共查询到20条相似文献,搜索用时 78 毫秒
1.
Joo Shun Tan Tau Chuan Ling Shuhaimi Mustafa Yew Joon Tam Ramakrishnan Nagasundara Ramanan Arbakariya B. Ariff 《Process Biochemistry》2013,48(4):551-558
A stirred tank bioreactor (STB) integrated with an expanded bed adsorption (EBA) system containing anion-exchange resin (Diaion WA30) was developed for in situ removal of acetate to increase the production of α-interferon-2b (α-PrIFN-2b) by Escherichia coli (E. coli). Although the total acetate (9.79 g/L) secreted by E. coli in the integrated STB/EBA system was higher than that in a bioreactor with dispersed resin or a conventional batch bioreactor, cell growth (14.97 g/L) and α-PrIFN-2b production (867.4 μg/L) were significantly improved owing to the high efficiency of acetate removal from the culture. The production of α-PrIFN-2b in the integrated STB/EBA system was improved by 3-fold and 1.4-fold over that obtained in a conventional batch bioreactor and a bioreactor containing dispersed resins, respectively. 相似文献
2.
An Escherichia coli strain, JM109, was successfully engineered into an efficient hyaluronic acid (HA) producer by co-expressing the only known
class-II HA synthase from a Gram-negative bacterium (Pasteurella multocida) and uridine diphosphate-glucose dehydrogenase from E. coli K5 strain. The engineered strain produced about 0.5 g/L HA in shake flask culture and about 2.0–3.8 g/L in a fed-batch fermentation
process in a 1-L bioreactor. The sharp increase in viscosity associated with HA accumulation necessitated pure oxygen supplement
to maintain fermentation in aerobic regime. Precursor supply during HA synthesis was probed by glucosamine supplement, which
shortens biosynthesis pathway and eliminates one step requiring ATP. HA synthesis was increased with glucosamine supplement
from 2.7 to 3.7 g/L (37%), which was mirrored with a concomitant 42% decrease in pure oxygen input, suggesting a close connection
between energy metabolism and precursor supply. Decoupling HA synthesis from cell growth by using fosfomycin (an inhibitor
for cell wall synthesis) led to a 70% increase in HA synthesis, suggesting detrimental effects on HA synthesis from cell growth
via precursor competition. This study demonstrates a potentially viable process for HA based on a recombinant E. coli strain. In addition, the precursor supply limitation identified in this study suggests new engineering targets in subsequent
metabolic engineering efforts. 相似文献
3.
Manabu Kanno Taiki Katayama Hideyuki Tamaki Yasuo Mitani Xian-Ying Meng Tomoyuki Hori Takashi Narihiro Naoki Morita Tamotsu Hoshino Isao Yumoto Nobutada Kimura Satoshi Hanada Yoichi Kamagata 《Applied and environmental microbiology》2013,79(22):6998-7005
Despite their importance as a biofuel production platform, only a very limited number of butanol-tolerant bacteria have been identified thus far. Here, we extensively explored butanol- and isobutanol-tolerant bacteria from various environmental samples. A total of 16 aerobic and anaerobic bacteria that could tolerate greater than 2.0% (vol/vol) butanol and isobutanol were isolated. A 16S rRNA gene sequencing analysis revealed that the isolates were phylogenetically distributed over at least nine genera: Bacillus, Lysinibacillus, Rummeliibacillus, Brevibacillus, Coprothermobacter, Caloribacterium, Enterococcus, Hydrogenoanaerobacterium, and Cellulosimicrobium, within the phyla Firmicutes and Actinobacteria. Ten of the isolates were phylogenetically distinct from previously identified butanol-tolerant bacteria. Two relatively highly butanol-tolerant strains CM4A (aerobe) and GK12 (obligate anaerobe) were characterized further. Both strains changed their membrane fatty acid composition in response to butanol exposure, i.e., CM4A and GK12 exhibited increased saturated and cyclopropane fatty acids (CFAs) and long-chain fatty acids, respectively, which may serve to maintain membrane fluidity. The gene (cfa) encoding CFA synthase was cloned from strain CM4A and expressed in Escherichia coli. The recombinant E. coli showed relatively higher butanol and isobutanol tolerance than E. coli without the cfa gene, suggesting that cfa can confer solvent tolerance. The exposure of strain GK12 to butanol by consecutive passages even enhanced the growth rate, indicating that yet-unknown mechanisms may also contribute to solvent tolerance. Taken together, the results demonstrate that a wide variety of butanol- and isobutanol-tolerant bacteria that can grow in 2.0% butanol exist in the environment and have various strategies to maintain structural integrity against detrimental solvents. 相似文献
4.
Higher alcohols such as isobutanol possess several physical characteristics that make them attractive as biofuels such as higher energy densities and infrastructure compatibility. Here we have developed a rapid evolutionary strategy for isolating strains of Escherichia coli that effectively produce isobutanol from glucose utilizing random mutagenesis and a growth selection scheme. By selecting for mutants with the ability to grow in the presence of the valine analog norvaline, we obtained E. coli NV3; a strain with improved 24-h isobutanol production (8.0 g/L) in comparison with a productivity of 5.3 g/L isobutanol obtained with the parental wild type strain. Genomic sequencing of NV3 identified the insertion of a stop codon in the C-terminus of the RNA polymerase σs-factor, RpoS. Upon repair of this inhibitory mutation (strain NV3r1), a final isobutanol titer of 21.2 g/L isobutanol was achieved in 99 h with a yield of 0.31 g isobutanol/g glucose or 76% of theoretical maximum. Furthermore, a mutation in ldhA, encoding d-lactate dehydrogenase, was identified in NV3; however, repair of LdhA in NV3r1 had no affect on LdhA activity detected from cell extracts or on isobutanol productivity. Further study of NV3r1 may identify novel genotypes that confer improved isobutanol production. 相似文献
5.
In the present work, Bacillus subtilis was engineered as the cell factory for isobutanol production due to its high tolerance to isobutanol. Initially, an efficient
heterologous Ehrlich pathway controlled by the promoter P43 was introduced into B. subtilis for the isobutanol biosynthesis. Further, investigation of acetolactate synthase of B. subtilis, ketol-acid reductoisomerase, and dihydroxy-acid dehydratase of Corynebacterium glutamicum responsible for 2-ketoisovalerate precursor biosynthesis showed that acetolactate synthase played an important role in isobutanol
biosynthesis. The overexpression of acetolactate synthase led to a 2.8-fold isobutanol production compared with the control.
Apart from isobutanol, alcoholic profile analysis also confirmed the existence of 1.21 g/L ethanol, 1.06 g/L 2-phenylethanol,
as well as traces of 2-methyl-1-butanol and 3-methyl-1-butanol in the fermentation broth. Under microaerobic condition, the
engineered B. subtilis produced up to 2.62 g/L isobutanol in shake-flask fed-batch fermentation, which was 21.3% higher than that in batch fermentation. 相似文献
6.
Jianzhong Yang Shaozhao Wang Marie-Josée Lorrain Denis Rho Kofi Abokitse Peter C. K. Lau 《Applied microbiology and biotechnology》2009,84(5):867-876
Recombinant Escherichia coli whole-cell biocatalysts harboring either a Baeyer–Villiger monooxygenase or ferulic acid decarboxylase were employed in organic-aqueous
two-phase bioreactor systems. The feasibility of the bioproduction of water-insoluble products, viz., lauryl lactone from
cyclododecanone and 4-vinyl guaiacol from ferulic acid were examined. Using hexadecane as the organic phase, 10∼16 g of lauryl
lactone were produced in a 3-l bioreactor that operated in a semicontinuous mode compared to 2.4 g of product in a batch mode.
For the decarboxylation of ferulic acid, a new recombinant biocatalyst, ferulic acid decarboxylase derived from Bacillus pumilus, was constructed. Selected solvents as well as other parameters for in situ recovery of vinyl guaiacol were investigated.
Up to 13.8 g vinyl guaiacol (purity of 98.4%) were obtained from 25 g of ferulic acid in a 2-l working volume bioreactor by
using octane as organic phase. These selected examples highlight the superiority of the two-phase biotransformations systems
over the conventional batch mode. 相似文献
7.
Zheng-Jun Li Lei Cai Qiong Wu Guo-Qiang Chen 《Applied microbiology and biotechnology》2009,83(5):939-947
NAD kinase was overexpressed to enhance the accumulation of poly(3-hydroxybutyrate) (PHB) in recombinant Escherichia coli harboring PHB synthesis pathway via an accelerated supply of NADPH, which is one of the most crucial factors influencing
PHB production. A high copy number expression plasmid pE76 led to a stronger NAD kinase activity than that brought about by
the low copy number plasmid pELRY. Overexpressing NAD kinase in recombinant E. coli was found not to have a negative effect on cell growth in the absence of PHB synthesis. Shake flask experiments demonstrated
that excess NAD kinase in E. coli harboring the PHB synthesis operon could increase the accumulation of PHB to 16–35 wt.% compared with the controls; meanwhile,
NADP concentration was enhanced threefold to sixfold. Although the two NAD kinase overexpression recombinants exhibited large
disparity on NAD kinase activity, their influence on cell growth and PHB accumulation was not proportional. Under the same
growth conditions without process optimization, the NAD kinase-overexpressing recombinant produced 14 g/L PHB compared with
7 g/L produced by the control in a 28-h fermentor study. In addition, substrate to PHB yield Y
PHB/glucose showed an increase from 0.08 g PHB/g glucose for the control to 0.15 g PHB/g glucose for the NAD kinase-overexpressing strain,
a 76% increase for the Y
PHB/glucose. These results clearly showed that the overexpression of NAD kinase could be used to enhance the PHB synthesis. 相似文献
8.
The possibility of using in situ addition of anion-exchange resin for the removal of acetate in the culture aimed at improving growth of E. coli and expression of periplasmic human interferon-α2b (PrIFN-α2b) was studied in shake flask culture and stirred tank bioreactor. Different types of anion-exchange resin were evaluated and the concentration of anion-exchange resin was optimized using response surface methodology. The addition of anion-exchange resins reduced acetate accumulation in the culture, which in turn, improved growth of E. coli and enhanced PrIFN-α2b expression. The presence of anion-exchange resins did not influence the physiology of the cells. The weak base anion-exchange resins, which have higher affinity towards acetate, yielded higher PrIFN-α2b expression as compared to strong anion-exchange resins. High concentrations of anion-exchange resin showed inhibitory effect towards growth of E. coli as well as the expression of PrIFN-α2b. The maximum yield of PrIFN-α2b in shake flask culture (501.8 μg/L) and stirred tank bioreactor (578.8 μg/L) was obtained at ion exchange resin (WA 30) concentration of 12.2 g/L. The production of PrIFN-α2b in stirred tank bioreactor with the addition of ion exchange resin was about 1.8-fold higher than that obtained in fermentation without ion exchange resin (318.4 μg/L). 相似文献
9.
Phloroglucinol is a valuable chemical which has been successfully produced by metabolically engineered Escherichia coli. However, the low productivity remains a bottleneck for large-scale application and cost-effective production. In the present
work, we cloned the key biosynthetic gene, phlD (a type III polyketide synthase), into a bacterial expression vector to produce phloroglucinol in E. coli and developed different strategies to re-engineer the recombinant strain for robust synthesis of phloroglucinol. Overexpression
of E. coli marA (multiple antibiotic resistance) gene enhanced phloroglucinol resistance and elevated phloroglucinol production to 0.27 g/g
dry cell weight. Augmentation of the intracellular malonyl coenzyme A (malonyl-CoA) level through coordinated expression of
four acetyl-CoA carboxylase (ACCase) subunits increased phloroglucinol production to around 0.27 g/g dry cell weight. Furthermore, the coexpression of ACCase and marA caused another marked improvement in phloroglucinol production 0.45 g/g dry cell weight, that is, 3.3-fold to the original
strain. Under fed-batch conditions, this finally engineered strain accumulated phloroglucinol up to 3.8 g/L in the culture
12 h after induction, corresponding to a volumetric productivity of 0.32 g/L/h. This result was the highest phloroglucinol
production to date and showed promising to make the bioprocess economically feasible. 相似文献
10.
Artificial amplification of gluconeogenic phosphoenolpyruvate carboxykinase (PCK) under glycolytic conditions enables Escherichia coli to maintain a greater intracellular ATP concentration during its growth phase. To demonstrate the biotechnological benefit
of E. coli harboring a high intracellular ATP concentration, we compared the recombinant protein synthesis of a soluble protein (enhanced
green fluorescence protein, GFP) with that of a secretory protein (alkaline protease, AP), under control of the T7 promoter
in E. coli BL21(DE3) overexpressing PCK. According to the batch fermentations, the strain overexpressing PCK produced more GFP and AP
with a lower increase in biomass than the control strain. In a chemostat culture (D = 0.7 h−1), the GFP production in the PCK overexpressing strain was 99.0 ± 4.31 mg/g cell, with a biomass of 0.22 g/L, while that of
the control strain was 53.5 ± 3.07 mg/g cell, with a biomass of 0.35 g/L. These results indicate that the PCK overexpressing
E. coli strain harboring high intracellular levels of ATP can be useful as a protein-synthesizing host. The potential uses of the
strain and associated rationale are discussed. 相似文献
11.
Kevin Michael Smith Kwang-Myung Cho James C. Liao 《Applied microbiology and biotechnology》2010,87(3):1045-1055
The production of isobutanol in microorganisms has recently been achieved by harnessing the highly active 2-keto acid pathways.
Since these 2-keto acids are precursors of amino acids, we aimed to construct an isobutanol production platform in Corynebacterium glutamicum, a well-known amino-acid-producing microorganism. Analysis of this host’s sensitivity to isobutanol toxicity revealed that
C. glutamicum shows an increased tolerance to isobutanol relative to Escherichia coli. Overexpression of alsS of Bacillus subtilis, ilvC and ilvD of C. glutamicum, kivd of Lactococcus lactis, and a native alcohol dehydrogenase, adhA, led to the production of 2.6 g/L isobutanol and 0.4 g/L 3-methyl-1-butanol in 48 h. In addition, other higher chain alcohols
such as 1-propanol, 2-methyl-1-butanol, 1-butanol, and 2-phenylethanol were also detected as byproducts. Using longer-term
batch cultures, isobutanol titers reached 4.0 g/L after 96 h with wild-type C. glutamicum as a host. Upon the inactivation of several genes to direct more carbon through the isobutanol pathway, we increased production
by ∼25% to 4.9 g/L isobutanol in a ∆pyc∆ldh background. These results show promise in engineering C. glutamicum for higher chain alcohol production using the 2-keto acid pathways. 相似文献
12.
Escherichia coli was metabolically engineered using a new host-vector system to produce phloroglucinol. The key biosynthetic gene phlD (encoding a type III polyketide synthase) from Pseudomonas fluorescens was expressed in E. coli using the stationary-phase promoter of the fic gene and a high-copy plasmid. In shake-flasks, the engineered strain produced phloroglucinol up to 0.28 g/l with a productivity
of 0.014 g/l h. About 9.2% of the glucose consumed was converted to phloroglucinol after 20 h. Compared with the widely used
inducible T7 promoter system, this strain did not require IPTG induction and the final titer of phloroglucinol was 22% higher. 相似文献
13.
Coupling lactate fermentation with cell growth was investigated in shake-flask and bioreactor cultivation systems by increasing
aeration to improve lactate productivity in Escherichia coli CICIM B0013-070 (ackA
pta
pps
pflB
dld
poxB
adhE
frdA). In shake-flasks, cells reached 1 g dry wt/l then, cultivated at 100 rpm and 42°C, achieved a twofold higher productivity
of lactic acid compared to aerobic and O2-limited two-phase fermentation. The cells in the bioreactor yielded an overall volumetric productivity of 5.5 g/l h and a
yield of 86 g lactic acid/100 g glucose which were 66% higher and the same level compared to that of the aerobic and O2-limited two-phase fermentation, respectively, using scaled-up conditions optimized from shake-flask experiments. These results
have revealed an approach for improving production of fermentative products in E. coli. 相似文献
14.
Raisa Vermasvuori Jani Koskinen Katri Salonen Noora Sirén Jan Weegar John Dahlbacka Nisse Kalkkinen Niklas von Weymarn 《Biotechnology progress》2009,25(1):95-102
Three popular expression host systems Escherichia coli, Pichia pastoris and Drosophila S2 were analyzed techno‐economically using HIV‐1 Nef protein as the model product. On scale of 100 mg protein, the labor costs corresponded to 52–83% of the manufacturing costs. When analyzing the cost impact of the different phases (strain/cell line construction, bioreactor production, and primary purification), we found that with the microbial host systems the strain construction phase was most significant generating 56% (E. coli) and 72% (P. pastoris) of the manufacturing costs, whereas with the Drosophila S2 system the cell line construction and bioreactor production phases were equally significant (46 and 47% of the total costs, respectively). With different titers and production goal of 100 mg of Nef protein, the costs of P. pastoris and Drosophila S2 systems were about two and four times higher than the respective costs of the E. coli system. When equal titers and bioreactor working volumes (10 L) were assumed for all three systems, the manufacturing costs of the bioreactor production of the P. pastoris and Drosophila S2 systems were about two and 2.5 times higher than the respective costs of the E. coli system. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 相似文献
15.
Ling Ding Xi Li Peng Liu Shiqian Li Jiliang Lv 《Biological trace element research》2010,137(3):364-372
The biological effect of Se and Cu2+ on Escherichia coli (E. coli) growth was studied by using a 3114/3236 TAM Air Isothermal Calorimeter, ampoule method, at 37°C. From the thermogenesis
curves, the thermokinetic equations were established under different conditions. The kinetics showed that a low concentration
of Se (1–10 μg/mL) promoted the growth of E. coli, and a high concentration of Se (>10 μg/mL) inhibited the growth, but the Cu2+ was always inhibiting the growth of E. coli. Moreover, there was an antagonistic or positive synergistic effect of Se and Cu2+ on E. coli in the different culture medium when Se was 1–10 μg/ml and Cu2+ was 1–20 μg/ml. There was a negative synergistic effect of Se and Cu2+ on E. coli when Se was higher than 10 μg/ml and Cu2+ was higher than 20 μg/ml. The antagonistic or synergistic effect between Se and Cu2+ on E. coli was related to the formation of Cu–Se complexes under the different experimental conditions chosen. 相似文献
16.
17.
The gene encoding malate dehydrogenase (MDH) was overexpressed in a pflB ldhA double mutant of Escherichia coli, NZN111, for succinic acid production. With MDH overexpression, NZN111/pTrc99A-mdh restored the ability to metabolize glucose anaerobically and 0.55 g/L of succinic acid was produced from 3 g/L of glucose
in shake flask culture. When supplied with 10 g/L of sodium bicarbonate (NaHCO3), the succinic acid yield of NZN111/pTrc99A-mdh reached 1.14 mol/mol glucose. Supply of NaHCO3 also improved succinic acid production by the control strain, NZN111/pTrc99A. Measurement of key enzymes activities revealed
that phosphoenolpyruvate (PEP) carboxykinase and PEP carboxylase in addition to MDH played important roles. Two-stage culture
of NZN111/pTrc99A-mdh was carried out in a 5-L bioreactor and 12.2 g/L of succinic acid were produced from 15.6 g/L of glucose. Fed-batch culture
was also performed, and the succinic acid concentration reached 31.9 g/L with a yield of 1.19 mol/mol glucose. 相似文献
18.
José Utrilla Guillermo Gosset Alfredo Martinez 《Journal of industrial microbiology & biotechnology》2009,36(8):1057-1062
A derivative strain of Escherichia coli MG1655 for d-lactate production was constructed by deleting the pflB, adhE and frdA genes; this strain was designated “CL3.” Results show that the CL3 strain grew 44% slower than its parental strain under
nonaerated (fermentative) conditions due to the inactivation of the main acetyl-CoA production pathway. In contrast to E. coli B and W3110 pflB derivatives, we found that the MG1655 pflB derivative is able to grow in mineral media with glucose as the sole carbon source under fermentative conditions. The glycolytic
flux was 2.8-fold higher in CL3 when compared to the wild-type strain, and lactate yield on glucose was 95%. Although a low
cell mass formed under fermentative conditions with this strain (1.2 g/L), the volumetric productivity of CL3 was 1.31 g/L h.
In comparison with the parental strain, CL3 has a 22% lower ATP/ADP ratio. In contrast to wild-type E. coli, the ATP yield from glucose to lactate is 2 ATP/glucose, so CL3 has to improve its glycolytic flux in order to fulfill its
ATP needs in order to grow. The aceF deletion in strains MG1655 and CL3 indicates that the pyruvate dehydrogenase (PDH) complex is functional under glucose-fermentative
conditions. These results suggest that the pyruvate to acetyl-CoA flux in CL3 is dependent on PDH activity and that the decrease
in the ATP/ADP ratio causes an increase in the flux of glucose to lactate. 相似文献
19.
Boghigian BA Myint M Wu J Pfeifer BA 《Journal of industrial microbiology & biotechnology》2011,38(11):1809-1820
Natural products have long served as rich sources of drugs possessing a wide range of pharmacological activities. The discovery
and development of natural product drug candidates is often hampered by the inability to efficiently scale and produce a molecule
of interest, due to inherent qualities of the native producer. Heterologous biosynthesis in an engineering and process-friendly
host emerged as an option to produce complex natural products. Escherichia coli has previously been utilized to produce complex precursors to two popular natural product drugs, erythromycin and paclitaxel.
These two molecules represent two of the largest classes of natural products, polyketides and isoprenoids, respectively. In
this study, we have developed a platform E. coli strain capable of simultaneous production of both product precursors at titers greater than 15 mg l−1. The utilization of a two-phase batch bioreactor allowed for very strong in situ separation (having a partitioning coefficient
of greater than 5,000), which would facilitate downstream purification processes. The system developed here could also be
used in metagenomic studies to screen environmental DNA for natural product discovery and preliminary production experiments. 相似文献
20.