acid-forming strain SAW041 showed a small decrease in overall qCO2 at this cell density.Fig. 1

Bottom Line: Also a general decrease in abundance of the protein synthesizing machinery of the cells and a specific induction of an oxidative stress response were observed in this mutant.In the lactic acid overproducing mutant, that expresses part of the heterologous l-lactate dehydrogenase from a self-replicating plasmid, specific activation of two CRISPR associated proteins, encoded on the endogenous pSYSA plasmid, was observed.For selected, limited, number of genes a striking correlation between the respective mRNA- and the corresponding protein expression level was observed, suggesting that for the expression of these genes regulation takes place primarily at the level of gene transcription.

View Article: PubMed Central - PubMed

Affiliation: Molecular Microbial Physiology, Swammerdam Institute for Life Sciences, and Netherlands Institute for System Biology, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands.

ABSTRACT

Background: This study aimed at exploring the molecular physiological consequences of a major redirection of carbon flow in so-called cyanobacterial cell factories: quantitative whole-cell proteomics analyses were carried out on two (14)N-labelled Synechocystis mutant strains, relative to their (15)N-labelled wild-type counterpart. Each mutant strain overproduced one specific commodity product, i.e. ethanol or lactic acid, to such an extent that the majority of the incoming CO2 in the organism was directly converted into the product.

Results: In total, 267 proteins have been identified with a significantly up- or down-regulated expression level. In the ethanol-producing mutant, which had the highest relative direct flux of carbon-to-product (>65%), significant up-regulation of several components involved in the initial stages of CO2 fixation for cellular metabolism was detected. Also a general decrease in abundance of the protein synthesizing machinery of the cells and a specific induction of an oxidative stress response were observed in this mutant. In the lactic acid overproducing mutant, that expresses part of the heterologous l-lactate dehydrogenase from a self-replicating plasmid, specific activation of two CRISPR associated proteins, encoded on the endogenous pSYSA plasmid, was observed. RT-qPCR was used to measure, of nine of the genes identified in the proteomics studies, also the adjustment of the corresponding mRNA level.

Conclusion: The most striking adjustments detected in the proteome of the engineered cells were dependent on the specific product formed, with, e.g. more stress caused by lactic acid- than by ethanol production. Up-regulation of the total capacity for CO2 fixation in the ethanol-producing strain was due to hierarchical- rather than metabolic regulation. Furthermore, plasmid-based expression of heterologous gene(s) may induce genetic instability. For selected, limited, number of genes a striking correlation between the respective mRNA- and the corresponding protein expression level was observed, suggesting that for the expression of these genes regulation takes place primarily at the level of gene transcription.

No MeSH data available.


Related in: MedlinePlus