Strains and culture conditions
Strains of E. coli were grown on Lysogeny Broth (LB) liquid medium at 37 °C in a shaking incubator at 200 rpm, or on the solid LB plates with 1.5% agar. Antibiotics were added to LB liquid medium or to solid plates, with the appropriate concentration as follows: kanamycin (50 µg mL-1) or ampicillin (100 µg mL-1), either separately or in combination.
Synechocystis, a glucose-tolerant wild type, obtained from D. Bhaya, University of Stanford, Stanford CA, was cultivated in a modified BG-11 medium enriched with 25 mM PIPPS buffer (pH 8.0) (27) at 30 °C, either in a shaking incubator at 120 rpm, or on solid BG-11 plates, supplemented with 1.5% agar and 0.3% (w/v) sodium thiosulphate. The cells were grown with white light of moderate intensity (~50 µmol photons m-2 s-1), except when indicated. To construct Synechocystis mutants, kanamycin or nickel were added separately into BG-11 liquid medium or solid plates, with the appropriate concentration as follows: kanamycin (50 µg mL-1) and/or nickel (20 µg mL-1). The culture density was monitored by determining the optical density at a wavelength of 730 nm (OD730).
Gene synthesis with codon optimization
The gene sequence of mannitol-1-phosphate-5-dehydrogenase (mtlD) from E. coli (NCBI Reference Sequence: NC_000913.3) and mannitol-1-phosphatase (m1p) from Eimeria tenella (NCBI Reference Sequence: AF032462.1) were taken directly from the NCBI database. Codon optimization was performed based on the codon usage table compiled for Synechocystis (https://www.kazusa.or.jp/codon/). The genes were synthesized by GenScript Biotech Corp, and were ligated to the pHKH and pUC57 plasmids, respectively (28) resulting in pHKHmtlD and pUCm1p.
Plasmids and Synechocystis mutant construction
To obtain marker-less deletion strains, the genes encoding sucrose phosphate synthase (sps) and glucosyl-glycerol phosphate synthase (ggpS) in the genome of Synechocystis, were deleted with a counter-selection approach (29). For each gene deletion, two plasmids are needed: The first one contains only the upstream and the downstream sequences of the region to be deleted, hereafter referred as the homologous regions, while the second plasmid contains an extra selection cassette flanked by both homologous regions. The selection cassette consists of a gene conferring kanamycin resistance to the host, as well as a toxic gene (mazF), under transcriptional control of the tightly regulated nickel-inducible promoter PnrsB. To construct each of the two plasmids, ~ 1 kb of each upstream and downstream homologous region of either sps or ggpS was individually PCR-amplified from the genome of Synechocystis, using Herculase II Fusion DNA Polymerase (Agilent Technologies). After gel purification, each set of fragments of an upstream and downstream homologous region was fused by overlap PCR, and the entire fused fragment was then further amplified by PCR. After the fused fragment was gel purified, an extra adenosine was added to the 3’ ends of these fragments and it was then ligated to the pFL-AN-T vector (30), resulting in plasmids pFL-AN2 (Δsps) and pFL-AN4 (ΔggpS), respectively. Because an XbaI restriction site was introduced via the primers during overlap PCR, the selection cassette, if provided with an XbaI site on both ends, can be easily inserted into pFL-AN2 (Δsps) and pFL-AN4 (ΔggpS), resulting in pFL-AN1 (Δsps) and pFL-AN3 (ΔggpS), respectively.
The mtlD and m1p genes were PCR amplified from pHKHmtlD and pUCm1p, respectively. Initially, we attempted to clone these genes using E. coli as a shuttle host. In order to correctly express mtlD in Synechocystis, the weaker promoter PnrsB was used to control the expression level of mtlD in Synechocystis, but repeatedly failed. This result surprised us, as PnrsB is from the nickel response system (nrs (31)), and is regarded as one of the weakest promoters in this host in the absence of an inducer. This result indicated the difficulty of high expression levels of MtlD, and that this might become a bottleneck for the synthesis of large amounts of mannitol in Synechocystis. So we decided to bypass the usage of a shuttle host. Instead we chose to fuse these two fragments together with the kanamycin resistance gene, and the upstream and downstream homologous regions of slr0168 (neutral site), via overlap PCR. The resulting mannitol cassette plus resistance marker was placed under control of the Ptrc1 promoter. The fused fragment was sequenced to check for the absence of mutations and then used directly for transformation of the chromosome of Synechocystis at the neutral site present in locus slr0168.
It takes two rounds of natural transformation of Synechocystis to achieve a markerless gene deletion of either sps or ggpS. The first round is to fully integrate the selection cassette into the chromosome through homologous recombination, while the second round is to completely remove the selection cassette. The method used for natural transformation was essentially as described previously (32). Briefly, 1 ml Synechocystis cultures grown in a shake flask to an OD730 of ~0.4 were harvested and concentrated by centrifugation at 5000 rpm for 5 min to a volume of 200 µL. Then, plasmid was added to the concentrated cells at 10 µg mL-1, followed by 5 hours’ incubation in moderate white light, in a shaking incubator at 150 rpm. After incubation, cells were spread onto a commercial membrane (Pall Corporation, USA) resting on an BG-11 plate without antibiotic pressure. After a 24-hour incubation in the 30 °C incubator under constant white light illumination, the membrane was transferred onto a new BG-11 plate with 50 µg mL-1 kanamycin. Single colonies appeared after approximately 12 days. The segregation status of mutants was confirmed by PCR, using the appropriate primers. When a mutant was confirmed to be fully segregated, a second round of transformation with a plasmid containing only the upstream and downstream homologous region was performed. The selection was then based on the resistance to nickel as only the colonies with the selection cassette fully removed can survive on the plates with nickel. The protocol for transformation of the DNA fragment containing the mannitol cassette and the kanamycin resistance fragment integrated at the slr0168 site was similar to the protocol mentioned above. Full segregation of this construct was achieved by propagations in the presence of kanamycin. All the mutants were confirmed by PCR and the primers that were used are listed in the Additional file 1, Table S3. The confirmed mutants were routinely stored at −80 °C in BG-11 medium supplemented with 20% (v/v) glycerol.
Growth rate determination under salt stress
To measure the growth rate for each strain under salt stress, we monitored the growth of each strain in a 96-well plate under a range of NaCl concentrations, from 0 to 500 mM. A preculture was prepared by inoculating cells from glycerol stocks directly into shake flasks containing liquid BG-11 medium and cultivated in the incubator with shaking of 120 rpm under continuously white light of moderate intensity (~50 µmol photons m-2 s-1). Once the precultures reached OD730 = 1, a total volume of 1 mL of a pre-culture was harvested and inoculated in each well of a 48-well plate, supplemented with 50 mM NaHCO3 plus increasing salt concentrations, ranging from 0 to 500 mM. After 2 days, the final optical density of the cultures in the 48-well plate was measured using a SPECTROstar Nano Microplate Photometer (BMG LABTECH GmbH, Germany) at 730 nm. To initiate experiments with a 96-well plate, pre-cultures acclimated to the corresponding salt stress in the 48-well plate were used for inoculation. This is to prevent the prolonging of the lag-phase of growth, resulting from the addition of salt, such that the same stage of cell growth under each condition can be extracted from the data for growth rate calculation. Pre-cultures were then diluted using 50 mM NaHCO3 and the respective concentration of NaCl in BG-11 to an initial OD of 0.05. Plates were incubated under constant white light illumination, with shaking at 600 rpm. Growth was monitored every 2 hours within the 36 hours incubation in the plate reader, to reliably calculate the maximum growth rate under each condition, the first 6 consecutive data points (from time 0 to 10 hours) were used by fitting a linear function through the natural logarithm of the OD. The slope of the linear function was computed and designated as the growth rate. A representative set of growth curves in the 96 well plate together with the data points for growth rate calculation for all the strains was presented in the Fig. S1 of the Additional file 1.
Plate assay with a linear NaCl gradient
The method for making linear salt gradient plates has been described previously by (33). Briefly, BG-11 media containing agar, with and without NaCl (either 0.5 M or 1 M), were individually prepared. When making linear salt gradient plates, one side of a square petri dish was lifted and the plate was filled with BG-11 agar without NaCl. After the agar solidified, the plate was placed in a horizontal position and BG-11 agar with NaCl was poured on top of the first layer. To test the salt tolerance of all the mutants, the cells in Synechocystis cultures were first counted using a Casy 1 Model TTC cell counter (Schärfe System GmbH, Reutlingen, Germany) with a 60 µm diameter capillary, and diluted to a total cell number of 12,500 cells µL-1. A 5 µL culture from each mutant, grown with 200 mM salt, was spotted on the linear gradient plates containing a 0 to 0.5 M or a 0 to 1 M NaCl concentration gradient. Visible, green colonies appeared within one week.
Measurements of extracellular mannitol concentrations
Extracellular mannitol concentrations were determined in the supernatant collected from Synechocystis cultures using a D-Mannitol-L-Arabitol Assay Kit (Megazyme) (9). Cells from shake-flask cultures were removed by centrifugation at 12,000 rpm for 1 minute. Then 100 µL of the supernatant samples was used for mannitol measurement according to manufacturer’s instructions. In this assay, the conversion of the mannitol present in the sample to mannose - catalyzed by mannitol dehydrogenase - is stoichiometrically coupled to the conversion of NAD+ to NADH. This leads to an increase of absorbance at 340 nm that can be measured using a plate reader (BMG FLUOstar OPTIMA Microplate Reader). For mannitol quantification, the assay was calibrated with a standard curve (from 3 to 100 µM mannitol) obtained under the same conditions.
The phenotypic stability of mannitol-producing strains was studied with the turbidostat mode of a Multi-Cultivator (MC1000-OD, PSI, Czech Republic). In this cultivation mode, Synechocystis populations can be kept at a fixed biomass density by continuously diluting cultures with fresh BG-11 medium without antibiotic, while simultaneously taking out an identical volume of cultured cells. Accordingly, cells in a turbidostat are under continuous selection for maximal specific growth rate. For this we used a modified Multi-Cultivator with additional pumps (Reglo ICC, ISMATEC, Germany) and controlled by the “pycultivator” software package (13). Pre-cultured cells were transferred into 8 independent cylindrical vessels of a multi-cultivator, filled with BG-11 medium with 0, 200 mM or 420 mM NaCl to an initial OD730 of 0.05. OD730 was measured every 5 minutes. Once the threshold of OD730 was reached (OD730 = 1) cell cultures were automatically diluted by 5% (v/v) with fresh BG-11 medium and the same volume of culture as the volume of medium just added, was discarded; all under control of “pycultivator”. All the cultures in the Multi-Cultivator were exposed to continuous white light with an intensity of 100 µmol photons m-2 s-1 OD-1. The genetic stability of each strain was assessed by mannitol production. Growth rate was calculated by fitting a linear function through the natural logarithm of the OD730 during each “growth-dilution” cycle. Samples for extracellular mannitol quantification were periodically taken during the cultivation period. The variation of growth rate and mannitol productivity throughout the whole experiment was then calculated relative to the initial values obtained at the beginning of each specific experiment.
Sequencing of the mannitol cassette
To check the sequence, the mannitol cassette for the occurrence of (a) spontaneous mutation(s), single colonies from each independent Multi-Cultivator culture were first isolated. This is achieved by taking 5 µL of culture and re-streaking the cells on BG-11 agar plates. After picking a single colony and inoculating it into liquid BG-11 medium, genomic DNA was extracted as previously described (34) and used as a template to amplify the mannitol cassette by PCR, using the high-fidelity Herculase II Fusion DNA Polymerase. The PCR product was then purified using MSB Spin PCRapace (STRATEC Molecular, Germany) and sent for sequencing (Macrogen) using the primers listed in Additional file 1, Table S3.