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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

Predicted interaction of differentially expressed proteins identified in the lactate-producing mutant, SAW041. Protein interaction was analyzed and reconstructed using STRING and Cytoscape, respectively. a Up-regulated proteins (α < 0.01). b Down-regulated proteins (α < 0.01).
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Fig5: Predicted interaction of differentially expressed proteins identified in the lactate-producing mutant, SAW041. Protein interaction was analyzed and reconstructed using STRING and Cytoscape, respectively. a Up-regulated proteins (α < 0.01). b Down-regulated proteins (α < 0.01).

Mentions: The distribution of the normalized protein abundance of the three biological replicates of WT, SAA012, and SAW041 is depicted in Fig. 2. As shown, the protein isotopic ratio of the WT is normally distributed around 1, further emphasizing the reproducibility of the measurements with the reference- and the WT cultures. This allows calculation of significant changes in relative protein expression level of the two product-forming strains, by using the z test and the protein ratio distribution of the WT as a reference, followed by Bonferroni correction. This resulted in significance thresholds for the two product-forming strains as described in: “Methods”; “Statistical analysis of the protein quantifications”. Using these boundary conditions a total number of 168 and 153 proteins, respectively, showed a significantly altered abundance in mutant strains SAA012 and SAW041. The two lists of these proteins, including their calculated p value, can be found in Additional file 1: Tables S4, S5, respectively. The numbers of differentially expressed proteins listed in these tables are visualized in Fig. 3. Furthermore, to establish the connection between the up-regulated proteins and the down-regulated proteins of the two product-forming strains, STRING v9.1 [34] plus KEGG pathway [35] enrichment analysis was used to predict the underlying protein interaction network. The protein interaction networks that have resulted from this analysis were reconstructed by Cytoscape v3.1.1.1 [36], and are shown in Figs. 4 and 5, for strain SAA012 and SAW041, respectively. The results of the KEGG pathway enrichment analyses showed that in SAA012, the up-regulated pathways were syn00710—carbon fixation in photosynthetic organisms, and syn00480—glutathione metabolism, while the down-regulated pathways found were syn03010—ribosome, and syn00196—photosynthesis-antenna proteins (p < 0.05). In contrast to SAA012, the enrichment analysis of SAW041 did not reveal specific up-regulated pathways, but only down-regulated pathways. Amongst others, photosynthesis (syn00195), photosynthesis-antenna proteins (syn00196), and glycolysis/gluconeogenesis (syn00010) were identified. An overview of the results of the KEGG pathway enrichment analyses of the two mutants is provided in Additional file 1: Table S6. Some of the observed proteins with significantly altered abundances are discussed in more detail below.Fig. 2


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)

Predicted interaction of differentially expressed proteins identified in the lactate-producing mutant, SAW041. Protein interaction was analyzed and reconstructed using STRING and Cytoscape, respectively. a Up-regulated proteins (α < 0.01). b Down-regulated proteins (α < 0.01).
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: Predicted interaction of differentially expressed proteins identified in the lactate-producing mutant, SAW041. Protein interaction was analyzed and reconstructed using STRING and Cytoscape, respectively. a Up-regulated proteins (α < 0.01). b Down-regulated proteins (α < 0.01).
Mentions: The distribution of the normalized protein abundance of the three biological replicates of WT, SAA012, and SAW041 is depicted in Fig. 2. As shown, the protein isotopic ratio of the WT is normally distributed around 1, further emphasizing the reproducibility of the measurements with the reference- and the WT cultures. This allows calculation of significant changes in relative protein expression level of the two product-forming strains, by using the z test and the protein ratio distribution of the WT as a reference, followed by Bonferroni correction. This resulted in significance thresholds for the two product-forming strains as described in: “Methods”; “Statistical analysis of the protein quantifications”. Using these boundary conditions a total number of 168 and 153 proteins, respectively, showed a significantly altered abundance in mutant strains SAA012 and SAW041. The two lists of these proteins, including their calculated p value, can be found in Additional file 1: Tables S4, S5, respectively. The numbers of differentially expressed proteins listed in these tables are visualized in Fig. 3. Furthermore, to establish the connection between the up-regulated proteins and the down-regulated proteins of the two product-forming strains, STRING v9.1 [34] plus KEGG pathway [35] enrichment analysis was used to predict the underlying protein interaction network. The protein interaction networks that have resulted from this analysis were reconstructed by Cytoscape v3.1.1.1 [36], and are shown in Figs. 4 and 5, for strain SAA012 and SAW041, respectively. The results of the KEGG pathway enrichment analyses showed that in SAA012, the up-regulated pathways were syn00710—carbon fixation in photosynthetic organisms, and syn00480—glutathione metabolism, while the down-regulated pathways found were syn03010—ribosome, and syn00196—photosynthesis-antenna proteins (p < 0.05). In contrast to SAA012, the enrichment analysis of SAW041 did not reveal specific up-regulated pathways, but only down-regulated pathways. Amongst others, photosynthesis (syn00195), photosynthesis-antenna proteins (syn00196), and glycolysis/gluconeogenesis (syn00010) were identified. An overview of the results of the KEGG pathway enrichment analyses of the two mutants is provided in Additional file 1: Table S6. Some of the observed proteins with significantly altered abundances are discussed in more detail below.Fig. 2

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