Ethylene Synthesis and Regulated Expression of Recombinant Protein in Synechocystis sp. PCC 6803

The ethylene-forming enzyme (EFE) from Pseudomonas syringae catalyzes the synthesis of ethylene which can be easily detected in the headspace of closed cultures. A synthetic codon-optimized gene encoding N-terminal His-tagged EFE (EFEh) was expressed in Synechocystis sp. PCC 6803 (Synechocystis) and Escherichia coli (E. coli) under the control of diverse promoters in a self-replicating broad host-range plasmid. Ethylene synthesis was stably maintained in both organisms in contrast to earlier work in Synechococcus elongatus PCC 7942. The rate of ethylene accumulation was used as a reporter for protein expression in order to assess promoter strength and inducibility with the different expression systems. Several metal-inducible cyanobacterial promoters did not function in E. coli but were well-regulated in cyanobacteria, albeit at a low level of expression. The E. coli promoter P_trc resulted in constitutive expression in cyanobacteria regardless of whether IPTG was added or not. In contrast, a Lac promoter variant, P_A1lacO-1, induced EFE-expression in Synechocystis at a level of expression as high as the Trc promoter and allowed a fine level of IPTG-dependent regulation of protein-expression. The regulation was tight at low cell density and became more relaxed in more dense cultures. A synthetic quorum-sensing promoter system was also constructed and shown to function well in E. coli, however, only a very low level of EFE-activity was observed in Synechocystis, independent of cell density.     

Premethylation of Foreign DNA Improves Integrative Transformation Efficiency in Synechocystis sp. Strain PCC 6803

Restriction digestion of foreign DNA is one of the key biological barriers against genetic transformation in microorganisms. To establish a high-efficiency transformation protocol in the model cyanobacterium, Synechocystis sp. strain PCC 6803 (Synechocystis 6803), we investigated the effects of premethylation of foreign DNA on the integrative transformation of this strain. In this study, two type II methyltransferase-encoding genes, i.e., sll0729 (gene M) and slr0214 (gene C), were cloned from the chromosome of Synechocystis 6803 and expressed in Escherichia coli harboring an integration plasmid. After premethylation treatment in E. coli, the integration plasmid was extracted and used for transformation of Synechocystis 6803. The results showed that although expression of methyltransferase M had little impact on the transformation of Synechocystis 6803, expression of methyltransferase C resulted in 11- to 161-fold-higher efficiency in the subsequent integrative transformation of Synechocystis 6803. Effective expression of methyltransferase C, which could be achieved by optimizing the 5′ untranslated region, was critical to efficient premethylation of the donor DNA and thus high transformation efficiency in Synechocystis 6803. Since premethylating foreign DNA prior to transforming Synechocystis avoids changing the host genetic background, the study thus provides an improved method for high-efficiency integrative transformation of Synechocystis 6803.     

Recombinant oxyntomodulin

An artificial gene encoding oxyntomodulin was obtained using chemical and enzymatic methods and cloned into Escherichia coli. A recombinant plasmid was constructed containing a hybrid oxyntomodulin gene and Ssp dnaB intein from Synechocystis sp. The expression of the resulting hybrid gene in E. coli, its properties, and the conditions of its autocatalytic cleavage to oxyntomodulin were studied.     

The Proteolytic Activation of the <Emphasis Type="Italic">relNEs</Emphasis> (<Emphasis Type="Italic">ssr1114/slr0664</Emphasis>) Toxin–Antitoxin System by Both Proteases Lons and ClpP2s/Xs of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

The proteolytic regulation of toxin–antitoxin (TA) systems has been well studied in Escherichia coli but remains unclear in other bacteria. A chromosomal gene pair ssr1114/slr0664, named relNEs, of Synechocystis sp. PCC 6803 forms a TA system belonging to rel family. Here, we used E. coli strain BL21 (DE3) as a host to characterize the proteolytic regulation of relNEs. The proteases of this strain could not degrade the antitoxin RelN, and the ectopic production of the ATP-dependant protease Lons or ClpP2s/Xs of Synechocystis sp. PCC6803 did not affect E. coli growth. Either Lons or ClpP2s/Xs was able to degrade RelN resulting in growth arrest of E. coli due to the activation of RelEs’s toxicity, and the presence of RelEs could protect RelN to a certain extent against Lons and ClpP2s/Xs. Our observations suggest that both Lons and ClpP2s/Xs are responsible for RelN proteolysis in the native host under certain conditions. RelN is the first protein substrate identified for cyanobacterial ATP-dependent proteases.     

High-Throughput Screen for Poly-3-Hydroxybutyrate in Escherichia coli and Synechocystis sp. Strain PCC6803

A novel, quantitative method for detecting poly-3-hydroxybutyrate (PHB) amounts in viable cells was developed to allow for high-throughput screening of mutant libraries. The staining technique was demonstrated and optimized for the cyanobacterium Synechocystis sp. strain PCC6803 and the eubacterium Escherichia coli to maximize the fluorescence difference between PHB-accumulating and control cells by flow cytometry. In Synechocystis, the level of nonspecific dye binding was reduced by using nonionic stain buffer that allowed quantitation of fluorescence levels. In E. coli, the use of a mild sucrose shock facilitated uptake of Nile red without significant loss of viability. The optimized staining protocols yielded a linear response for the mean fluorescence against (chemically measured) PHB. The staining protocols are novel methods useful in the high-throughput evaluation of combinatorial libraries of Synechocystis and E. coli using fluorescence-activated cell sorting to identify mutants with increased PHB-accumulating properties.     

Production of optically pure d-lactate from CO2 by blocking the PHB and acetate pathways and expressing d-lactate dehydrogenase in cyanobacterium Synechocystis sp. PCC 6803

Lactate is an important industrial material with numerous potential applications, and its production from carbon dioxide is very attractive. d-Lactate is an essential monomer for production of thermostable polylactide. The photoautotrophic prokaryote cyanobacterium Synechocystis sp. PCC 6803 represents a promising host for biosynthesis of d-lactate from CO₂ as it only contains d-lactate dehydrogenase. The production of d-lactate from CO₂ by an engineered strain of Synechocystis sp. PCC 6803 with overexpressing d-lactate dehydrogenase and a soluble transhydrogenase has been reported recently. Here, we report an alternative engineering strategy to produce d-lactate from CO₂. This strategy involves blocking two competitive pathways, the native poly-3-hydroxybutyrate and acetate pathways from the acetyl-CoA node, and introducing a more efficient d-lactate dehydrogenase into Synechocystis sp. PCC 6803. The engineered strain of Synechocystis sp. PCC 6803 was capable of producing 1.06 g/L of d-lactate from CO₂. This alternative strategy for the production of optically pure d-lactate could also be used to produce other acetyl-CoA-derived chemicals from CO₂ by using engineered cyanobacteria.     

Production of Sepiapterin in Escherichia coli by Coexpression of Cyanobacterial GTP Cyclohydrolase I and Human 6-Pyruvoyltetrahydropterin Synthase

Synechocystis sp. strain PCC 6803 GTP cyclohydrolase I and human 6-pyruvoyltetrahydropterin synthase were coexpressed in Escherichia coli. The E. coli transformant produced sepiapterin, which was identified by high-performance liquid chromatography and enzymatically converted to dihydrobiopterin by sepiapterin reductase. Aldose reductase, another indispensable enzyme for sepiapterin production, may be endogenous in E. coli.     

Cellular physiology controls photoautotrophic production of 1,2-propanediol from pools of CO2 and glycogen

Synechocystis sp. PCC 6803 PG is a cyanobacterial strain capable of synthesizing 1,2-propanediol from carbon dioxide (CO₂) via a heterologous three-step pathway and a methylglyoxal synthase (MGS) originating from Escherichia coli as an initial enzyme. The production window is restricted to the late growth and stationary phase and is apparently coupled to glycogen turnover. To understand the underlying principle of the carbon partitioning between the Calvin-Benson-Bassham (CBB) cycle and glycogen in the context of 1,2-propanediol production, experiments utilizing ¹³C labeled CO₂ have been conducted. Carbon fluxes and partitioning between biomass, storage compounds, and product have been monitored under permanent illumination as well as under dark conditions. About one-quarter of the carbon incorporated into 1,2-propanediol originated from glycogen, while the rest was derived from CO₂ fixed in the CBB cycle during product formation. Furthermore, 1,2-propanediol synthesis was depending on the availability of photosynthetic active radiation and glycogen catabolism. We postulate that the regulation of the MGS from E. coli conflicts with the heterologous reactions leading to 1,2-propanediol in Synechocystis sp. PCC 6803 PG. Additionally, homology comparison of the genomic sequence to genes encoding for the methylglyoxal bypass in E. coli suggested the existence of such a pathway also in Synechocystis sp. PCC 6803. These findings are critical for all heterologous pathways coupled to the CBB cycle intermediate dihydroxyacetone phosphate via a MGS and reveal possible engineering targets for rational strain optimization.     

Recombinant polyamine-binding protein of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 specifically binds to and is induced by polyamines

His-tagged Synechocystis sp. PCC 6803 PotD protein (rPotD) involved in polyamine transport was overexpressed in Escherichia coli. The purified rPotD showed saturable binding kinetics with radioactively labeled polyamines. The rPotD exhibited a similar binding characteristic for three polyamines, with putrescine having less preference. The K _d values for putrescine, spermine, and spermidine were 13.2, 8.3, and 7.8 μM, respectively. Binding of rPotD with polyamines was maximal at pH 8.0. Docking of these polyamines into the homology model of Synechocystis PotD showed that all three polyamines are able to interact with Synechocystis PotD. The binding modes of the docked putrescine and spermidine in Synechocystis are similar to those of PotF and PotD in E. coli, respectively. Competition experiments showed specific binding of rPotD with polyamines. The presence of putrescine and spermidine in the growth medium could induce an increase in PotD contents, suggesting the role of PotD in mediating the transport of polyamine in Synechocystis sp. PCC 6803.     

The tRNA Required for in Vitro delta-Aminolevulinic Acid Formation from Glutamate in Synechocystis Extracts : Determination of Activity in a Synechocystis in Vitro Protein Synthesizing System

RNA is an essential component for the enzymic conversion of glutamate to δ-aminolevulinic acid (ALA), the universal heme and chlorophyll precursor, as carried out in plants, algae, and some bacteria. The RNA required in this process was reported to bear a close structural resemblance to tRNA^Glu(UUC), and it can be isolated by affinity chromatography directed against the UUC anticodon. Affinity-purified tRNA^Glu(UUC) from the cyanobacterium Synechocystis sp. PCC 6803 was resolved into two major subfractions by reverse-phase HPLC. Only one of these was effectively charged with glutamate in enzyme extract from Synechocystis, but both were charged in Chlorella vulgaris enzyme extract. When charged with glutamate, the two glutamyl-tRNA^Glu(UUC) species produced were equally effective in supporting both ALA formation and protein synthesis in vitro, as measured by label transfer from [³H]glutamyl-tRNA to ALA and protein. These results indicate that one of the two tRNA^Glu(UUC) species is used by Synechocystis for both protein biosynthesis and ALA formation. Both of the tRNA^Glu(UUC) subfractions from Synechocystis supported ALA formation in Chlorella enzyme extract. Escherichia coli tRNA^Glu(UUC) was charged with glutamate, but did not support ALA formation in Synechocystis enzyme extract. Unfractionated tRNA from Chlorella, pea, and E. coli, having been charged with [³H] glutamate by Chlorella enzyme extract and then re-isolated, were all able to transfer label to proteins in the Synechocystis enzyme extract.     

Increased Lysine Content Is the Main Characteristic of the Soluble Form of the Polyamide Cyanophycin Synthesized by Recombinant Escherichia coli

Cyanophycin, a polyamide of cyanobacterial or noncyanobacterial origin consisting of aspartate, arginine, and lysine, was synthesized in different recombinant strains of Escherichia coli expressing cphA from Synechocystis sp. strain PCC 6308 or PCC 6803, Anabaena sp. strain PCC 7120, or Acinetobacter calcoaceticus ADP1. The molar aspartate/arginine/lysine ratio of the water-soluble form isolated from a recombinant strain expressing CphA₆₃₀₈ was 1:0.5:0.5, with a lysine content higher than any ever described before. The water-insoluble form consisted instead of mainly aspartate and arginine residues and had a lower proportion of lysine, amounting to a maximum of only 5 mol%. It could be confirmed that the synthesis of soluble cyanobacterial granule polypeptide (CGP) is independent of the origin of cphA. Soluble CGP isolated from all recombinant strains contained a least 17 mol% lysine. The total CGP portion of cell dry matter synthesized by CphA₆₃₀₈ from recombinant E. coli was about 30% (wt/wt), including 23% (wt/wt) soluble CGP, by using terrific broth complex medium for cultivation at 30°C for 72 h. Enhanced production of soluble CGP instead of its insoluble form is interesting for further application and makes recombinant E. coli more attractive as a suitable source for the production of polyaspartic acid or dipeptides. In addition, a new low-cost, time-saving, effective, and common isolation procedure for mainly soluble CGP, suitable for large-scale application, was established in this study.     

Accumulation of Inorganic Polyphosphate in phoU Mutants of Escherichia coli and Synechocystis sp. Strain PCC6803

The biological process for phosphate (P_i) removal is based on the use of bacteria capable of accumulating inorganic polyphosphate (polyP). We obtained Escherichia coli mutants which accumulate a large amount of polyP. The polyP accumulation in these mutants was ascribed to a mutation of the phoU gene that encodes a negative regulator of the P_i regulon. Insertional inactivation of the phoU gene also elevated the intracellular level of polyP in Synechocystis sp. strain PCC6803. The mutant could remove fourfold more P_i from the medium than the wild-type strain removed.     

Protein trans-Splicing To Produce Herbicide-Resistant Acetolactate Synthase

Protein splicing in trans has been demonstrated both in vivo and in vitro by biochemical and immunological analyses, but in vivo production of a functional protein by trans-splicing has not been reported previously. In this study, we used the DnaE intein from Synechocystis sp. strain PCC6803, which presumably reconstitutes functional DnaE protein by trans-splicing in vivo, to produce functional herbicide-resistant acetolactate synthase II (ALSII) from two unlinked gene fragments in Escherichia coli. The gene for herbicide-resistant ALSII was fused in frame to DnaE intein segments capable of promoting protein splicing in trans and was expressed from two compatible plasmids as two unlinked fragments. Cotransformation of E. coli with the two plasmids led to production of a functional enzyme that conferred herbicide resistance to the host E. coli cells. These results demonstrate the feasibility of expressing functional genes from two unlinked DNA loci and provide a model for the design of nontransferable transgenes in plants.     

Using photosystem I as a reporter protein for C analysis in a coculture containing cyanobacterium and a heterotrophic bacterium

¹³C metabolism analysis of a microbial community is often hindered by the time-consuming and complicated separation procedure for a single species. However, a “reporter protein,” produced uniquely by one cell type, retains ¹³C fingerprint information in microbial consortia. This study describes the use of photosystem I (PSI), a multi-subunit protein complex universally found in oxygenic phototrophs, as a reliable reporter protein to probe microalgal metabolism (i.e., cyanobacterium Synechocystis sp. PCC 6803) in a mixed culture with heterotrophic bacteria (i.e., Escherichia coli). We demonstrate that efficient purification of PSI and subsequent ¹³C-based amino acid analyses may decipher photomixotrophic metabolism of Synechocystis 6803 in the coculture. This study also indicates that a supplement of NaHCO₃ at high concentration could significantly improve the robustness of cyanobacterial growth against bacterial contamination.     

Development of a Native Escherichia coli Induction System for Ionic Liquid Tolerance

The ability to solubilize lignocellulose makes certain ionic liquids (ILs) very effective reagents for pretreating biomass prior to its saccharification for biofuel fermentation. However, residual IL in the aqueous sugar solution can inhibit the growth and function of biofuel-producing microorganisms. In E. coli this toxicity can be partially overcome by the heterologous expression of an IL efflux pump encoded by eilA from Enterobacter lignolyticus. In the present work, we used microarray analysis to identify native E. coli IL-inducible promoters and develop control systems for regulating eilA gene expression. Three candidate promoters, PmarR’, PydfO’, and PydfA’, were selected and compared to the IPTG-inducible PlacUV5 system for controlling expression of eilA. The PydfA’ and PmarR’ based systems are as effective as PlacUV5 in their ability to rescue E. coli from typically toxic levels of IL, thereby eliminating the need to use an IPTG-based system for such tolerance engineering. We present a mechanistic model indicating that inducible control systems reduce target gene expression when IL levels are low. Selected-reaction monitoring mass spectrometry analysis revealed that at high IL concentrations EilA protein levels were significantly elevated under the control of PydfA’ and PmarR’ in comparison to the other promoters. Further, in a pooled culture competition designed to determine fitness, the strain containing pPmarR’-eilA outcompeted strains with other promoter constructs, most significantly at IL concentrations above 150 mM. These results indicate that native promoters such as PmarR’ can provide effective systems for regulating the expression of heterologous genes in host engineering and simplify the development of industrially useful strains.     

Cloning,sequence and overexpression of the thermophilic cyanobacterium gene for the ribulose-1,5-bisophosphate carboxylase/oxygenase

The genes for the large (rbcL) and small (rbcS) subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) were cloned from a thermophilic cyanobacterium, Synechococcus sp. strain a-1. These two genes were located tandemly on the same strand of chromosomal DNA with a 467 bp spacer region. The rbcL gene codes for 474 amino acid residues (1,422 bp), and rbcS for 118 residues (354 bp). The deduced amino acid sequence for the large subunit was highly similar to those of other cyanobacteria and higher plants. The RubisCO genes were overexpressed (>30% of total soluble protein) in E. coli cells by using pKK223-3 as an expression vector. The overexpressed RubisCO formed hexademers (L₈S₈ form) in E. coli cells, the same form as in Synechococcus cells. The RubisCO was easily purified from E. coli cell free extract using the advantage of its high thermostability, and the purified RubisCO had almost the same characteristics as the native RubisCO purified from Synechococcus sp. strain a-1.     

Chromosomal Sil system contributes to silver resistance in <Emphasis Type="Italic">E. coli</Emphasis> ATCC 8739

The rise of antibiotic resistance in pathogenic bacteria is endangering the efficacy of antibiotics, which consequently results in greater use of silver as a biocide. Chromosomal mapping of the Cus system or plasmid encoded Sil system and their relationship with silver resistance was studied for several gram-negative bacteria. However, only few reports investigated silver detoxification mediated by the Sil system integrated in Escherichia coli chromosome. Accordingly, this work aimed to study the Sil system in E. coli ATCC 8739 and to produce evidence for its role in silver resistance development. Silver resistance was induced in E. coli ATCC 8739 by stepwise passage in culture media containing increasing concentrations of AgNO₃. The published genome of E. coli ATCC 8739 contains a region showing strong homology to the Sil system genes. The role of this region in E. coli ATCC 8739 was assessed by monitoring the expression of silC upon silver stress, which resulted in a 350-fold increased expression. De novo sequencing of the whole genome of a silver resistant strain derived from E. coli ATCC 8739 revealed mutations in ORFs putative for SilR and CusR. The silver resistant strain (E. coli AgNO₃R) showed constitutive expression of silC which posed a cost of fitness resulting in retarded growth. Furthermore, E. coli AgNO₃R exhibited cross-resistance to ciprofloxacin and a slightly increased tolerance to ampicillin. This study demonstrates that E. coli is able to develop resistance to silver, which may pose a threat towards an effective use of silver compounds as antiseptics.     

Biotechnological Method for Production of Recombinant Peptide Analgesic (Purotoxin-1) from <Emphasis Type="Italic">Geolycosa</Emphasis> sp. Spider Poison

Severe chronic and sometimes incurable diseases are frequently accompanied by a pain syndrome. Purotoxin-1, isolated from the poison of the Central Asian Geolycosa sp. spider and selectively inhibiting the purinergic P2X3 receptor (which is considered as a target for the control of the pain states), is one potentially highly effective drug with an analgesic effect. To produce the recombinant purotoxin-1, we created four genetically engineered constructions with different carrier proteins for the expression in E. coli: pTRX-PT1, pCBD-PT1, pGyrA-PT1, pDnaB-PT1. The construction with mini-intein DnaB from the Synechocystis sp. was the most efficient. Using the E. coli C3030/pDnaB-PT1 producer strain, the laboratory method, based on which a pilot technology of recombinant purotoxin-1 production was developed as a result of optimization and scaling, was created. Six grams of recombinant PT1 preparation with the confirmed pharmacological purity was developed for preclinical trials.