Transcriptomic response to prolonged ethanol production in the cyanobacterium Synechocystis sp. PCC6803
© Dienst et al.; licensee BioMed Central Ltd. 2014
Received: 3 September 2013
Accepted: 17 January 2014
Published: 6 February 2014
The production of biofuels in photosynthetic microalgae and cyanobacteria is a promising alternative to the generation of fuels from fossil resources. To be economically competitive, producer strains need to be established that synthesize the targeted product at high yield and over a long time. Engineering cyanobacteria into forced fuel producers should considerably interfere with overall cell homeostasis, which in turn might counteract productivity and sustainability of the process. Therefore, in-depth characterization of the cellular response upon long-term production is of high interest for the targeted improvement of a desired strain.
The transcriptome-wide response to continuous ethanol production was examined in Synechocystis sp. PCC6803 using high resolution microarrays. In two independent experiments, ethanol production rates of 0.0338% (v/v) ethanol d-1 and 0.0303% (v/v) ethanol d-1 were obtained over 18 consecutive days, measuring two sets of biological triplicates in fully automated photobioreactors. Ethanol production caused a significant (~40%) delay in biomass accumulation, the development of a bleaching phenotype and a down-regulation of light harvesting capacity. However, microarray analyses performed at day 4, 7, 11 and 18 of the experiment revealed only three mRNAs with a strongly modified accumulation level throughout the course of the experiment. In addition to the overexpressed adhA (slr1192) gene, this was an approximately 4 fold reduction in cpcB (sll1577) and 3 to 6 fold increase in rps8 (sll1809) mRNA levels. Much weaker modifications of expression level or modifications restricted to day 18 of the experiment were observed for genes involved in carbon assimilation (Ribulose bisphosphate carboxylase and Glutamate decarboxylase). Molecular analysis of the reduced cpcB levels revealed a post-transcriptional processing of the cpcBA operon mRNA leaving a truncated mRNA cpcA* likely not competent for translation. Moreover, western blots and zinc-enhanced bilin fluorescence blots confirmed a severe reduction in the amounts of both phycocyanin subunits, explaining the cause of the bleaching phenotype.
Changes in gene expression upon induction of long-term ethanol production in Synechocystis sp. PCC6803 are highly specific. In particular, we did not observe a comprehensive stress response as might have been expected.
KeywordsBiofuel Cyanobacteria Ethanol production Synechocystis Metabolic engineering Synthetic biology Transcription
Cyanobacteria are considered to be important and promising resources for the production of biofuels, such as hydrogen , ethanol , isobutyraldehyde and isobutanol , ethylene , volatile isoprene hydrocarbons  and alkanes . Several commercial companies have begun working toward the metabolic remodeling of genetically modified cyanobacteria . To achieve economically feasible production rates, the following two goals need to be addressed: (i) the yield of the intended product is to be maximized, and (ii) the producer strains should be of considerable robustness to tolerate the product, which is frequently alien to their metabolism.
Indeed, genetic instability and the onset of severe stress responses have been reported. Thus far, two unicellular model strains of cyanobacteria have mainly been used in these studies, Synechococcus sp. PCC7942 and Synechocystis sp. PCC6803 (from now on Synechocystis 6803). A depressed growth rate and a yellow-green phenotype interpreted as severe metabolic stress was reported for an ethylene-producing strain of Synechococcus sp. PCC7942 . A substantial and unspecific general stress response was found upon the external application of ethanol both at proteome , as well as transcriptome level in Synechocystis 6803 .
To be meaningful for the optimization of biofuel production from cyanobacteria, the actual response to the internal production of a metabolite should be analyzed. Here we focused on an engineered strain of Synechocystis 6803, which synthesizes ethanol from pyruvate by the sequential activity of overexpressed pyruvate decarboxylase (PDC) from Zymomonas mobilis and alcohol dehydrogenase (ADH) from Synechocystis 6803. Employing high-resolution microarrays we identified a remarkably focused remodeling of the transcriptome in the course of 18 days of continuous ethanol production. The response included a discoordinated operon expression between the phycocyanin cpcB and cpcA genes, fully consistent with the observed bleaching phenotype.
Characterization of Synechocystis 6803 upon long-term ethanol production
Engineering cyanobacteria to produce ethanol from pyruvate is accomplished by coupled overexpression of the cytosolic enzymes PDC and ADH. The synthesized ethanol further accumulates in the growth medium, most likely as a result of diffusion from the interior of the cells .
The OD in the controls continued to increase during the whole course of the experiment at a steady pace (Figure 1A,C). An increase in OD was also observed for the ethanol producer strain but at a slower pace, and growth started to level off after approximately 2 weeks.
The production rates were quite similar in both Pr cultivations, with rates of 0.0338 ± 0.002% (v/v) EtOH d-1 (266.7 mg L-1 d-1) in cultivation A and 0.0303 ± 0.002% (v/v) EtOH d-1 (239.1 mg L-1 d-1) in cultivation B. These productivities were comparable to recently published data on a similar Synechocystis 6803 system (212 mg L-1 d-1; ) and several orders of magnitude higher than demonstrated for the pioneering Synechococcus PCC 7942 system (4.3 μg L-1 d-1; ).
These characteristics were clearly linked to the development of a bleaching phenotype.
Microarray analysis of ethanol producer strains
List of transcripts exhibiting the strongest fold changes in transcript accumulation in response to ethanol production (expressed as log 2 difference producer-control strain)
(log2) fold change
Iron transport system (ferric ions)
Iron transport system (ferric ions)
sll1198-as1 trmD (3′ extension of futA1 transcript)
Biopolymer transport ExbB-like protein
Phycocyanin beta subunit
Ribosomal protein S8
Periplasmic sugar-binding protein of ABC transporter
An overview on the most strongly responding transcripts has been compiled in Table 1. The complete set of microarray data is visualized in Additional file 1 and can be downloaded from the database [GEO: GSE49552].
Ethanol production induces discoordinated expression of the cpcBA operon
Ethanol production induces the transcription of a small part of a ribosomal gene cluster
The generation of microbial producer strains for the sustainable and economically feasible production of biofuels through photosynthetic processes is considered a challenging topic of research. Here we used a full transcriptome microarray developed on the basis of previous RNAseq and dRNAseq analyses  for the model cyanobacterium Synechocystis 6803. In contrast to previous studies in which a massive stress response was reported upon the external application of ethanol [8, 9] we used a producer strain in which the ethanol was produced by an intracellular metabolic process. Our results demonstrate the host response on the internal ethanol synthesis to be unexpectedly narrow. In contrast to a comprehensive stress response, we identified mainly minor changes in transcript levels.
We detected a post-transcriptional regulatory component, involving a previously unknown RNA processing event in the cpcBA operon, leading to the generation of a truncated version of the cpcA transcript (cpcA*) by cleavage of the longer transcript at a specific position. According to its sequence, cpcA* is most likely not coding for a protein as it is 5′-truncated with regard to the cpcA reading frame and is interrupted by multiple stop codons in the other two reading frames. Discoordinated operon expression is frequently linked to the activity of regulatory small RNAs [18, 19]. Recently, the successful metabolic engineering of E. coli was reported using synthetic small regulatory RNAs . However, a native process that induces specific processing of the cpcBA operon mRNA and is leading to a translational nonfunctional cpcA* transcript is currently not known, nor is the possible function of cpcA*.
The second truncated mRNA that appeared specific for the ethanologenic conditions was rps8*. The protein Rps8 plays a major role in assembly of the 30S ribosomal subunit through interaction with 16S rRNA  as well as in the autoregulatory control of ribosomal protein expression from the spc operon in E. coli[22–24]. Although this operon is much longer in Synechocystis 6803 (effectively constituting a fusion of the S10 and spc operons known from enterobacteria), it is tempting to speculate that it plays a regulatory role as well. If so, the preceding intergenic spacer appears conspicuous with its length of 91 nt being by far the longest spacer in this 18-gene operon and constituting the 5′ UTR of rps8. One could speculate that this long 5′ UTR is the autoregulatory target of Rps8 in Synechocystis 6803 and that rps8* serves as competitive binding partner for surplus Rps8 subunits, in this way bypassing the default mechanism for autoregulatory control and allowing further Rps8 production.
In addition to the strongly responding mRNAs for rps8 and cpcB, analyzed here in more detail, in total 240 transcripts were identified that showed mainly minor, but significant expression changes at some point during the experiment. Among these transcripts are many newly discovered transcripts not coding for protein. Some of these transcripts might be regulated by promoters that become induced or repressed at different stages of the production process. Therefore, this dataset can be used in conjunction with our previous genome-wide mapping of TSS  to construct expression cassettes that become active or repressed during different stages of the ethanol producing process.
High ethanol production rates were obtained in engineered strains of Synechocystis 6803 over 18 consecutive days in fully automated PBRs. The physiological effects of high ethanol production include a delay in biomass accumulation, downregulation of light-harvesting capacity and the development of a bleaching phenotype. Microarray-based RNA profiling revealed a highly specific stress response, involving differential accumulation levels of only 31 mRNAs and a small number of non-coding RNAs. The molecular basis for the observed physiological effects of ethanol overproduction consists of a specific RNA processing event in the major light-harvesting operon encoding the phycocyanin subunits α and β. Thus, the molecular responses of engineered cyanobacteria upon sustained ethanol production are specific and appear well manageable for desired long-term cultivation.
Materials and methods
Culture media and growth conditions
The ethanologenic Pr strain #309 of Synechocystis 6803 and the isogenic wild-type Co strain #621 were cultivated in triplicate for 19 days in optimized PBRs containing 0.5 L BG11 medium  supplemented with 2 mM TES, 35 g/L instant-ocean seawater salts (Aquarium Systems Inc., France) and 10 μg/mL gentamycin. The lid was fitted with ports for incoming pH-, dissolved oxygen- and temperature-sensors as well as sampling ports and connections to in- and out-gas. Dissolved oxygen, pH and temperature were monitored by three-channel MultiMeter 44 devices (Crison Instruments, S. A., Barcelona, Spain). Cells were grown under day-night cycle conditions with a 12-h photoperiod. The light intensity was successively adapted to the increasing cell density (approximately 100 μmol photons m-2 s-1 per OD750 unit) and reached a maximum value of 1,000 μmol photons m-2 s-1. The culture temperature was controlled in a day-night cycle with 35°C daytime and 25°C night-time temperature. During the 12-h photoperiod, the liquid phase was discontinuously aerated with CO2-enriched air (10% CO2), pH-dependent and computer-controlled. At a culture pH above 7.35, the aeration started and incoming air flow ceased at a pH below 7.25. There was no aeration of the culture at night. Cells were constantly mixed by stirring with a magnetic stir bar (7 cm length) at 250 rpm. Samples from discrete stages of cultivation were subsequently subjected to microarray (transcriptome) analysis. Furthermore, growth, ethanol accumulation and pigment profiles were monitored over the cultivation period.
Induction of ethanol synthesis from the petJ promoter in #309 was triggered by centrifugation and resuspension of pre-cultures in copper-free medium. Thereby, pre-cultures of OD750 = 7 to 8 were diluted to a final OD750 of 2 (equivalent to about 10 mg chlorophyll * L-1) and subsequently divided into triplicates. In order to maintain maximal ethanol production, nutrient limitations were counter-steered by proportionate supplementation of a 100× nutrient concentrate when the nitrate concentration was below 50% of the BG11 concentration (determined with Quantofix Nitrate/Nitrite, Macherey-Nagel, Düren, Germany).
Ethanol producer strain and quantification of ethanol accumulation
For generation of the ethanologenic strain #309, initially the dicistronic pdc-adhII cassette was Eco RI/Bam HI cut from plasmid pCB4-LR(TF)pa  and fused at its 5′ end (via Eco RI) to the promoter PpetJ from Synechocystis 6803. The Z. mobilis adhII gene was replaced by the AdhA-encoding ORF slr1192 from Synechocystis 6803 (synADH) via Sac I/Pst I. In the final construct [see Additional file 2], the ethanologenic cassette is integrated via Sal I/ Pst I into the self-replicating plasmid pVZ325, which is a derivative of pVZ321  with an additional spectinomycin/streptomycin (Sp/Sm) resistance cassette (from pRL277 ), introduced into the Xba I site (resulting in pVZ321B) and a gentamycin (Gm) resistance cassette (from pVZ322 ), replacing the original kanamycin resistance cassette via Cla I/ Xho I. Plasmid pVZ325 was used for generating the empty-vector-control strain #621.
Primers used for cloning were:
synADH-fw: 5′-ATGAGCTCTCTGGATAAAACTAATAAAC -3′
synADH-rev: 5′- ATCTGCAGATCGAATGTCAAGCTTTCC -3′
PpetJ-fw: 5′- GTCGACGGGAATTGCTCTGGCAAC -3′
PpetJ-rev: 5′- GAATTCATTAGTTCTCCTTTCAAGG -3′
Gm-fw: 5′- ATCGATGCTCGAATTGACATAAGC -3′
Gm-rev: 5′- ATCGATGCTCGAATTGACATAAGC -3′
Quantification of ethanol in the liquid phase was accomplished by head-space gas chromatography (GC) using a Shimadzu GC-20104 gas chromatograph, with a medium-bore capillary column (FS-CS-624, length 30 m; I.D. 0.32 mm; film 1.8 μm; Chromatographie Service GmbH, Germany) and a flame ionization detector (FID). For analysis, 0.5 mL of culture were transferred into 20-mL GC vials for headspace autosampling (Shimadzu PAL LHS2-SHIM/AOC-5000) with screwed silicone-septum caps. For generation of a calibration curve, 0.5 mL calibrator solutions of 0.0125, 0.025, 0.059, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 and 10.0 mg*mL-1 ethanol were measured.
Absorption spectra and determination of the chlorophyll content
Absorption spectra of whole cells were recorded using an UV-2450 PC UV–vis spectrophotometer (Shimadzu Deutschland GmbH, Duisburg, Germany). Chlorophyll contents were measured by spectrophotometry after extraction in 90% methanol .
RNA preparation and northern blot hybridization
Samples from discrete stages of cultivation (as labelled in Figure 1) in PBRs were immediately quenched on ice and spun down at 0°C. RNA isolation and northern blot hybridization were performed essentially as described previously . For analysis of the approximately 300-nt rps8 transcript, total RNA was separated by electrophoresis using urea-polyacrylamide gels (8% acrylamide-bisacrylamide, 19:1; 8.3 M urea; 1× TBE (Tris-Borate-EDTA buffer) and transferred to nylon+ membranes using the Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Biorad, Munich, Germany). The RNA probe for detection of rps8-specific transcripts was prepared using in vitro transcription with the MAXIscript kit (Invitrogen, Darmstadt, Germany) from a T7 promoter containing PCR fragment, which was amplified with the primer pair rps8-S-for 5′-ATGGCTTCAACAGACACAATTTC-3′ and T7-rps8-S-rev 5′-TAATACGACTCACTATAGGGACCAAATGTAACAAAGGAT-3′. The respective probe for the detection of cpcA transcripts was generated using the primers cpcA-fw: 5′- CAAACCCAAGGCAACAACTT −3′ and cpcA-T7: 5′- TAATACGACTCACTATAGGGGCCGTGGTTAGCTTTGATGT - 3′.
Analyses by 5′RACE
The analyses of RNA primary and secondary 5′ ends followed previously established protocols  with the following modifications. For determination of TSS and RNA 5′ ends, 0.65 μg (for cpcA*) and 2.00 μg (for rps8*) of total RNA were subjected to Turbo DNase (Life Technologies, Darmstadt, Germany) digestion, followed by tobacco acid pyrophosphatase (TAP) treatment (Epicentre) and 5′-RNA linker addition using T4 RNA ligase (Epicentre, Madison, Wisconsin, U.S.). Two different oligonucleotides were used as 5′-RNA linkers, li1 in the case of cpcA* and adapterB in the case of rps8*. Synthesis of cDNA was performed with Superscript III reverse transcriptase (Life Technologies) using primers cpcA_R1 or rps8-R1, respectively. For the PCR amplification the RNA-linker-specific primers, Anchor-P1a’ (for cpcA*) or antiadapterB-fw (for rps8*) as well as primer cpcA_R2 or rps8-R1 were used. For the rps8* amplification, a second PCR with nested primers rps8-R2 and antiadapterBII-fw was performed. All reactions were carried out in accordance with the manufacturers’ recommendations.
The following oligonucleotides were used:
li1: 5′- GAUAUGCGCGAAUUCCUGUAGAACGAACACUAGAAGAAA −3′
adapterB: 5′ GUGAUCCAACCGACGCGACAAGCUAAUGCAAGANNN-3′
cpcA_R1: 5′- ATTGTCGGTCAGAGCTTTAG −3′,
cpcA_R2: 5′- TGCAAACCAGCATTAGCTTG −3′,
rps8-R1: 5′- ACCAAATGTAACAAAGGATTTCGCC
rps8-R2: 5′- CCTTCGCCGGTTTCAGAGT
antiadapterB-fw: 5′- TGATCCAACCGACGCGAC
antiadapterBII-fw: 5′- ACCGACGCGACAAGCTAATGC
Gene expression microarray
SDS PAGE and immunoblot analyses
Soluble extracts of Synechocystis 6803 were prepared as described . Proteins were separated by Tricine SDS-PAGE  using gels containing 6 M urea and transferred by electrophoresis onto nitrocellulose membranes. Blot membranes were incubated with specific primary antibodies and then with a secondary antibody (goat anti-rabbit IgG-peroxidase conjugate) (Sigma). Immunolabelled bands were visualized using the Immobilon western membrane chemiluminescence system (Millipore, Bedford, MA, USA). For detection of Zn2+ -induced fluorescence a 16% Tricine SDS-PAGE without urea containing 1 mM zinc acetate was used.
- Chl a:
open reading frame
Tobacco acid pyrophosphatase
transcriptional start site
This work was supported by the German Ministry for Education and Research (BMBF) program Cyanosys to AW, HE and WRH (grant number 0316183) and FORSYS Partner (grant number 0315274) to AW, HE, TB and WRH. We thank Gudrun Krüger, Juliane Wambutt and Andrea Voigt for technical assistance as well as Anne Karradt for providing plasmid pVZ321B.
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