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1.
Kuit W Minton NP López-Contreras AM Eggink G 《Applied microbiology and biotechnology》2012,94(3):729-741
In microorganisms, the enzyme acetate kinase (AK) catalyses the formation of ATP from ADP by de-phosphorylation of acetyl phosphate into acetic acid. A mutant strain of Clostridium acetobutylicum lacking acetate kinase activity is expected to have reduced acetate and acetone production compared to the wild type. In this work, a C. acetobutylicum mutant strain with a selectively disrupted ack gene, encoding AK, was constructed and genetically and physiologically characterized. The ack (-) strain showed a reduction in acetate kinase activity of more than 97% compared to the wild type. The fermentation profiles of the ack (-) and wild-type strain were compared using two different fermentation media, CGM and CM1. The latter contains acetate and has a higher iron and magnesium content than CGM. In general, fermentations by the mutant strain showed a clear shift in the timing of peak acetate production relative to butyrate and had increased acid uptake after the onset of solvent formation. Specifically, in acetate containing CM1 medium, acetate production was reduced by more than 80% compared to the wild type under the same conditions, but both strains produced similar final amounts of solvents. Fermentations in CGM showed similar peak acetate and butyrate levels, but increased acetoin (60%), ethanol (63%) and butanol (16%) production and reduced lactate (-50%) formation by the mutant compared to the wild type. These findings are in agreement with the proposed regulatory function of butyryl phosphate as opposed to acetyl phosphate in the metabolic switch of solventogenic clostridia. 相似文献
2.
3.
Ai-Yong He Chun-Yan Yin Hao Xu Xiang-Ping Kong Jia-Wei Xue Jing Zhu Min Jiang Hao Wu 《Bioprocess and biosystems engineering》2016,39(2):245-254
Reducing power such as NADH is an essential factor for acetone/butanol/ethanol (ABE) fermentation using Clostridium spp. The objective of this study was to increase available NADH in Clostridium beijerinckii IB4 by a microbial electrolysis cell (MEC) with an electron carrier to enhance butanol production. First of all, a MEC was performed without electron carrier to study the function of cathodic potential applying. Then, various electron carriers were tested, and neutral red (NR)-amended cultures showed an increase of butanol concentration. Optimal NR concentration (0.1 mM) was used to add in a MEC. Electricity stimulated the cell growth obviously and dramatically diminished the fermentation time from 40 to 28 h. NR and electrically reduced NR improved the final butanol concentration and inhibited the acetone generation. In the MEC with NR, the butanol concentration, yield, proportion and productivity were increased by 12.2, 17.4, 7.2 and 60.3 %, respectively. To further understand the mechanisms of NR, cathodic potential applying and electrically reduced NR, NADH and NAD+ levels, ATP levels and hydrogen production were determined. NR and electrically reduced NR also improved ATP levels and the ratio of NADH/NAD+, whereas they decreased hydrogen production. Thus, the MEC is an efficient method for enhancing the butanol production. 相似文献
4.
5.
Inui M Suda M Kimura S Yasuda K Suzuki H Toda H Yamamoto S Okino S Suzuki N Yukawa H 《Applied microbiology and biotechnology》2008,77(6):1305-1316
A recombinant butanol pathway composed of Clostridium acetobutylicum ATCC 824 genes, thiL, hbd, crt, bcd-etfB-etfA, and adhe1 (or adhe) coding for acetyl-CoA acetyltransferase (THL), β-hydroxybutyryl-CoA dehydrogenase (HBD), 3-hydroxybutyryl-CoA dehydratase
(CRT), butyryl-CoA dehydrogenase (BCD), butyraldehyde dehydrogenase (BYDH), and butanol dehydrogenase (BDH), under the tac promoter control was constructed and was introduced into Escherichia coli. The functional expression of these six enzymes was proved by demonstrating the corresponding enzyme activities using spectrophotometric,
high performance liquid chromatography and gas chromatography analyses. The BCD activity, which was not detected in E. coli previously, was shown in the present study by performing the procedure from cell extract preparation to activity measurement
under anaerobic condition. Moreover, the etfA and etfB co-expression was found to be essential for the BCD activity. In the case of BYDH activity, the adhe gene product was shown to have higher specificity towards butyryl-CoA compared to the adhe1 product. Butanol production from glucose was achieved by the highly concentrated cells of the butanologenic E. coli strains, BUT1 with adhe1 and BUT2 with adhe, under anaerobic condition, and the BUT1 and BUT2 strains were shown to produce 4 and 16-mM butanol with 6- and 1-mM butyrate
as a byproduct, respectively. This study reports the novel butanol production by an aerobically pregrown microorganism possessing
the genes of a strict anaerobe, Clostridium acetobutylicum. 相似文献
6.
In the past, it has been difficult to discriminate between hydrogen synthesis and uptake for the three active hydrogenases
in Escherichia coli (hydrogenase 1, 2, and 3); however, by combining isogenic deletion mutations from the Keio collection, we were able to see
the role of hydrogenase 3. In a cell that lacks hydrogen uptake via hydrogenase 1 (hyaB) and via hydrogenase 2 (hybC), inactivation of hydrogenase 3 (hycE) decreased hydrogen uptake. Similarly, inactivation of the formate hydrogen lyase complex, which produces hydrogen from formate
(fhlA) in the hyaB hybC background, also decreased hydrogen uptake; hence, hydrogenase 3 has significant hydrogen uptake activity. Moreover, hydrogen
uptake could be restored in the hyaB hybC hycE and hyaB hybC fhlA mutants by expressing hycE and fhlA, respectively, from a plasmid. The hydrogen uptake results were corroborated using two independent methods (both filter plate
assays and a gas-chromatography-based hydrogen uptake assay). A 30-fold increase in the forward reaction, hydrogen formation
by hydrogenase 3, was also detected for the strain containing active hydrogenase 3 activity but no hydrogenase 1 or 2 activity
relative to the strain lacking all three hydrogenases. These results indicate clearly that hydrogenase 3 is a reversible hydrogenase. 相似文献
7.
Feng Qi Chandresh Thakker Fayin Zhu Matthew Pena Ka-Yiu San George N. Bennett 《Journal of industrial microbiology & biotechnology》2018,45(11):993-1002
Clostridium acetobutylicum is a natural producer of butanol, butyrate, acetone and ethanol. The pattern of metabolites reflects the partitioning of redox equivalents between hydrogen and carbon metabolites. Here the exogenous genes of ferredoxin-NAD(P)+ oxidoreductase (FdNR) and trans-enoyl-coenzyme reductase (TER) are introduced to three different Clostridium acetobutylicum strains to investigate the distribution of redox equivalents and butanol productivity. The FdNR improves NAD(P)H availability by capturing reducing power from ferredoxin. A butanol production of 9.01 g/L (36.9% higher than the control), and the highest ratios of butanol/acetate (7.02) and C4/C2 (3.17) derived metabolites were obtained in the C acetobutylicum buk- strain expressing FdNR. While the TER functions as an NAD(P)H oxidase, butanol production was decreased in the C. acetobutylicum strains containing TER. The results illustrate that metabolic flux can be significantly changed and directed into butanol or butyrate due to enhancement of NAD(P)H availability by controlling electron flow through the ferredoxin node. 相似文献
8.
To understand the metabolic characteristics of Clostridium acetobutylicum and to examine the potential for enhanced butanol production, we reconstructed the genome-scale metabolic network from its
annotated genomic sequence and analyzed strategies to improve its butanol production. The generated reconstructed network
consists of 502 reactions and 479 metabolites and was used as the basis for an in silico model that could compute metabolic and growth performance for comparison with fermentation data. The in silico model successfully predicted metabolic fluxes during the acidogenic phase using classical flux balance analysis. Nonlinear
programming was used to predict metabolic fluxes during the solventogenic phase. In addition, essential genes were predicted
via single gene deletion studies. This genome-scale in silico metabolic model of C. acetobutylicum should be useful for genome-wide metabolic analysis as well as strain development for improving production of biochemicals,
including butanol.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
J. L. and H. Y. equally contributed to this work. 相似文献
9.
A salt-tolerant alkaliphilic actinomycete, Mit-1 was isolated from Mithapur, coastal region of Gujarat, India. The strain
was identified as Streptomyces clavuligerus and based on 16S rRNA gene sequence (EU146061) homology; it was related to Streptomyces sp. (AY641538.1). The organism could grow with up to 15% salt and pH 11, optimally at 5% and pH 9. It was able to tolerate
and secrete alkaline protease in the presence of a number of organic solvents including xylene, ethanol, acetone, butanol,
benzene and chloroform. Besides, it could also utilize these solvents as the sole source of carbon with significant enzyme
production. However, the organism produced spongy cell mass with all solvents and an orange brown soluble pigment was evident
with benzene and xylene. Further, the enzyme secretion increased by 50-fold in the presence of butanol. With acetone and ethanol;
the enzyme was highly active at 60–80°C and displayed optimum activity at 70°C. The protease was significantly stable and
catalyzed the reaction in the presence of xylene, acetone and butanol. However, ethanol and benzene affected the catalysis
of the enzyme adversely. Crude enzyme preparation was more stable at 37°C in solvents as compared to partially purified and
purified enzymes. The study holds significance as only few salt-tolerant alkaliphilic actinomycetes are explored and information
on their enzymatic potential is still scares. To the best of our knowledge this is the first report on organic solvent tolerant
protease from salt-tolerant alkaliphilic actinomycetes. 相似文献
10.
Daniel Ng Hing Kah Chin Victor Vai Tak Wong 《Journal of industrial microbiology & biotechnology》2009,36(11):1345-1351
11.
Guo T Tang Y Zhang QY Du TF Liang DF Jiang M Ouyang PK 《Journal of industrial microbiology & biotechnology》2012,39(3):401-407
Clostridium beijerinckii mutant strain IB4, which has a high level of inhibitor tolerance, was screened by low-energy ion implantation and used for
butanol fermentation from a non-detoxified hemicellulosic hydrolysate of corn fiber treated with dilute sulfuric acid (SAHHC).
Evaluation of toxicity showed C. beijerinckii IB4 had a higher level of tolerance than parent strain C. beijerinckii NCIMB 8052 for five out of six phenolic compounds tested (the exception was vanillin). Using glucose as carbon source, C. beijerinckii IB4 produced 9.1 g l−1 of butanol with an acetone/butanol/ethanol (ABE) yield of 0.41 g g−1. When non-detoxified SAHHC was used as carbon source, C. beijerinckii NCIMB 8052 grew well but ABE production was inhibited. By contrast, C. beijerinckii IB4 produced 9.5 g l−1 of ABE with a yield of 0.34 g g−1, including 2.2 g l−1 acetone, 6.8 g l−1 butanol, and 0.5 g l−1 ethanol. The remarkable fermentation and inhibitor tolerance of C. beijerinckii IB4 appears promising for ABE production from lignocellulosic materials. 相似文献
12.
Gutiérrez-Lomelí M Torres-Guzmán JC González-Hernández GA Cira-Chávez LA Pelayo-Ortiz C Ramírez-Córdova Jde J 《Antonie van Leeuwenhoek》2008,93(4):363-371
This work assessed the effect of the overexpression of ADH1 and HXT1 genes in the Saccharomyces cerevisiae AR5 strain during fermentation of Agave tequilana Weber blue variety must. Both genes were cloned individually and simultaneously into a yeast centromere plasmid. Two transformant
strains overexpressing ADH1 and HXT1 individually and one strain overexpressing both genes were randomly selected and named A1, A3 and A5 respectively. Overexpression
effect on growth and ethanol production of the A1, A3 and A5 strains was evaluated in fermentative conditions in A. tequilana Weber blue variety must and YPD medium. During growth in YPD and Agave media, all the recombinant strains showed lower cell
mass formation than the wild type AR5 strain. Adh enzymatic activity in the recombinant strains A1 and A5 cultivated in A. tequilana and YPD medium was higher than in the wild type. The overexpression of both genes individually and simultaneously had no
significant effect on ethanol formation; however, the fermentative efficiency of the A5 strain increased from 80.33% to 84.57%
and 89.40% to 94.29% in YPD and Agave medium respectively. 相似文献
13.
Development of a butanologenic strain with high selectivity for butanol production is often proposed as a possible route for
improving the economics of biobutanol production by solventogenic Clostridium species. The acetoacetate decarboxylase (aadc) gene encoding acetoacetate decarboxylase (AADC), which catalyzes the decarboxylation of acetoacetate into acetone and CO2, was successfully disrupted by homologous recombination in solventogenic Clostridium beijerinckii NCIMB 8052 to generate an aadc
−
mutant. Our fermentation studies revealed that this mutant produces a maximum acetone concentration of 3 g/L (in P2 medium),
a value comparable to that produced by wild-type C. beijerinckii 8052. Therefore, we postulated that AADC-catalyzed decarboxylation of acetoacetate is not the sole means for acetone generation.
Our subsequent finding that non-enzymatic decarboxylation of acetoacetate in vitro, under conditions similar to in vivo acetone–butanol–ethanol
(ABE) fermentation, produces 1.3 to 5.2 g/L acetone between pH 6.5 and 4 helps rationalize why various knock-out and knock-down
strategies designed to disrupt aadc in solventogenic Clostridium species did not eliminate acetone production during ABE fermentation. Based on these results, we discuss alternatives to
enhance selectivity for butanol production. 相似文献
14.
Shewanella decolorationis S12, a representative dissimilatory azo-reducing bacterium of Shewanella genus, can grow by coupling the oxidation of hydrogen to the reduction of azo compounds as the sole electron acceptor, indicating that an uptake hydrogenase is an important component for electron transfer for azoreduction. For searching to the uptake hydrogenase in the genome of S. decolorationis, two operons, hyd and hya, were cloned and sequenced, which encode periplasmically oriented Fe-only hydrogenase and a Ni-Fe hydrogenase, respectively, according to the homologous comparison with other bacterial hydrogenases. In order to assess the roles of these two enzymes in hydrogen-dependent azoreduction and growth, hyd- and hya-deficient mutants were generated by gene replacement. Hya was found to be required for hydrogen-dependent reduction of azo compound by resting cell suspensions and to be essential for growth with hydrogen as electron donor and azo compound as electron acceptor. Hyd, in contrast, was not. These findings suggest that Hya is an essential respiratory hydrogenase of dissimilatory azoreduction in S. decolorationis. 相似文献
15.
Background
The maturation of hydrogenases into active enzymes is a complex process and e.g. a correctly assembled active site requires the involvement of at least seven proteins, encoded by hypABCDEF and a hydrogenase specific protease, encoded either by hupW or hoxW. The N2-fixing cyanobacterium Nostoc sp. strain PCC 7120 may contain both an uptake and a bidirectional hydrogenase. The present study addresses the presence and expression of hyp-genes in Nostoc sp. strain PCC 7120. 相似文献16.
Objectives
To improve H2 production, the green algae Chlamydomonas reinhardtii cc849 was co-cultured with Azotobacter chroococcum.Results
The maximum H2 production of the co-culture was 350% greater than that of the pure algal cultures under optimal H2 production conditions. The maximum growth and the respiratory rate of the co-cultures were about 320 and 300% of the controls, and the dissolved O2 of co-cultures was decreased 74%. Furthermore, the in vitro maximum hydrogenase activity of the co-culture was 250% greater than that of the control, and the in vivo maximum hydrogenase activity of the co-culture was 1.4-fold greater than that of the control. In addition, the maximum starch content of co-culture was 1400% that of the control.Conclusions
Azotobacter chroococcum improved the H2 production of the co-cultures by decreasing the O2 content and increasing the growth and starch content of the algae and the hydrogenase activity of the co-cultures relative to those of pure algal cultures.17.
Sang-Hyun Lee Moon-Ho Eom Jin-Dal-Rae Choi Sooah Kim Jungyeon Kim Yong-An Shin Kyoung Heon Kim 《Bioprocess and biosystems engineering》2016,39(5):695-702
In situ butanol recovery fermentation has been intensively studied as an effective alternative to conventional butanol production, which is limited due to the cellular toxicity of butanol. However, the low biocompatibility of adsorbents often leads to failure of in situ recovery fermentations. In this study, Clostridium beijerinckii NCIMB 8052 was cultured in flasks without shaking and in situ recovery fermentation was performed by using an adsorbent L493. The amounts of acetone, butanol, and ethanol (ABE) increased by 34.4 % in the presence of the adsorbent. In contrast, cell growth and production of organic acids and ABE were retarded in the 7-L batch fermentations with in situ butanol recovery. Cell damage occurred in the fermentor upon agitation in the presence of the adsorbent, unlike in static flask cultures with in situ recovery. Ex situ recovery fermentation using circulation of fermentation broth after mid-exponential phase of cell growth was developed to avoid adsorbent-cell incompatibility. No apparent cell damage was observed and 25.7 g/L of ABE was produced from 86.2 g/L glucose in the fed-batch mode using 7 L fermentors. Thus, ex situ recovery fermentation with C. beijerinckii is effective for enhancing butanol fermentation. 相似文献
18.
Scotcher MC Huang KX Harrison ML Rudolph FB Bennett GN 《Journal of industrial microbiology & biotechnology》2003,30(7):414-420
The high solvent phenotype of Clostridium acetobutylicum mutants B and H was complemented by the introduction of a plasmid that contains either an intact or partially-deleted copy
of solR, restoring acetone and butanol production to wild-type levels. This demonstrates that the solR open reading frame on pSOLThi is not required to restore solvent levels. The promoter region upstream of alcohol dehydrogense
E (adhE) was examined in efforts to identify sites that play major roles in the control of expression. A series of adhE promoter fragments was constructed and the expression of each in acid- and solvent-phases of growth was analyzed using a
chloramphenicol acetyl-transferase reporter system. Our results show that a region beyond the 0A box is needed for full induction
of the promoter. Additionally, we show that the presence of sequences around a possible processing site designated S2 may
have a negative role in the regulation of adhE expression. 相似文献
19.
To utilize fermentative bacteria for producing the alternative fuel hydrogen, we performed successive rounds of P1 transduction
from the Keio Escherichia coli K-12 library to introduce multiple, stable mutations into a single bacterium to direct the metabolic flux toward hydrogen
production. E. coli cells convert glucose to various organic acids (such as succinate, pyruvate, lactate, formate, and acetate) to synthesize
energy and hydrogen from formate by the formate hydrogen-lyase (FHL) system that consists of hydrogenase 3 and formate dehydrogenase-H.
We altered the regulation of FHL by inactivating the repressor encoded by hycA and by overexpressing the activator encoded by fhlA, removed hydrogen uptake activity by deleting hyaB (hydrogenase 1) and hybC (hydrogenase 2), redirected glucose metabolism to formate by using the fdnG, fdoG, narG, focA, focB, poxB, and aceE mutations, and inactivated the succinate and lactate synthesis pathways by deleting frdC and ldhA, respectively. The best of the metabolically engineered strains, BW25113 hyaB hybC hycA fdoG frdC ldhA aceE, increased hydrogen production 4.6-fold from glucose and increased the hydrogen yield twofold from 0.65 to 1.3 mol H2/mol glucose (maximum, 2 mol H2/mol glucose). 相似文献
20.
The structural gene for sphingomyelinase (SMase) from Streptomyces
griseocarneus, was introduced into Streptomyces lividans using a shuttle vector, pUC702, for Escherichia coli/S. lividans. High-level secretory production of SMase was achieved using the promoter, signal sequence and terminator regions of phospholipase
D from Streptoverticillium cinnamoneum. The transformant constitutively expressed a high specific activity of SMase extracellularly during batch culture. Maximum
SMase activity (555 ± 114 U/mg protein) was with 1.75 M MgCl2 which was about 50-fold more than that with 10 mM MgCl2. 相似文献