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.     

In silico prediction and validation of the importance of the Entner-Doudoroff pathway in poly(3-hydroxybutyrate) production by metabolically engineered Escherichia coli

The metabolic network of Escherichia coli was constructed and was used to simulate the distribution of metabolic fluxes in wild-type E. coli and recombinant E. coli producing poly(3-hydroxybutyrate) [P(3HB)]. The flux of acetyl-CoA into the tricarboxylic acid (TCA) cycle, which competes with the P(3HB) biosynthesis pathway, decreased significantly during P(3HB) production. It was notable to find from in silico analysis that the Entner-Doudoroff (ED) pathway flux increased significantly under P(3HB)-accumulating conditions. To prove the role of ED pathway on P(3HB) production, a mutant E. coli strain, KEDA, which is defective in the activity of 2-keto-3-deoxy-6-phosphogluconate aldolase (Eda), was examined as a host strain for the production of P(3HB) by transforming it with pJC4, a plasmid containing the Alcaligenes latus P(3HB) biosynthesis operon. The P(3HB) content obtained with KEDA (pJC4) was lower than that obtained with its parent strain KS272 (pJC4). The reduced P(3HB) biosynthetic capacity of KEDA (pJC4) could be restored by the co-expression of the E. coli eda gene, which proves the important role of ED pathway on P(3HB) synthesis in recombinant E. coli as predicted by metabolic flux analysis.     

Recovery of poly(3-hydroxybutyrate) from high cell density culture of Ralstonia eutropha by direct addition of sodium dodecyl sulfate

A simple and effective method for the recovery poly(3-hydroxybutyrate) [P(3HB)] directly from high cell density culture broth with no pretreatment steps has been developed. This method consists of direct addition of sodium dodecyl sulfate (SDS) to the culture broth, shaking, heat treatment, and washing steps. When the SDS/biomass ratio was higher than 0.4, the purity of recovered P(3HB) was over 95% for various cell concentrations of 50–300 g dry cell l^–1, with the highest value of 97%. The recovery of P(3HB) was over 90% regardless of cell concentration and SDS dosage (SDS/biomass ratios, 0.1–0.7). One g SDS digests 0.72 g non-P(3HB) cell materials. The reduction in molecular weight, due to degradation of P(3HB) by SDS, was negligible.     

En route to economical eco‐friendly solvent system in enhancing sustainable recovery of poly(3‐hydroxybutyrate‐co‐4‐hydroxybutyrate) copolymer

Separation of poly(3‐hydroxybutyrate‐co‐4‐hydroxybutyrate) [P(3HB‐co‐4HB)] from bacterial cell matter is a critical step in the downstream process with respect to material quality and eco‐balance as P(3HB‐co‐4HB) is widely used for biomedical applications. Therefore, an efficient and eco‐based extraction of P(3HB‐co‐4HB) using a combination of NaOH and Lysol in digesting the non‐polymeric cell material (NPCM) digestion is developed. The NaOH and Lysol show synergistic influence on the copolymer extraction at a high purity and recovery of 97 and 98 wt% respectively. The optimized cell digestion method was found applicable to a vast batch of cells containing copolymers from various 4HB monomer compositions. At the largest extraction volume of 100 L, P(3HB‐co‐4HB) with a purity of 89 wt% was extracted with a maximum recovery of 90 wt%. The method developed has also eliminated the cell pretreatment step. The extraction method developed in this research has not only produced an economic and efficient copolymer recovery but has also retained the copolymer quality, in term of its molecular weight and thermal properties. It demonstrates a practical and promising downstream processing method in recovering the copolymer effectively from the bacterial biomass.     

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.     

Chemo-microbial conversion of cellulose into polyhydroxybutyrate through ruthenium-catalyzed hydrolysis of cellulose into glucose

Cellulose-derived glucose generated using the supported ruthenium catalyst was applied to poly(3-hydroxybutyrate) [P(3HB)] production in recombinant Escherichia coli. By the reaction with the catalyst at 220°C, 15-20 carbon mol% of cellulose was converted into glucose. The hydrolysate also contained byproducts such as fructose, mannose, levoglucosan, oligomeric cellulose, 5-hydroxymethylfurfural (5-HMF), and furfural together with unidentified compounds. Setting the reaction temperature lower (215°C) improved the ratio of glucose to 5-HMF, which was a main inhibiting factor for the cell growth. Indeed, the recombinant E. coli exhibited better performance on the hydrolysate generated at 215°C and accumulated P(3HB) up to 42 wt%, which was the same as the case of the same concentration of analytical grade glucose. The result indicated that the ruthenium-mediated cellulose hydrolysis has a potency as a useful biorefinery process for production of bio-based plastic from cellulosic biomass.     

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.     

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°C was higher than 10⁴ 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.     

Metabolic engineering of Escherichia coli for enhanced biosynthesis of poly(3-hydroxybutyrate) based on proteome analysis

We have previously analyzed the proteome of recombinant Escherichia coli producing poly(3-hydroxybutyrate) [P(3HB)] and revealed that the expression level of several enzymes in central metabolism are proportional to the amount of P(3HB) accumulated in the cells. Based on these results, the amplification effects of triosephosphate isomerase (TpiA) and fructose-bisphosphate aldolase (FbaA) on P(3HB) synthesis were examined in recombinant E. coli W3110, XL1-Blue, and W lacI mutant strains using glucose, sucrose and xylose as carbon sources. Amplification of TpiA and FbaA significantly increased the P(3HB) contents and concentrations in the three E. coli strains. TpiA amplification in E. coli XL1-Blue lacI increased P(3HB) from 0.4 to 1.6 to g/l from glucose. Thus amplification of glycolytic pathway enzymes is a good strategy for efficient production of P(3HB) by allowing increased glycolytic pathway flux to make more acetyl-CoA available for P(3HB) biosynthesis.     

Synthesis of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) by recombinant Escherichia coli

Several recombinant Escherichia coli strains, including XL1-Blue, JM109, HB101, and DH5alpha harboring a stable high-copynumber plasmid pSYL105 containing the Alcaligenes eutrophus polyhydroxyalkanoate (PHA) biosynthesis genes were constructed. These recombinant strains were examined for their ability to synthesize and accumulate poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymer from glucose and either propionate or valerate. All recombinant E. coli strains could synthesize the P(3HB-co-3HV) copolymer in the medium containing glucose and propionate. However, only the homopolymer poly-(3-hydroxybutyrate) [P(3HB)] was synthesized from glucose and valerate. The PHA concentration and the 3HV fraction could be increased by inducing with acetate and/or oleate. When supplemented with oleate, the 3HV fraction increased by fourfold compared with that obtained without induction. Induction with propionate resulted in lower PHA concentration due to the inhibitory effect, but an 3HV fraction of as high as 33.0% could be obtained. These results suggest that P(3HB-co-3HV) can be efficiently produced from propionate by recombinant E. coli by inducing with acetate, propionate, or oleate. (c) 1996 John Wiley & Sons, Inc.     

Biosynthesis of synthons in two-liquid-phase media

The Pseudomonas oleovorans alkane hydroxylase and xylene oxygenase from Pseudomonas putida are versatile mono-oxygenases for stereo- and regioselective oxidation of aliphatic and aromatic hydrocarbons. Pseudomonas oleovorans and alkanol dehydrogenase deficient mutants of Pseudomonas have previously been used to produce alkanols from various alkanes and optically active epoxides from alkenes. Similarly, P. putida strains have been used to produce aromatic alcohols, aromatic acids, and optically active styrene oxides. A limitation in the use of Pseudomonas strains for bioconversions is that these strains can degrade some of the products formed. To counter this problem, we have constructed Escherichia coli recombinants, which contain the alk genes from the OCT plasmid of P. oleovorans [E. coli HB101 (pGEc47)] and the xylMA genes from the TOL plasmid of P. putida mt-2 [E. coli HB101 (pGB63)], encoding alkane hydroxylase and xylene oxygenase, respectively. Escherichia coli HB101 (pGEc47) was used to produce octanoic acid from n-octane and E. coli HB101 (pBG63) was put to use for the oxidation of styrene to styrene oxide in two-liquid phase biocatalysis at high cell densities. The alk(+) recombinant strain E. coli HB101 (pGEc47) was grown to 40 g/L cell dry mass in the presence of n-octane, which was converted to octanoic acid by the alkane oxidation system, the product accumulating in the aqueous phase. The xyl(+) recombinant E. coli HB101 (pBG63) was grown to a cell density of 26 g/L cell dry mass in the presence of around 7% (v/v) n-dodecane, which contained 2% (v/v) styrene. The recombinant E. coli (xyl(+)) converted styrene to (S)-(+)-styrene oxide at high enantiomeric excess (94% ee) and this compound partitioned almost exclusively into the organic phase. Using these high-cell-density two-liquid-phase cultures, the products accumulated rapidly, yielding high concentrations of products (50 mM octanoic acid and 90 mM styrene oxide) in the respective phases. (c) 1996 John Wiley & Sons, Inc.     

Synergistic effects of Glu130Asp substitution in the type II polyhydroxyalkanoate (PHA) synthase: enhancement of PHA production and alteration of polymer molecular weight

In vitro evolution of the polyhydroxyalkanoate (PHA) synthase gene from Pseudomonas sp. 61-3 (phaC1(Ps)) has been performed to generate highly active enzymes. In this study, a positive mutant of PHA synthase, Glu130Asp (E130D), was characterized in detail in vivo and in vitro. Recombinant Escherichia coli strain JM109 harboring the E130D mutant gene accumulated 10-fold higher (1.0 wt %) poly(3-hydroxybutyrate) [P(3HB)] from glucose, compared to recombinant E. coli harboring the wild-type PHA synthase gene (0.1 wt %). Recombinant E. coli strain LS5218 harboring the E130D PHA synthase gene grown on dodecanoate produced more poly(3HB-co-3-hydroxyalkanoate) [P(3HB-co-3HA)] (20 wt %) copolymer than an LS5218 strain harboring the wild-type PHA synthase gene (13 wt %). The E130D mutation also resulted in the production of copolymer with a slight increase in 3HB composition, compared to copolymer produced by the wild-type PHA synthase. In vitro enzyme activities of the E130D PHA synthase toward various 3-hydroxyacyl-CoAs (4-10 carbons in length) were all higher than those of the wild-type enzyme. The combination of the E130D mutation with other beneficial mutations, such as Ser325Thr and Gln481Lys, exhibited a synergistic effect on in vivo PHA production and in vitro enzyme activity. Interestingly, gel-permeation chromatography analysis revealed that the E130D mutation also had a synergistic effect on the molecular weight of polymers produced in vivo.     

Properties and biodegradability of ultra-high-molecular-weight poly[(R)-hydroxybutyrate] produced by a recombinant Escherichia coli.

Ultra-high-molecular-weight poly[(R)-3-hydroxybutyrate] (P(3HB)) (Mw = 3-11 x 10(6)) was produced from glucose by a recombinant Escherichia coli XL1-Blue (pSYL105) harboring Ralstonia eutropha H16 polyhydroxyalkanoate (PHA) biosynthesis genes. Morphology of ultra-high-molecular-weight P(3HB) granules in the recombinant cells was studied by transmission electron microscopy. The recombinant E. coli contained several P(3HB) granules within a cell. Freeze-fracture morphology of ultra-high-molecular-weight P(3HB) granules showed the needle-type as that of P(3HB) granules in R. eutropha. Both the P(3HB) granules in wet cells and wet native granules isolated from the recombinant cells proved to be amorphous on the X-ray diffraction patterns. Mechanical properties of ultra-high-molecular-weight P(3HB) films were markedly improved by stretching over 400%, resulting from high crystallinity and highly oriented crystal regions. Biodegradability of the films of ultra-high-molecular-weight P(3HB) was tested with an extracellular polyhydroxybutyrate depolymerase from Alcaligenes faecalis T1. The rate of enzymatic erosion of P(3HB) films was not dependent of the molecular weight but was dependent of the crystallinity. In addition, it is demonstrated that all ultra-high-molecular-weight P(3HB) films were completely degraded at 25 degrees C in a natural river freshwater within 3 weeks.     

Expression of a cloned 987P adhesion-antigen fimbrial determinant in Escherichia coli K-12 strain HB101

The properties of three independent enterotoxigenic Escherichia coli isolates known to express 987P adhesion fimbriae in a manner subject to phase variation were examined. Phase variation could not be correlated with any major changes in the plasmid DNA content of these strains or with readily detectable changes in any other tested phenotypic markers. The 987P genetic determinant from one of these strains, E. coli 987, was cloned into the non-fimbriated E. coli K-12 strains HB101, and expressed, using the cosmid vector system. 987P fimbriae produced by cells harbouring these recombinant plasmids (987P+ phenotype) could not be distinguished from 987P fimbriae produced by strain 987. Expression of 987P fimbriae from some recombinant plasmids was unstable but none of the recombinants exhibited the phase variation phenotype displayed by the parental strain. One recombinant plasmid, pPM200, contained an insert of strain 987 DNA of ca. 33 kb. The HB101[pPM200] displayed a rather stable 987P+ phenotype, but this was not true for several hosts, since pPM200 acquired approx. 20-kb deletions following transformations of E. coli K-12 strains other than HB101. The deletions mapped to the same region of pPM200 irrespective of the host strain transformed. Cells harbouring the deleted plasmids did not express 987P fimbriae (987P- phenotype).     

Rearrangement of gene order in the phaCAB operon leads to effective production of ultrahigh-molecular-weight poly[(R)-3-hydroxybutyrate] in genetically engineered Escherichia coli

Ultrahigh-molecular-weight poly[(R)-3-hydroxybutyrate] [UHMW-P(3HB)] synthesized by genetically engineered Escherichia coli is an environmentally friendly bioplastic material which can be processed into strong films or fibers. An operon of three genes (organized as phaCAB) encodes the essential proteins for the production of P(3HB) in the native producer, Ralstonia eutropha. The three genes of the phaCAB operon are phaC, which encodes the polyhydroxyalkanoate (PHA) synthase, phaA, which encodes a 3-ketothiolase, and phaB, which encodes an acetoacetyl coenzyme A (acetoacetyl-CoA) reductase. In this study, the effect of gene order of the phaCAB operon (phaABC, phaACB, phaBAC, phaBCA, phaCAB, and phaCBA) on an expression plasmid in genetically engineered E. coli was examined in order to determine the best organization to produce UHMW-P(3HB). The results showed that P(3HB) molecular weights and accumulation levels were both dependent on the order of the pha genes relative to the promoter. The most balanced production result was achieved in the strain harboring the phaBCA expression plasmid. In addition, analysis of expression levels and activity for P(3HB) biosynthesis enzymes and of P(3HB) molecular weight revealed that the concentration of active PHA synthase had a negative correlation with P(3HB) molecular weight and a positive correlation with cellular P(3HB) content. This result suggests that the level of P(3HB) synthase activity is a limiting factor for producing UHMW-P(3HB) and has a significant impact on P(3HB) production.     

人乳头瘤病毒16型湖北株E7基因的克隆和高效表达

利用基因克隆技术,将ＨＰＶ１６湖北株完整的Ｅ７基因克隆到含乳糖操纵子的表达载体ｐＷＲ５９０１上,经限制性酶切分析获得重组质粒ｐＷＨＢＥ７。ｐＷＨＢＥ７转化大肠杆菌后表达产生分子量为７０ｋＤ的融合蛋白ｌａｃＥ７,该蛋白在免疫印迹实验中可被标准Ｅ７单抗识别。经ＩＰＴＧ诱导后,Ｅ７融合蛋白产量可达细菌总蛋白含量的３０％以上。利用ｌａｃＥ７蛋白在细菌胞浆中形成包含体的性质,简便地提取并纯化了该蛋白质。结果为从免疫学角度探讨ＨＰＶ１６与宫颈癌的关系以及ＨＰＶ疫苗的研制打下基础。     

Recent advances in polyhydroxyalkanoate production by bacterial fermentation: mini-review.

Poly(3-hydroxybutyrate) [P(3HB)] and other polyhydroxyalkanoates (PHAs) have been drawing much attention as biodegradable substitutes for conventional nondegradable plastics. For the economical production of P(3HB), various bacterial strains, either wild-type or recombinant, and new fermentation strategies were developed for the production of P(3HB) with high concentration and productivity. To reduce the cost of carbon substrate, several processes for P(3HB) production from cheap carbon sources were also developed. P(3HB) can now be produced to a content of 80% of cell dry weight with the productivity greater than 4 g/l per h. Fermentation strategy was also developed for the efficient production of medium chain length PHA by high cell density culture. With all these advances, P(3HB) and PHAs can be produced by bacterial fermentation at a cost (ca. $2/kg) similar to that of other biodegradable polymers under development.     

利用重组大肠杆菌产聚-4-羟基丁酸的研究

重组大肠杆菌 E.coli XL-1 Blue（pKSSE5.3）携带Ralstonia　eutropha　H16的 PHA聚合酶基因（phaC）和Clostridium kluyveri的4-羟基丁酸:CoA转移酶基因（orfZ）,可以利用葡萄糖和4-羟基丁酸为碳源合成均聚的聚-4-羟基丁酸[P（4HB）]。优化培养基和培养条件后,进行了补料分批培养。结果表明,经68h左右培养,E.coli XL-1 Blue（pKSSE5.3）的发酵液中菌体干重达13g/L,P（4HB）的密度达5g/L,P（4HB）百分含量为36％。从收获的冻干细胞中提纯得到40g均聚的P（4HB）,为进一步分析检测P（4HB）生物、理化、加工特性及其应用价值成为可能。     

A new pathway for poly(3-hydroxybutyrate) production in Escherichia coli and Corynebacterium glutamicum by functional expression of a new acetoacetyl-coenzyme A synthase

A biosynthetic pathway for poly(3-hydroxybutyrate) [P(3HB)] was developed in Escherichia coli and Corynebacterium glutamicum by an acetoacetyl-coenzyme A (CoA) synthase (AACS) recently isolated from terpenoid-producing Streptomyces sp. strain CL190. Expression of AACS led to significant productions of P(3HB) in E. coli (10.5 wt %) and C. glutamicum (19.7 wt %).