High cell density culture of metabolically engineered Escherichia coli for the production of poly(3-hydroxybutyrate) in a defined medium

A recombinant Escherichia coli strain XL1-Blue harboring a stable high-copy-number plasmid pSYL107 containing the Alcaligenes eutrophus polyhydroxyalkanoate biosynthesis genes and the Escherichia coli ftsZ gene was employed for the production of poly(3-hydroxybutyrate) (PHB) by fed-batch culture in a defined medium. Suppression of filamentation by overexpressing the cell division protein FtsZ allowed production of PHB to a high concentration (77 g/L) with high productivity (2 g/L/h) in a defined medium, which was not possible with the recombinant E. coli that underwent filamentation. Further optimization of fed-batch culture condition resulted in PHB concentration of 104 g/L in a defined medium, which was the highest value reported to date by employing recombinant E. coli.     

Optimization of microbial poly(3-hydroxybutyrate) recover using dispersions of sodium hypochlorite solution and chloroform

Optimization was carried out for the recovery of microbiol poly(3-hydroxybutyrate) (PHB) from Alcaligenes eutrophus. This process involved the use of a dispersion made of sodium hypochlorite solution and chloroform. The dispersion enabled us to take advantage of both differential digestion by hypochlorite and solvent extraction by chloroform. The PHB recovery (%) from cell powder was maximized using a 30% hypochlorite concentration, a 90-min treatment time, and a 1:1 (v/v) chloroform-to-aqueous-phase ratio. Under these optimal conditions, the recovery was about 91% and the purity of recovered PHB was higher than 97%. The number average molecular weight, M(n) of recovered PHB was about 300,000 and the weight average molecular weight M(w) was about 1,020,000, compared to the original M(n) of 530,000 and M(w) of 1,272,000. The moderate decrease in both M(n) and M(w) might be ascribed to the shielding effect of chloroform. In addition, the relatively small decrease in M(w) probably resulted from the loss of short PHB chains which might be water soluble. The crystallinity of recovered PHB was in the range of 60 to 65%although a slightly higher crystallinity was observed when the dispersion was used. (c) 1994 John Wiley & Sons, Inc.     

Production of poly(3-hydroxybutyrate) by fed-batch culture of filamentation-suppressed recombinant Escherichia coli.

Recombinant Escherichia coli XL1-Blue harboring a high-copy-number plasmid containing the Alcaligenes eutrophus polyhydroxyalkanoate synthesis genes could efficiently synthesize poly(3-hydroxybutyrate) (PHB) in a complex medium containing yeast extract and tryptone but not in a defined medium. One of the reasons for the reduced PHB production in a defined medium was thought to be severe filamentation of cells in this medium. By overexpressing an essential cell division protein, FtsZ, in recombinant E. coli producing PHB, filamentation could be suppressed and PHB could be efficiently produced in a defined medium. A high PHB concentration of 149 g/liter, with high productivity of 3.4 g of PHB/liter/h, could be obtained by the pH-stat fed-batch culture of the filamentation-suppressed recombinant E. coli in a defined medium. It was also found that insufficient oxygen supply at a dissolved oxygen concentration (DOC) of 1 to 3% of air saturation during active PHB synthesis phase did not negatively affect PHB production. By growing cells to the concentration of 110 g/liter and then controlling the DOC in the range of 1 to 3% of air saturation, a PHB concentration of 157 g/liter and PHB productivity of 3.2 g of PHB/liter/h were obtained. For the scale-up studies, fed-batch culture was carried out in a 50-liter stirred tank fermentor, in which the DOC decreased to zero when cell concentration reached 50 g/liter. However, a relatively high PHB concentration of 101 g/liter and PHB productivity of 2.8 g of PHB/liter/h could still be obtained, which demonstrated the possibility of industrial production of PHB in a defined medium by employing the filamentation-suppressed recombinant E. coli.     

Structure of native poly(3-hydroxybutyrate) granules characterized by X-ray diffraction

The structure of native poly(3-hydroxybutyrate) (PHB) granules of Alcaligenes eutrophus was characterized in wet cells or wet granules by analysis of X-ray diffraction. The PHB granules in intact cells were completely amorphous, but became crystalline after treatment with alkali or sodium hypochlorite. The native PHB granules were isolated from the cells by treatment with enzymes and sonic oscillation. The isolated PHB granules remained amorphous in suspension. The PHB granules were crystallized by various treatments with aqueous acetone, alkaline solution (of either NaOH or sodium hypochlorite), and lipase in an aqueous environment. These results suggest that crystallization of PHB molecules is started by the removal of a lipid component from native granules by various treatments.     

Identification of the region of a 14-kilodalton protein of Rhodococcus ruber that is responsible for the binding of this phasin to polyhydroxyalkanoic acid granules.

The function of the polyhydroxyalkanoic acid (PHA) granule-associated GA14 protein of Rhodococcus ruber was investigated in Escherichia coli XL1-Blue, which coexpressed this protein with the polyhydroxybutyric acid (PHB) biosynthesis operon of Alcaligenes eutrophus. The GA14 protein had no influence on the biosynthesis rate of PHB in E. coli XL1-Blue(pSKCO7), but this recombinant E. coli strain formed smaller PHB granules than were formed by an E. coli strain that expressed only the PHB operon. Immunoelectron microscopy with GA14-specific antibodies demonstrated the binding of GA14 protein to these mini granules. In a previous study, two hydrophobic domains close to the C terminus of the GA14 protein were analyzed, and a working hypothesis that suggested an anchoring of the GA14 protein in the phospholipid monolayer surrounding the PHA granule core by these hydrophobic domains was developed (U. Pieper-Fürst, M. H. Madkour, F. Mayer, and A. Steinbüchel, J. Bacteriol. 176:4328-4337, 1994). This hypothesis was confirmed by the construction of C-terminally truncated variants of the GA14 protein lacking the second or both hydrophobic domains and by the demonstration of their inability to bind to PHB granules. Further confirmation of the hypothesis was obtained by the construction of a fusion protein composed of the acetaldehyde dehydrogenase II of A. eutrophus and the C terminus of the GA14 protein containing both hydrophobic domains and by its affinity to native and artificial PHB granules.     

Regulatory effects of cellular nicotinamide nucleotides and enzyme activities on poly(3-hydroxybutyrate) synthesis in recombinant Escherichia coli

Regulatory roles of nicotinamide nucleotides and three key enzymes, beta-ketothiolase (KT), NADPH-dependent acetoacetyl-CoA reductase (AAR), and citrate synthase (CS), on poly(3-hydroxybutyrate) (PHB) synthesis in recombinant Escherichia coli harboring a plasmid containing the Alcaligenes eutrophus polyhydroxyalkanoate (PHA) biosynthesis genes were examined. Cells were grown in various media and were subsequently compared for PHB concentration, PHB content, the activities of the key enzymes, and the levels of nicotinamide nucleotides. Cells of recombinant E. coli accumulated the largest amount of PHB in LB+glucose medium among those tested. PHB synthesis was not enhanced by limiting inorganic ions. The activity of CS, which competes with KT for acetyl-CoA, was lower when cells were grown in LB+glucose compared with other media. The NADPH level and the NADPH/NADP ratio were high in LB+glucose. Examination of the time profiles of the specific PHB synthesis rate, key enzyme activities, and the levels of nicotinamide nucleotides showed that PHB synthesis is most significantly affected by the NADPH level. Even though the NADH level and the NADH/NAD ratio were also high during the synthesis of PHB, no direct evidence of their positive effect on PHB synthesis was found. Low activity of CS was beneficial for PHB synthesis due to the availability of more acetyl-CoA to PHB biosynthetic pathway. In recombinant E. coli, the level of NADPH and/or the NADPH/NADP ratio seem to be the most critical factor regulating the activity of AAR and, subsequently, PHB synthesis. (c) 1996 John Wiley & Sons, Inc.     

Recovery of poly(3-hydroxybutyrate) from coagulated Ralstonia eutropha using a chemical digestion method

For economic recovery of poly(3-hydroxybutyrate) (PHB) from culture broths of Ralstonia eutropha containing PHB, Al-based and Fe-based coagulants were used in the pretreatment step. The coagulated cells were then separated by centrifugation, and PHB was extracted by chemical digestion with a sodium hypochlorite/chloroform dispersion solution. The practical upper limits of dosage were found to be 1, 500 mg-Al/L and 1,000 mg-Fe/L, respectively, for Al- and Fe-based coagulants. When the harvested cells were treated with a 50% sodium hypochlorite/chloroform dispersion solution, PHB recovery and purity were 90-94% and 98-99%, respectively. The influence of the use of coagulants on the PHB recovery process was found to be insignificant. Despite the residual Al and Fe in the recovered PHB (less than 450 mg-Al/kg-PHB and 750 mg-Fe/kg-PHB, respectively), no detectable amounts of Al and Fe were leached from films made of the recovered PHB under acidic conditions. The use of Fe-based coagulants is less recommended because the Fe impurity can cause an unwanted colorization problem in the final product.     

羟基脂肪酸聚酯制备与热性质研究

不同的碳源条件下,真养产碱杆菌可在胞内积累聚羟丁酸(PHB)或含羟基戊酸单体(HV)比例不等的聚羟丁戊酸共聚物(PHBv)。利用次氯酸钠,氯仿混合液体系提取上述羟基脂肪酸聚酯(PHA),提取率为85％,纯度达97．O％。以差示扫描热分析法对PHB和PHBV材料进行热性质研究,发现材料中的HV组分逐渐增加．材料的熔点Tm,熔化焓Hm逐渐下降。热分解峰值逐渐向低温区偏移。但含HV为6lmol％的PHBV材料有关热性质出现回升现象。     

Identification of the intracellular polyhydroxyalkanoate depolymerase gene of Paracoccus denitrificans and some properties of the gene product

Paracoccus denitrificans degraded poly(3-hydroxybutyrate) (PHB) in the cells under carbon source starvation. Intracellular poly(3-hydroxyalkanoate) (PHA) depolymerase gene (phaZ) was identified near the PHA synthase gene (phaC) of P. denitrificans. Cell extract of Escherichia coli carrying lacZ--phaZ fusion gene degraded protease-treated PHB granules. Reaction products were thought to be mainly D(--)-3-hydroxybutyrate (3HB) dimer and 3HB oligomer. Diisopropylfluorophosphonate and Triton X-100 exhibited an inhibitory effect on the degradation of PHB granules. When cell extract of the recombinant E. coli was used, Mg(2+) ion inhibited PHB degradation. However, the inhibitory effect by Mg(2+) ion was not observed using the cell extract of P. denitrificans.     

Production of Poly(3-hydroxybutyrate) by fed-batch culture of recombinant Escherichia coli with a highly concentrated whey solution

Fermentation strategies for the production of poly(3-hydroxybutyrate) (PHB) from whey by recombinant Escherichia coli strain CGSC 4401 harboring the Alcaligenes latus polyhydroxyalkanoate (PHA) biosynthesis genes were developed. The pH-stat fed-batch cultures of E. coli CGSC 4401 harboring pJC4, a stable plasmid containing the A. latus PHA biosynthesis genes, were carried out with a concentrated whey solution containing 280 g of lactose equivalent per liter. Final cell and PHB concentrations of 119.5 and 96.2 g/liter, respectively, were obtained in 37.5 h, which resulted in PHB productivity of 2.57 g/liter/h.     

The recovery of poly(3-hydroxybutyrate) by using dispersions of sodium hypochlorite solution and chloroform

Summary To take advantage of both differential digestion by hypochlorite and solvent extraction, we used dispersions of sodium hypochlorite solution and chloroform in the recovery of microbial PHB. The treatment with hypochlorite alone caused such severe degradation and the molecular weight decreased drastically with increasing hypochlorite concentration. However, using the dispersion, the degradation of PHB was markedly diminished owing to theshielding effect of chloroform. In this case, we could obtain PHB of above 97% purity with a number average molecular weight of 1,000,000 comparable to the original molecular weight of 1,200,000.     

Hyperproduction of poly(4-hydroxybutyrate) from glucose by recombinant Escherichia coli

ABSTRACT: BACKGROUND: Poly(4-hydroxybutyrate) [poly(4HB)] is a strong thermoplastic biomaterial with remarkable mechanical properties, biocompatibility and biodegradability. However, it is generally synthesized when 4-hydroxybutyrate (4HB) structurally related substrates such as gamma-butyrolactone, 4-hydroxybutyrate or 1,4-butanediol (1,4-BD) are provided as precursor which are much more expensive than glucose. At present, high production cost is a big obstacle for large scale production of poly(4HB). RESULTS: Recombinant Escherichia coli strain was constructed to achieve hyperproduction of poly(4-hydroxybutyrate) [poly(4HB)] using glucose as a sole carbon source. An engineering pathway was established in E. coli containing genes encoding succinate degradation of Clostridium kluyveri and PHB synthase of Ralstonia eutropha. Native succinate semialdehyde dehydrogenase genes sad and gabD in E. coli were both inactivated to enhance the carbon flux to poly(4HB) biosynthesis. Four PHA binding proteins (PhaP or phasins) including PhaP1, PhaP2, PhaP3 and PhaP4 from R. eutropha were heterologously expressed in the recombinant E. coli, respectively, leading to different levels of improvement in poly(4HB) production. Among them PhaP1 exhibited the highest capability for enhanced polymer synthesis. The recombinant E. coli produced 5.5 g L-1 cell dry weight containing 35.4% poly(4HB) using glucose as a sole carbon source in a 48 h shake flask growth. In a 6-L fermentor study, 11.5 g L-1 cell dry weight containing 68.2% poly(4HB) was obtained after 52 h of cultivation. This was the highest poly(4HB) yield using glucose as a sole carbon source reported so far. Poly(4HB) was structurally confirmed by gas chromatographic (GC) as well as 1H and 13C NMR studies. CONCLUSIONS: Significant level of poly(4HB) biosynthesis from glucose can be achieved in sad and gabD genes deficient strain of E. coli JM109 harboring an engineering pathway encoding succinate degradation genes and PHB synthase gene, together with expression of four PHA binding proteins PhaP or phasins, respectively. Over 68% poly(4HB) was produced in a fed-batch fermentation process, demonstrating the feasibility for enhanced poly(4HB) production using the recombinant strain for future cost effective commercial development.     

Fermentation characteristics and protein expression patterns in a recombinant Escherichia coli mutant lacking phosphoglucose isomerase for poly(3-hydroxybutyrate) production

For the efficient production of poly(3-hydroxybutyrate) (PHB) using recombinant Escherichia coli, it is of primal importance to overproduce NADPH, which is necessary for the PHB synthetic pathway. In order to overproduce NADPH in the pentose phosphate (PP) pathway, a recombinant E. coli was constructed in which the phosphoglucose isomerase ( pgi) gene was knocked out to force the carbon flow into the PP pathway. The fermentation characteristics of the recombinant E. coli mutant lacking pgi were then investigated to determine the effect of overproduction of NADPH on efficient PHB production. It was found that, compared with the parent strain ( E. coli JM109), growth of the E. coli mutant lacking pgi ( E. coli DF11) is repressed due to NADPH overproduction in the PP pathway. Furthermore, repressed cell growth can be recovered to some extent by introducing a NADPH-consuming pathway, such as the PHB synthetic pathway. Efficient PHB production using such recombinant E. coli (DF11/pAeKG1) could be attained by appropriately controlling the glucose concentration in the fermentor. Total gene expression was investigated at the protein level by two-dimensional electrophoresis. Out of 22 differentially expressed proteins, 12 were identified with the aid of MALDI-TOF mass spectrometry. Variations in the accumulation of PHB in the recombinant pgi mutant carrying phb (E. coli DF11/pAeKG1) corresponded to the expression of proteins encoded by rpsA, znuA, fabD, potD, fkpA, gapA, ynaF and ibpA. The unfavorable conditions generated by PHB accumulation in the pgi mutant carrying phb resulted in the highest expression of 30S ribosomal protein S1, which ultimately caused a further increase in soluble protein synthesis.     

Removal of endotoxin during purification of poly(3-hydroxybutyrate) from gram-negative bacteria.

Poly(3-hydroxybutyrate) (PHB) was produced by cultivating several gram-negative bacteria, including Ralstonia eutropha, Alcaligenes latus, and recombinant Escherichia coli. PHB was recovered from these bacteria by two different methods, and the endotoxin levels were determined. When PHB was recovered by the chloroform extraction method, the endotoxin level was less than 10 endotoxin units (EU) per g of PHB irrespective of the bacterial strains employed and the PHB content in the cell. The NaOH digestion method, which was particularly effective for the recovery of PHB from recombinant E. coli, was also examined for endotoxin removal. The endotoxin level present in PHB recovered by 0.2 N NaOH digestion for 1 h at 30 degrees C was higher than 10(4) EU/g of PHB. Increasing the digestion time or NaOH concentration reduced the endotoxin level to less than 1 EU/g of PHB. It was concluded that PHB with a low endotoxin level, which can be used for various biomedical applications, could be produced by chloroform extraction. Furthermore, PHB with a much lower endotoxin level could be produced from recombinant E. coli by simple NaOH digestion.     

Production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) in a recombinant Escherichia coli strain.

An Escherichia coli strain has been constructed that produces the copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) P(HB-co-HV). This has been accomplished by placing the PHB biosynthetic genes from Alcaligenes eutrophus into an E. coli fadR atoC(Con) mutant and culturing the strain in M9 minimal medium containing glucose and propionate. 3-Hydroxyvalerate incorporation is absolutely dependent on the presence of both glucose and propionate, and 3-hydroxybutyrate-3-hydroxyvalerate ratios in the copolymer can be manipulated by altering the propionate concentration and/or the glucose concentration in the culture. P(HB-co-HV) production can be accomplished by using a wide variety of feeding regimens, but the most efficient is to allow the culture to grow to late log phase in minimal medium containing acetate and then add glucose and propionate to initiate copolymer production. A broad range of propionate concentrations can be used in the culture to stimulate 3-hydroxyvalerate incorporation; however, the most efficient utilization of propionate occurs at concentrations below 10 mM. 3-Hydroxyvalerate molar percentages in the copolymer are relatively constant over the course of growth. The copolymer has been purified and confirmed to be P(HB-co-HV) by gas chromatography/mass spectrometry and differential scanning calorimetry.     

Production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) in a recombinant Escherichia coli strain.

An Escherichia coli strain has been constructed that produces the copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) P(HB-co-HV). This has been accomplished by placing the PHB biosynthetic genes from Alcaligenes eutrophus into an E. coli fadR atoC(Con) mutant and culturing the strain in M9 minimal medium containing glucose and propionate. 3-Hydroxyvalerate incorporation is absolutely dependent on the presence of both glucose and propionate, and 3-hydroxybutyrate-3-hydroxyvalerate ratios in the copolymer can be manipulated by altering the propionate concentration and/or the glucose concentration in the culture. P(HB-co-HV) production can be accomplished by using a wide variety of feeding regimens, but the most efficient is to allow the culture to grow to late log phase in minimal medium containing acetate and then add glucose and propionate to initiate copolymer production. A broad range of propionate concentrations can be used in the culture to stimulate 3-hydroxyvalerate incorporation; however, the most efficient utilization of propionate occurs at concentrations below 10 mM. 3-Hydroxyvalerate molar percentages in the copolymer are relatively constant over the course of growth. The copolymer has been purified and confirmed to be P(HB-co-HV) by gas chromatography/mass spectrometry and differential scanning calorimetry.     

Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-beta-hydroxybutyrate biosynthetic pathway.

The poly-beta-hydroxybutyrate (PHB) biosynthetic pathway from Alcaligenes eutrophus H16 has been cloned and expressed in Escherichia coli. Initially, an A. eutrophus H16 genomic library was constructed by using cosmid pVK102, and cosmid clones that encoded the PHB biosynthetic pathway were sought by assaying for the first enzyme of the pathway, beta-ketothiolase. Six enzyme-positive clones were identified. Three of these clones manifested acetoacetyl coenzyme A reductase activity, the second enzyme of the biosynthetic pathway, and accumulated PHB. PHB was produced in the cosmid clones at approximately 50% of the level found in A. eutrophus. One cosmid clone was subjected to subcloning experiments, and the PHB biosynthetic pathway was isolated on a 5.2-kilobase KpnI-EcoRI fragment. This fragment, when cloned into small multicopy vectors, can direct the synthesis of PHB in E. coli to levels approaching 80% of the bacterial cell dry weight.     

Process analysis and economic evaluation for Poly(3-hydroxybutyrate) production by fermentation

Several processes for the production and recovery of poly(3-hydroxybutyrate) (PHB) by Alcaligenes eutrophus, Alcaligenes latus, Methylobacterium organophilum, and recombinant Escherichia coli were designed based on the previously reported data and analyzed by computer-aided bioprocess design. PHB productivity, content, and yield significantly affected the final price of PHB. For the annual production of 2,850 tonnes of purified PHB, the process employing A. eutrophus with the recovery method of surfactant-hypochlorite digestion resulted in lowest price of PHB, $ 5.58/kg. As the production scale increased to one million tonnes per year, the price of PHB dropped to $ 4.75/kg. The cost of carbon substrate significantly affected the overall economics in large production scale. Therefore, the production cost can be considerably lowered when agricultural wastes, such as whey and molasses, are used.     

phbB,phbC Gene Expression in E. coli and Their Transformation and Identification in Potato

Using NADPH-dependent acetoacetyl-CoA reductase gene (phbB) and poly-β-hydroxybutyrate (PHB) synthase gene (phbC) cloned from Alcaligenes eutrophus H16 and expression vector pKK223-3, the authors constructed an E. coli expression vector pKCB containing independent phbB and phbC operators, respectively, and transfered it into E. coli JM109. The microscopy and GC analysis indicated that E. coli JM109 (containing pKCB) induced by IPTG could synthesize poly-β- hydroxybutyrate (PHB). By DNA processing, three tuber-specific plant expression vectors, pP- SAGB (containing phbB), pBIBGC ( containing phbC) and pPSAGCB ( containing both phbB and phbC), were successfully constructed. In 5 transformed potato cuhivars, the authors screened 20 positive lines.