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

Biotechnology for Biofuels

Orawan Borirak³  Leo J de Koning⁴  Aniek D van der Woude²  Huub C J Hoefsloot¹  Henk L Dekker⁴  Winfried Roseboom⁴  Chris G de Koster⁴  Klaas J Hellingwerf³ 

[1] Biosystems Data Analysis, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands

[2] Photanol B.V., Science Park 408, Amsterdam, 1098 XH, The Netherlands

[3] Molecular Microbial Physiology, Swammerdam Institute for Life Sciences, and Netherlands Institute for System Biology, University of Amsterdam, Science Park 904, Amsterdam, 1098 XH, The Netherlands

[4] Mass Spectrometry of Biomacromolecules, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands

关键词: Optimization of product formation;    CRISPR/CAS system;    Lactate dehydrogenase;    Alcohol dehydrogenase;    Pyruvate decarboxylase;    Metabolism;    Synthetic biology;   

Others  :  1225776
DOI  :  10.1186/s13068-015-0294-z

 received in 2015-03-10, accepted in 2015-07-24,  发布年份 2015

【 摘 要 】

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 two¹⁴ N-labelled Synechocystis mutant strains, relative to their¹⁵ 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 CO ₂in 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 CO ₂fixation 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 CO ₂fixation 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.

【 授权许可】

   
2015 Borirak et al.

【 图 表 】

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