Abstract
BackgroundThis 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 14N-labelled Synechocystis mutant strains, relative to their 15N-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.ResultsIn 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.ConclusionThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0294-z) contains supplementary material, which is available to authorized users.
Highlights
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 14N-labelled Synechocystis mutant strains, relative to their 15N-labelled wild-type counterpart
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
Due to the differences in growth rate between the strains, the qCO2 is here plotted as a function of biomass density in gDW l−1 (Fig. 1b)
Summary
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 14N-labelled Synechocystis mutant strains, relative to their 15N-labelled wild-type counterpart. Whereas the first generation approach would use key products from the food sector, like sucrose or starch, to produce ethanol [8, 15], in the fourth generation approach— referred to as ‘direct conversion’—one aims at the direct conversion of CO2 into the desired product, without the need to form biomass (and all the minerals required to form it) as an obligatory intermediate [6, 7, 9,10,11] This latter approach preferably uses cyanobacteria, to engineer them towards highly efficient ‘production systems’ for the desired product, because these organisms combine the highest efficiency in oxygenic photosynthesis [16] with straightforward application of synthetic systems biology, both in terms of molecular genetic intervention and in terms of the required computational analyses (e.g. review [17]). The product titre may increase to levels that change the physico-chemical conditions of the growth medium and/or the cell, such that it causes growth inhibition
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
