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Engineering of a green-light inducible gene expression system in Synechocystis sp. PCC6803.

ABSTRACT: In order to construct a green-light-regulated gene expression system for cyanobacteria, we characterized a green-light sensing system derived from Synechocystis sp. PCC6803, consisting of the green-light sensing histidine kinase CcaS, the cognate response regulator CcaR, and the promoter of cpcG2 (PcpcG 2 ). CcaS and CcaR act as a genetic controller and activate gene expression from PcpcG 2 with green-light illumination. The green-light induction level of the native PcpcG 2 was investigated using GFPuv as a reporter gene inserted in a broad-host-range vector. A clear induction of protein expression from native PcpcG 2 under green-light illumination was observed; however, the expression level was very low compared with Ptrc , which was reported to act as a constitutive promoter in cyanobacteria. Therefore, a Shine-Dalgarno-like sequence derived from the cpcB gene was inserted in the 5' untranslated region of the cpcG2 gene, and the expression level of CcaR was increased. Thus, constructed engineered green-light sensing system resulted in about 40-fold higher protein expression than with the wild-type promoter with a high ON/OFF ratio under green-light illumination. The engineered green-light gene expression system would be a useful genetic tool for controlling gene expression in the emergent cyanobacterial bioprocesses.

SUBMITTER: Abe K

PROVIDER: S-EPMC3937721 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Engineering of a green-light inducible gene expression system in Synechocystis sp. PCC6803.

Abe Koichi K Miyake Kotone K Nakamura Mayumi M Kojima Katsuhiro K Ferri Stefano S Ikebukuro Kazunori K Sode Koji K

Microbial biotechnology 20131212 2

In order to construct a green-light-regulated gene expression system for cyanobacteria, we characterized a green-light sensing system derived from Synechocystis sp. PCC6803, consisting of the green-light sensing histidine kinase CcaS, the cognate response regulator CcaR, and the promoter of cpcG2 (PcpcG 2 ). CcaS and CcaR act as a genetic controller and activate gene expression from PcpcG 2 with green-light illumination. The green-light induction level of the native PcpcG 2 was investigated usin ...[more]

PMID: 24330635

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Project description:BackgroundMolecular engineering of the intermediary physiology of cyanobacteria has become important for the sustainable production of biofuels and commodity compounds from CO2 and sunlight by "designer microbes." The chemical commodity product L-lactic acid can be synthesized in one step from a key intermediary metabolite of these organisms, pyruvate, catalyzed by a lactate dehydrogenase. Synthetic biology engineering to make "designer microbes" includes the introduction and overexpression of the product-forming biochemical pathway. For further optimization of product formation, modifications in the surrounding biochemical network of intermediary metabolism have to be made.ResultsTo improve light-driven L-lactic acid production from CO2, we explored several metabolic engineering design principles, using a previously engineered L-lactic acid producing mutant strain of Synechocystis sp. PCC6803 as the benchmark. These strategies included: (i) increasing the expression level of the relevant product-forming enzyme, lactate dehydrogenase (LDH), for example, via expression from a replicative plasmid; (ii) co-expression of a heterologous pyruvate kinase to increase the flux towards pyruvate; and (iii) knockdown of phosphoenolpyruvate carboxylase to decrease the flux through a competing pathway (from phosphoenolpyruvate to oxaloacetate). In addition, we tested selected lactate dehydrogenases, some of which were further optimized through site-directed mutagenesis to improve the enzyme's affinity for the co-factor nicotinamide adenine dinucleotide phosphate (NADPH). The carbon partitioning between biomass and lactic acid was increased from about 5% to over 50% by strain optimization.ConclusionAn efficient photosynthetic microbial cell factory will display a high rate and extent of conversion of substrate (CO2) into product (here: L-lactic acid). In the existing CO2-based cyanobacterial cell factories that have been described in the literature, by far most of the control over product formation resides in the genetically introduced fermentative pathway. Here we show that a strong promoter, in combination with increased gene expression, can take away a significant part of the control of this step in lactic acid production from CO2. Under these premises, modulation of the intracellular precursor, pyruvate, can significantly increase productivity. Additionally, production enhancement is achieved by protein engineering to increase co-factor specificity of the heterologously expressed LDH.

Project description:BackgroundNADPH is used as a reductant in various biosynthetic reactions. Cell-free bio-systems have gained considerable attention owing to their high energy utilization and time efficiency. Efforts have been made to continuously supply reducing power to the reaction mixture in a cyclical manner. The thylakoid membrane (TM) is a promising molecular energy generator, producing NADPH under light. Thus, TM sustainability is of major relevance for its in vitro utilization.ResultsOver 70% of TMs prepared from Synechocystis sp. PCC6803 existed in a sealed vesicular structure, with the F1 complex of ATP synthase facing outward (right-side-out), producing NADPH and ATP under light. The NADPH generation activity of TM increased approximately two-fold with the addition of carbonyl cyanide-p-(trifluoromethoxy) phenylhydrazone (FCCP) or removal of the F1 complex using EDTA. Thus, the uncoupling of proton translocation from the electron transport chain or proton leakage through the Fo complex resulted in greater NADPH generation. Biosilicified TM retained more than 80% of its NADPH generation activity after a week at 30°C in the dark. However, activity declined sharply to below 30% after two days in light. The introduction of engineered water-forming NADPH oxidase (Noxm) to keep the electron transport chain of TM working resulted in the improved sustainability of NADPH generation activity in a ratio (Noxm to TM)-dependent manner, which correlated with the decrease of singlet oxygen generation. Removal of reactive oxygen species (ROS) by catalase further highlighted the sustainable NADPH generation activity of up to 80% in two days under light.ConclusionReducing power generated by light energy has to be consumed for TM sustainability. Otherwise, TM can generate singlet oxygen, causing oxidative damage. Thus, TMs should be kept in the dark when not in use. Although NADPH generation activity by TM can be extended via silica encapsulation, further removal of hydrogen peroxide results in an improvement of TM sustainability. Therefore, as long as ROS formation by TM in light is properly handled, it can be used as a promising source of reducing power for in vitro biochemical reactions.

Dataset Information

Engineering of a green-light inducible gene expression system in Synechocystis sp. PCC6803.

Publications

Engineering of a green-light inducible gene expression system in Synechocystis sp. PCC6803.

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