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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

Altered mRNA- and protein expression level of selected genes of the two product-forming mutants, SAA012 and SAW041, relative to the WT strain. a Relative fold change of mRNA and protein level of the selected genes. Data are mean ± SEM of 3 biological- and 3 technical-replicates. Genes that were changed significantly in their level of expression are indicated with an asterisk. b Changes in relative protein abundance of the selected set of genes. Proteins with significantly changed expression level are marked with an asterisk (α < 0.01). Data are mean ± SEM of at least two biological replicates. Proteins that are shown without error bars, i.e. GabD in SAA012 and KaiA in SAW041, were quantified in only one biological replicate.
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Fig6: Altered mRNA- and protein expression level of selected genes of the two product-forming mutants, SAA012 and SAW041, relative to the WT strain. a Relative fold change of mRNA and protein level of the selected genes. Data are mean ± SEM of 3 biological- and 3 technical-replicates. Genes that were changed significantly in their level of expression are indicated with an asterisk. b Changes in relative protein abundance of the selected set of genes. Proteins with significantly changed expression level are marked with an asterisk (α < 0.01). Data are mean ± SEM of at least two biological replicates. Proteins that are shown without error bars, i.e. GabD in SAA012 and KaiA in SAW041, were quantified in only one biological replicate.

Mentions: To verify and further characterize the significance of the quantitative proteomics results, nine genes were chosen for analysis with RT-qPCR (see Fig. 6a; “Methods”). These genes were chosen, based on their corresponding protein levels, for which a wide range of abundances was observed. This set includes genes that may play an important role in the regulation of biofuel production. Among them, three proteins did not change significantly in relative abundance, neither in SAA012, nor in SAW041 (i.e. Pgl, GabD, and Gap2). Two proteins were chosen that are similarly down-regulated (i.e. CpcG2 and Gap1) in the two mutant strains, and one protein was selected that is up-regulated in both (i.e. KaiA). Also included were CbbL and CcmK1, which were up-regulated only in SAA012, and Sll7087 (i.e. the CRISPR3-associated protein Cmr4) which was only up-regulated in SAW041.Fig. 6


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)

Altered mRNA- and protein expression level of selected genes of the two product-forming mutants, SAA012 and SAW041, relative to the WT strain. a Relative fold change of mRNA and protein level of the selected genes. Data are mean ± SEM of 3 biological- and 3 technical-replicates. Genes that were changed significantly in their level of expression are indicated with an asterisk. b Changes in relative protein abundance of the selected set of genes. Proteins with significantly changed expression level are marked with an asterisk (α < 0.01). Data are mean ± SEM of at least two biological replicates. Proteins that are shown without error bars, i.e. GabD in SAA012 and KaiA in SAW041, were quantified in only one biological replicate.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig6: Altered mRNA- and protein expression level of selected genes of the two product-forming mutants, SAA012 and SAW041, relative to the WT strain. a Relative fold change of mRNA and protein level of the selected genes. Data are mean ± SEM of 3 biological- and 3 technical-replicates. Genes that were changed significantly in their level of expression are indicated with an asterisk. b Changes in relative protein abundance of the selected set of genes. Proteins with significantly changed expression level are marked with an asterisk (α < 0.01). Data are mean ± SEM of at least two biological replicates. Proteins that are shown without error bars, i.e. GabD in SAA012 and KaiA in SAW041, were quantified in only one biological replicate.
Mentions: To verify and further characterize the significance of the quantitative proteomics results, nine genes were chosen for analysis with RT-qPCR (see Fig. 6a; “Methods”). These genes were chosen, based on their corresponding protein levels, for which a wide range of abundances was observed. This set includes genes that may play an important role in the regulation of biofuel production. Among them, three proteins did not change significantly in relative abundance, neither in SAA012, nor in SAW041 (i.e. Pgl, GabD, and Gap2). Two proteins were chosen that are similarly down-regulated (i.e. CpcG2 and Gap1) in the two mutant strains, and one protein was selected that is up-regulated in both (i.e. KaiA). Also included were CbbL and CcmK1, which were up-regulated only in SAA012, and Sll7087 (i.e. the CRISPR3-associated protein Cmr4) which was only up-regulated in SAW041.Fig. 6

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