Enhanced poly-beta-hydroxybutyrate accumulation in a unicellular cyanobacterium, Synechocystis sp. PCC 6803

AIM: To stimulate poly-beta-hydroxybutyrate (PHB) accumulation in Synechocystis sp. PCC 6803 by manipulating culture conditions. METHODS AND RESULTS: Stationary phase cultures of Synechocystis sp. PCC 6803 were subjected to N- and P-deficiency, chemoheterotrophy and limitations of gas-exchange. Enhanced PHB accumulation was observed under all the above conditions. However, interaction of P-deficiency with gas-exchange limitation (GEL) in the presence of exogenous carbon boosted PHB accumulation maximally. CONCLUSIONS: Combined effects of P-deficiency and GEL boosted PHB accumulation up to 38% (w/w) of dry cell weight (dcw) in Synechocystis sp. PCC 6803 in the presence of fructose and acetate. This value is about eightfold higher as compared with the accumulation under photoautotrophic growth condition. SIGNIFICANCE AND IMPORTANCE OF THE STUDY: These results showed a good potential of Synechocystis sp. PCC 6803 in accumulating poly-beta-hydroxybutyrate, an appropriate raw material for biodegradable and biocompatible plastic. Poly-beta-hydroxybutyrate could be an important material for plastic and pharmaceutical industries.     

Sucrose accumulation in salt-stressed cells of agp gene deletion-mutant in cyanobacterium Synechocystis sp PCC 6803

The agp gene encoding the ADP-glucose pyrophosphorylase is involved in cyanobacterial glycogen synthesis and glucosylglycerol formation. By in vitro DNA recombination technology, a mutant with partial deletion of agp gene in the cyanobacterium Synechocystis sp. PCC 6803 was constructed. This mutant could not synthesize glycogen or the osmoprotective substance glucosylglycerol. In the mutant cells grown in the medium containing 0.9 M NaCl for 96 h, no glucosylglycerol was detected and the total amount of sucrose was 29 times of that of in wild-type cells. Furthermore, the agp deletion mutant could tolerate up to 0.9 M salt concentration. Our results suggest that sucrose might act as a similar potent osmoprotectant as glucosylglycerol in cyanobacterium Synechocystis sp. PCC 6803.     

A novel counter‐selection method for markerless genetic modification in Synechocystis sp. PCC 6803

The cyanobacterium Synechocystis sp. PCC 6803 is a photosynthetic organism capable of efficient harnessing of solar energy while capturing CO₂ from the environment. Methods to genetically alter its genomic DNA are essential for elucidating gene functions and are useful tools for metabolic engineering. In this study, a novel counter‐selection method for the genetic alteration of Synechocystis was developed. This method utilizes the nickel inducible expression of mazF, a general protein synthesis inhibitor, as a counter‐selection marker. Counter‐selection is particularly useful because the engineered strain is free of any markers which make further genetic modification independent of available antibiotic resistance genes. The usability of this method was further demonstrated by altering genes at several loci in two variants of Synechocystis. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Biochemical,mechanical, and spectroscopic analyses of genetically engineered flax fibers producing bioplastic (poly‐β‐hydroxybutyrate)

The interest in biofibers has grown in recent years due to their expanding range of applications in fields as diverse as biomedical science and the automotive industry. Their low production costs, biodegradability, physical properties, and perceived eco‐friendliness allow for their extensive use as composite components, a role in which they could replace petroleum‐based synthetic polymers. We performed biochemical, mechanical, and structural analyses of flax stems and fibers derived from field‐grown transgenic flax enriched with PHB (poly‐β‐hydroxybutyrate). The analyses of the plant stems revealed an increase in the cellulose content and a decrease in the lignin and pectin contents relative to the control plants. However, the contents of the fibers' major components (cellulose, lignin, pectin) remain unchanged. An FT‐IR study confirmed the results of the biochemical analyses of the flax fibers. However, the arrangement of the cellulose polymer in the transgenic fibers differed from that in the control, and a significant increase in the number of hydrogen bonds was detected. The mechanical properties of the transgenic flax stems were significantly improved, reflecting the cellulose content increase. However, the mechanical properties of the fibers did not change in comparison with the control, with the exception of the fibers from transgenic line M13. The generated transgenic flax plants, which produce both components of the flax/PHB composites (i.e., fibers and thermoplastic matrix in the same plant organ) are a source of an attractive and ecologically safe material for industry and medicine. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

A study of synthesis and properties of poly‐3‐hydroxybutyrate/diethylene glycol copolymers

This study investigates synthesis of poly(3‐hydroxybutyrate)/diethylene glycol copolymers (P3HB/DEG) by Cupriavidus eutrophus B‐10646 cells as related to DEG concentration in the medium and the time when it is added to the culture of cells synthesizing P3HB. The study determines the limits of physiological effect of DEG on C. eutrophus cells, showing that at DEG concentrations above 30 g/L, it inhibits cell growth, decreasing cell concentration and total P3HB/DEG yield and inducing an increase in the degree of saturation of fatty acids in lipids of cell cytoplasmic membrane. A series of copolymers containing different molar fractions of DEG (between 0.13 and 3.0 mol%) have been synthesized and their physicochemical, physical/mechanical, and biological properties have been investigated as related to the chemical composition and proportions of DEG monomers of the polymers. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1017–1028, 2016     

Production and characterization of poly‐3‐hydroxybutyrate from biodiesel‐glycerol by Burkholderia cepacia ATCC 17759

Glycerol, a byproduct of the biodiesel industry, can be used by bacteria as an inexpensive carbon source for the production of value‐added biodegradable polyhydroxyalkanoates (PHAs). Burkholderia cepacia ATCC 17759 synthesized poly‐3‐hydroxybutyrate (PHB) from glycerol concentrations ranging from 3% to 9% (v/v). Increasing the glycerol concentration results in a gradual reduction of biomass, PHA yield, and molecular mass (M_n and M_w) of PHB. The molecular mass of PHB produced utilizing xylose as a carbon source is also decreased by the addition of glycerol as a secondary carbon source dependent on the time and concentration of the addition. ¹H‐NMR revealed that molecular masses decreased due to the esterification of glycerol with PHB resulting in chain termination (end‐capping). However, melting temperature and glass transition temperature of the end‐capped polymers showed no significant difference when compared to the xylose‐based PHB. The fermentation was successfully scaled up to 200 L for PHB production and the yield of dry biomass and PHB were 23.6 g/L and 7.4 g/L, respectively. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Proteomic analysis of the heat shock response in Synechocystis PCC6803 and a thermally tolerant knockout strain lacking the histidine kinase 34 gene

Proteomic analysis of the heat shock response of wild type and a mutant of the histidine kinase 34 gene (Deltahik34), which shows increased thermal tolerance, has been performed in the cyanobacterium Synechocystis sp. PCC6803. In vivo radioactive labelling demonstrates that major proteomic changes occur within 1 h of heat shock. 2-D DIGE and MS have been used to quantify changes in specific proteins following heat shock in the wild type and the mutant. Over 100 spots, corresponding to 65 different proteins alter following heat shock. Changes occur not only in the classical heat shock proteins but also in the protein biosynthetic machinery, amino acid biosynthetic enzymes, components of the light and dark acts of photosynthesis and energy metabolism. The Deltahik34 cells have elevated levels of heat shock proteins under both non-heat shock and heat shock conditions, in comparison to the wild type, consistent with Hik34, or a down stream component, being a negative regulator of heat shock-responsive genes.     

Enhanced lipid production in Nannochloropsis sp. using fluorescence‐activated cell sorting

To advance the utilization of microalgae as a viable feedstock for biodiesel production, the intracellular lipid content of three strains of the marine microalgae Nannochloropsis sp. was enhanced using flow cytometry (FC) coupled with cell sorting. Total lipid content was doubled to 55% (biomass dry weight) in the sorted, daughter cells of Nannochloropsis (strain 47) after consecutive three rounds of cell sorting, and this trait was maintained for approximately 100 subsequent cell generations. In addition, daughter cells had a fatty acid profile similar to that of the parent, wild‐type strain. The study demonstrates that FC coupled with cell sorting is a powerful tool for the enhancement of intracellular lipid content in microalgae exploited for biodiesel feedstock.     

Genetic and biochemical evidence for distinct key functions of two highly divergent GAPDH genes in catabolic and anabolic carbon flow of the cyanobacterium Synechocystis sp. PCC 6803

Cyanobacterial genomes harbour two separate highly divergent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes, gap1 and gap2, which are closely related at the sequence level to the nuclear genes encoding cytosolic and chloroplast GAPDH of higher plants, respectively. Genes gap1 and gap2 of the unicellular cyanobacterium Synechocystis sp. PCC 6803 were cloned and sequenced and subsequently inactivated by insertional mutagenesis to understand their metabolic functions. We obtained homozygous gap1^- mutants which have lost the capacity to grow on glucose under dim light while growth on organic acids as well as photosynthetic growth under CO₂ and high light is not impaired. Homozygous gap2^- mutants show the reciprocal phenotype. Under dim light they only grow on glucose but not on organic acids nor do they survive under photosynthetic conditions. Measurements of the anabolic activities (reduction of 1,3-bisphosphoglycerate) in extracts from wild type and mutant cells show that Gap2 is a major enzyme with dual cosubstrate specificity for NAD and NADP, while Gap1 displays a minor NAD-specific GAPDH activity. However, if measured in the catabolic direction (oxidation of glyceraldehyde-3-phosphate) Gap2 activity is very low and increases three- to fivefold after gel filtration of extracts over Sephadex G25. Our results suggest that enzymes Gap1 and Gap2, although coexpressed in cyanobacterial wild-type cells, play distinct key roles in catabolic and anabolic carbon flow, respectively. While Gap2 operates in the photosynthetic Calvin cycle and in non-photosynthetic gluconeogenesis, Gap1 seems to be essential only for glycolytic glucose breakdown, conditions under which the catabolic activity of Gap2 seems to be repressed by a specific low-molecular-weight inhibitor.     

Low concentrations of NaHSO3 increase cyclic photophosphorylation and photosynthesis in cyanobacterium Synechocystis PCC6803

Application of NaHSO₃ solution at low concentrations (20–200 μM) to the culture medium enhanced photosynthetic oxygen evolution in cyanobacterium Synechocystis PCC6803 by more than 10%. The slow phase of ms-DLE was strengthened, showing that the transmembrane proton motive force related to photophosphorylation was enhanced. It was also observed that dry weight as well as ATP content under illuminated conditions were both increased after the treatment, indicating that low concentrations of NaHSO₃ could enhance the supply of ATP and thus increase biomass accumulation. In accord with the promotion in the photosynthetic oxygen evolution and ATP content, the transient increase in chlorophyll fluorescence after the termination of actinic light was increased; and meanwhile, the half-time of re-reduction of P700⁺ in the presence of DCMU after a pulse light under background far-red light was shortened by approximately 30%, indicating that cyclic electron flow around PS I was accelerated by the treatment. Based on these results it is suggested that the increase in photosynthesis in Synechocystis PCC6803 caused by low concentrations of NaHSO₃ solution might be due to the stimulation of the cyclic electron flow around PS I and thus the increase in photophosphorylation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Synthesis of poly(3‐hydroxybutyrate‐co‐4‐hydroxybutyrate)/chitosan/silver nanocomposite material with enhanced antimicrobial activity

This work aims to shed light in the fabrication of poly(3‐hydroxybutyrate‐co‐44%‐4‐hydroxybutyrate)[P(3HB‐co‐44%4HB)]/chitosan‐based silver nanocomposite material using different contents of silver nanoparticle (SNP); 1–9 wt%. Two approaches were applied in the fabrication; namely solvent casting and chemical crosslinking via glutaraldehyde (GA). A detailed characterization was conducted in order to yield information regarding the nanocomposite material. X‐ray diffraction analysis exhibited the nature of the three components that exist in the nanocomposite films: P(3HB‐co‐4HB), chitosan, and SNP. In term of mechanical properties, tensile strength, and elongation at break were significantly improved up to 125% and 22%, respectively with the impregnation of the SNP. The melting temperature of the nanocomposite materials was increased whereas their thermal stability was slightly changed. Scanning electron microscopy images revealed that incorporation of 9 wt% of SNP caused agglomeration but the surface roughness of the material was significantly improved with the loading. Staphylococcus aureus and Escherichia coli were completely inhibited by the nanocomposite films with 7 and 9 wt% of SNP, respectively. On the other hand, degradation of the nanocomposite materials outweighed the degradation of the pure copolymer. These bioactive and biodegradable materials stand a good chance to serve the vast need of biomedical applications namely management and care of wound as wound dressing. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1469–1479, 2014     

Effective recovery of poly‐β‐hydroxybutyrate (PHB) biopolymer from Cupriavidus necator using a novel and environmentally friendly solvent system

This work demonstrates a significant advance in bioprocessing for a high‐melting lipid polymer. A novel and environmental friendly solvent mixture, acetone/ethanol/propylene carbonate (A/E/P, 1:1:1 v/v/v) was identified for extracting poly‐hydroxybutyrate (PHB), a high‐value biopolymer, from Cupriavidus necator. A set of solubility curves of PHB in various solvents was established. PHB recovery of 85% and purity of 92% were obtained from defatted dry biomass (DDB) using A/E/P. This solvent mixture is compatible with water, and from non‐defatted wet biomass, PHB recovery of 83% and purity of 90% were achieved. Water and hexane were evaluated as anti‐solvents to assist PHB precipitation, and hexane improved recovery of PHB from biomass to 92% and the purity to 93%. A scale‐up extraction and separation reactor was designed, built and successfully tested. Properties of PHB recovered were not significantly affected by the extraction solvent and conditions, as shown by average molecular weight (1.4 × 10⁶) and melting point (175.2°C) not being different from PHB extracted using chloroform. Therefore, this biorenewable solvent system was effective and versatile for extracting PHB biopolymers. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:678–685, 2016     

Protein-protein interactions in carotenoid triggered quenching of phycobilisome fluorescence in Synechocystis sp. PCC 6803

An inquiry into the effect of temperature on carotenoid triggered quenching of phycobilisome (PBS) fluorescence in a photosystem II-deficient mutant of Synechocystis sp. results in identification of two temperature-dependent processes: one is responsible for the quenching rate, and one determines the yield of PBS fluorescence. Non-Arrhenius behavior of the light-on quenching rate suggests that carotenoid-absorbed light triggers a process that bears a strong resemblance to soluble protein folding, showing temperature-dependent enthalpy of activated complex formation. The response of PBS fluorescence yield to hydration changing additives and to passing of the membrane lipid phase transition point indicates that the pool size of PBSs subject to quenching depends on the state of some membrane component.     

Chromosomal integration of a synthetic expression control sequence achieves poly‐γ‐glutamate production in a Bacillus subtilis strain

Poly‐γ‐glutamate (γ‐PGA) has applications in food, medical, cosmetic, animal feed, and wastewater industries. Bacillus subtilis DB430, which possesses the γ‐PGA synthesis ywsC‐ywtAB genes in its chromosome, cannot produce γ‐PGA. An efficient synthetic expression control sequence (SECS) was introduced into the upstream region of the ywtABC genes, and this resulted in γ‐PGA‐producing B. subtilis mutant strains. Mutant B. subtilis PGA6‐2 stably produces high levels of γ‐PGA in medium A without supplementation of extra glutamic acid or ammonium chloride. The mutant B. subtilis PGA 6‐2 is not only a γ‐PGA producer, but it is also a candidate for the genetic and metabolic engineering of γ‐PGA production. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Bacillus subtilis pgsE (Formerly ywtC) stimulates poly‐γ‐glutamate production in the presence of zinc

Poly‐γ‐glutamate (PGA) is a versatile nylon‐like material, and enhanced production of PGA is required for various bio‐industrial applications. In this study, we first examined the effects of available sugars on the production of Bacillus subtilis PGA, and demonstrated the good applicability of pentoses (e.g., D ‐xylose). Then, we characterized the pgsE gene of B. subtilis, which encodes a 6.5‐kDa protein of 55 amino acids (PgsE), as a genetic tool for increasing the yield of PGA without changing its structural features (e.g., polymer stereochemistry and molecular size distribution). In the presence of Zn²⁺, the induction of PgsE tripled the PGA productivity of B. subtilis subsp. chungkookjang. This finding will contribute to the establishment of an improved PGA‐production system. Biotechnol. Bioeng. 2011; 108:226–230. © 2010 Wiley Periodicals, Inc.     

Isolation and characterization of new poly(3HB)‐accumulating star‐shaped cell‐aggregates‐forming thermophilic bacteria

Aims: This study aimed at isolating thermophilic bacteria that utilize cheap carbon substrates for the economically feasible production of poly(3‐hydroxybutyrate), poly(3HB), at elevated temperatures. Methods and Results: Thermophilic bacteria were enriched from an aerobic organic waste treatment plant in Germany, and from hot springs in Egypt. Using the viable colony staining method for hydrophobic cellular inclusions with Nile red in mineral salts medium (MSM) containing different carbon sources, six Gram‐negative bacteria were isolated. Under the cultivation conditions used in this study, strains MW9, MW11, MW12, MW13 and MW14 formed stable star‐shaped cell‐aggregates (SSCAs) during growth; only strain MW10 consisted of free‐living rod‐shaped cells. The phylogenetic relationships of the strains as derived from 16S rRNA gene sequence comparisons revealed them as members of the Alphaproteobacteria. The 16S rRNA gene sequences of the isolates were very similar (>99% similarity) and exhibited similarities ranging from 93 to 99% with the most closely related species that were Chelatococcus daeguensis, Chelatococcus sambhunathii , Chelatococcus asaccharovorans, Bosea minatitlanensis, Bosea thiooxidans and Methylobacterium lusitanum. Strains MW9, MW10, MW13 and MW14 grew optimally in MSM with glucose, whereas strains MW11 and MW12 preferred glycerol as sole carbon source for growth and poly(3HB) accumulation. The highest cell density and highest poly(3HB) content attained were 4·8 g l^?l (cell dry weight) and 73% (w/w), respectively. Cells of all strains grew at temperatures between 37 and 55°C with the optimum growth at 50°C. Conclusions: New PHA‐accumulating thermophilic bacterial strains were isolated and characterized to produce poly(3HB) from glucose or glycerol in MSM at 50°C. SSCAs formation was reported during growth. Significance and Impact of the Study: To the best of our knowledge, this is the first report on the formation of SSCAs by PHA‐accumulating bacteria and also by thermophilic bacteria. PHA‐producing thermophiles can significantly reduce the costs of fermentative PHA production.     

Application of flow cytometry for monitoring the production of poly(3‐hydroxybutyrate) by Halomonas boliviensis

In this study, a flow cytometry (FC) protocol was implemented to measure poly(3‐hydroxybutyrate) (PHB) content in a halophilic bacterium, to have a faster and easier control of the process. The halophilic bacterium Halomonas boliviensis was stained with BODIPY 493/503 and analyzed using FC. Bacterial polymer accumulation induced by two different nutrient limitations during the operation of a 2 L bioreactor was studied using traditional gas chromatography (GC) analysis and FC. The application of this rapid and straightforward method is useful to obtain complex and precise information about PHB accumulation that could be used for the monitoring, control and optimization of the production of PHB. A clear correlation between the PHB concentration determined by GC and the fluorescence signal obtained from stained bacteria by using FC was observed. Additionally, the heterogeneity of bacterial population as a function of PHB content was measured. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:276–284, 2017