title: Quantitative proteomics analysis of an ethanol- and a lactate-producing mutant strain of Synechocystis sp. PCC6803.
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.
  
  
  CONCLUSIONS
  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.
authors.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.
authors.forename: Orawan
authors.initials: O
authors.lastname: Borirak
authors.affiliation: Mass Spectrometry of Biomacromolecules, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Leo J
authors.initials: LJ
authors.lastname: de Koning
authors.affiliation: Photanol B.V., Science Park 408, 1098 XH Amsterdam, The Netherlands.
authors.forename: Aniek D
authors.initials: AD
authors.lastname: van der Woude
authors.affiliation: Biosystems Data Analysis, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Huub C J
authors.initials: HC
authors.lastname: Hoefsloot
authors.affiliation: Mass Spectrometry of Biomacromolecules, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Henk L
authors.initials: HL
authors.lastname: Dekker
authors.affiliation: Mass Spectrometry of Biomacromolecules, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Winfried
authors.initials: W
authors.lastname: Roseboom
authors.affiliation: Mass Spectrometry of Biomacromolecules, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Chris G
authors.initials: CG
authors.lastname: de Koster
authors.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.
authors.forename: Klaas J
authors.initials: KJ
authors.lastname: Hellingwerf
keywords: Alcohol dehydrogenase; CRISPR/CAS system; Lactate dehydrogenase; Metabolism; Optimization of product formation; Pyruvate decarboxylase; Synthetic biology