Fermentation characterization and flux analysis of recombinant strains of Clostridium acetobutylicum with an inactivated solR gene.

The effect of solR inactivation on the metabolism of Clostridium acetobutylicum was examined using fermentation characterization and metabolic flux analysis. The solR-inactivated strain (SolRH) of this study had a higher rate of glucose utilization and produced higher solvent concentrations (by 25%, 14%, and 81%, respectively, for butanol, acetone, and ethanol) compared to the wild type. Strain SolRH(pTAAD), carrying a plasmid-encoded copy of the bifunctional alcohol/aldehyde dehydrogenase gene (aad) used in butanol production, produced even higher concentrations of solvents (by 21%, 45%, and 62%, respectively, for butanol, acetone, and ethanol) than strain SolRH. Clarithromycin used for strain SolRH maintenance during SolRH(pTAAD) fermentations did not alter product formation; however, tetracycline used for pTAAD maintenance resulted in 90% lower solvent production.

Characterization of recombinant strains of the Clostridium acetobutylicum butyrate kinase inactivation mutant: need for new phenomenological models for solventogenesis and butanol inhibition?

Two metabolic engineering tools, namely gene inactivation and gene overexpression, were employed to examine the effects of two genetic modifications on the fermentation characteristics of Clostridium acetobutylicum. Inactivation of the butyrate kinase gene (buk) was examined using strain PJC4BK, while the combined effect of buk inactivation and overexpression of the aad gene-encoding the alcohol aldehyde dehydrogense (AAD) used in butanol formation-was examined using strain PJC4BK(pTAAD). The two strains were characterized in controlled pH > or = 5.0 fermentations, and by a recently enhanced method of metabolic flux analysis. Strain PJC4BK was previously genetically characterized, and fermentation experiments at pH > or = 5.5 demonstrated good, but not exceptional, solvent-production capabilities. Here, we show that this strain is a solvent superproducer in pH > or = 5.0 fermentations producing 225 mM (16.7 g/L) of butanol, 76 mM of acetone (4.4 g/L), and 57 mM (2.6 g/L) of ethanol. Strain PJC4BK(pTAAD) produced similar amounts of butanol and acetone but 98 mM (4.5 g/L) of ethanol. Both strains overcame the 180 mM (13 g/L) butanol toxicity limit, without any selection for butanol tolerance. Work with strain PJC4BK(pTAAD) is the first reported use of dual antibiotic selection in C. acetobutylicum. One antibiotic was used for selection of strain PJC4BK while the second antibiotic selected for the pTAAD presence. Overexpression of aad from pTAAD resulted in increased ethanol production but did not increase butanol titers, thus indicating that AAD did not limit butanol production under these fermentation conditions. Metabolic flux analysis showed a decrease in butyrate formation fluxes by up to 75% and an increase in acetate formation fluxes of up to 100% during early growth. The mean specific butanol and ethanol formation fluxes increased significantly in these recombinant strains, up to 300% and 400%, respectively. Onset of solvent production occurred during the exponential-growth phase when the culture optical density was very low and when total and undissociated butyric acid levels were <1 mM. Butyrate levels were low throughout all fermentations, never exceeding 20 mM. Thus, threshold butyrate concentrations are not necessary for solvent production in these stains, suggesting the need for a new phenomenological model to explain solvent formation.

Development and characterization of a gene expression reporter system for Clostridium acetobutylicum ATCC 824.

A gene expression reporter system (pHT3) for Clostridium acetobutylicum ATCC 824 was developed by using the lacZ gene from Thermoanaerobacterium thermosulfurogenes EM1 as the reporter gene. In order to test the reporter system, promoters of three key metabolic pathway genes, ptb (coding for phosphotransbutyrylase), thl (coding for thiolase), and adc (coding for acetoacetate decarboxylase), were cloned upstream of the reporter gene in pHT3 in order to construct vectors pHT4, pHT5, and pHTA, respectively. Detection of beta-galactosidase activity in time course studies performed with strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA) demonstrated that the reporter gene produced a functional beta-galactosidase in C. acetobutylicum. In addition, time course studies revealed differences in the beta-galactosidase specific activity profiles of strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA), suggesting that the reporter system developed in this study is able to effectively distinguish between different promoters. The stability of the beta-galactosidase produced by the reporter gene was also examined with strains ATCC 824(pHT4) and ATCC 824(pHT5) by using chloramphenicol treatment to inhibit protein synthesis. The data indicated that the beta-galactosidase produced by the lacZ gene from T. thermosulfurogenes EM1 was stable in the exponential phase of growth. In pH-controlled fermentations of ATCC 824(pHT4), the kinetics of beta-galactosidase formation from the ptb promoter and phosphotransbutyrylase formation from its own autologous promoter were found to be similar.

Metabolic flux analysis elucidates the importance of the acid-formation pathways in regulating solvent production by Clostridium acetobutylicum.

Metabolic flux analysis was used to investigate the roles of the acid formation pathways in Clostridium acetobutylicum. The acid formation pathways were revealed to serve different roles in wildtype fermentations than previously expected. Specifically, enzymes known to catalyze butyrate formation were found to uptake butyrate without concomitant production of acetone. This role was further corroborated by flux analysis of a recombinant strain overexpressing the butyrate formation enzymes. Analysis of wildtype fermentation data also revealed an important role for the acetate formation enzymes, namely the cycling of carbon between acetate and acetylCoA during the stationary phase. Next, metabolic flux analysis was used to compare the patterns of activity in two butyrate kinase deficient strains of C. acetobutylicum. The strain developed by gene inactivation, PJC4BK, exhibited a shift in acid formation fluxes toward acetate while the strain developed by antisense RNA strategies, 824(pRD4), did not exhibit such a shift. However, both strains exhibited altered solvent formation patterns. PJC4BK exhibited a strong transient enhancement of solvent formation fluxes. In contrast, 824(pRD4) exhibited relatively lower levels of solvent formation fluxes, although fluxes were sustained over a longer period of time.

Expression of Clostridium acetobutylicum ATCC 824 genes in Escherichia coli for acetone production and acetate detoxification.

A synthetic acetone operon (ace4) composed of four Clostridium acetobutylicum ATCC 824 genes (adc, ctfAB, and thl, coding for the acetoacetate decarboxylase, coenzyme A transferase, and thiolase, respectively) under the control of the thl promoter was constructed and was introduced into Escherichia coli on vector pACT. Acetone production demonstrated that ace4 is expressed in E. coli and resulted in the reduction of acetic acid levels in the fermentation broth. Since different E. coli strains vary significantly in their growth characteristics and acetate metabolism, ace4 was expressed in three E. coli strains: ER2275, ATCC 11303, and MC1060. Shake flask cultures of MC1060(pACT) produced ca. 2 mM acetone, while both strains ER2275(pACT) and ATCC 11303(pACT) produced ca. 40 mM acetone. Glucose-fed cultures of strain ATCC 11303(pACT) resulted in a 150% increase in acetone titers compared to those of batch shake flask cultures. External addition of sodium acetate to glucose-fed cultures of ATCC 11303(pACT) resulted in further increased acetone titers. In bioreactor studies, acidic conditions (pH 5.5 versus 6.5) improved acetone production. Despite the substantial acetone evaporation due to aeration and agitation in the bioreactor, 125 to 154 mM acetone accumulated in ATCC 11303(pACT) fermentations. These acetone titers are equal to or higher than those produced by wild-type C. acetobutylicum. This is the first study to demonstrate the ability to use clostridial genes in nonclostridial hosts for solvent production. In addition, acetone-producing E. coli strains may be useful hosts for recombinant protein production in that detrimental acetate accumulation can be avoided.

New shuttle vector for cloning in Bacillus stearothermophilus.

Cloning vector plasmid pRP9 was constructed on the basis of the broad host-range plasmid pLM6. pRP9 was a small plasmid (2.9 kb), possessed a convenient polyrestriction site sequence and efficiently transformed Bacillus subtilis, Bacillus stearothermophilus and Escherichia coli. Furthermore, pRP9 presented a very high segregational stability in Bacillus hosts. Also, the structural stability in Bacillus strains, grown under selective pressure, of pRP9 carrying a 3-kb fragment, was high. No single-stranded and high-molecular weight pRP9 DNA was found in B. stearothermophilus. The host/vector systems described possessed all the properties required for efficient gene cloning.

Expression of cloned homologous fermentative genes in Clostridium acetobutylicum ATCC 824.

We have previously cloned the acetone-formation pathway gene, encoding acetoacetate decarboxylase (adc), and butyrate-formation pathway gene, encoding phosphotransbutyrylase (ptb), of Clostridium acetobutylicum ATCC 824 in Escherichia coli. Here we report their subcloning in Bacillus subtilis and transfer to strain ATCC 824 via electrotransformation, where the corresponding enzyme activities were expressed at elevated levels, using pFNK1, a new B. subtilis/C. acetobutylicum shuttle vector. Plasmid pFNK1 was used because shuttle vectors that function in E. coli were unable to electrotransform ATCC 824 unless they became deleted in the E. coli-plasmid regions. The difficulties with shuttle vectors that function in E. coli are probably due to the presence of a restriction endonuclease in ATCC 824. This endonuclease recognizes the sequence 5'-GCNGC-3', which is prevalent in E. coli plasmids but occurs infrequently in pFNK1 and C. acetobutylicum genes. Cloning of genes in C. acetobutylicum is critical for redirecting the cellular metabolism (metabolic engineering) as well as for genetic studies of this industrial organism.

Temperature-induced protein synthesis in Bacillus stearothermophilus NUB36.

Cultures of Bacillus stearothermophilus subjected to a temperature shift-up or shift-down of 15 degrees C within the normal temperature range of growth (45 to 65 degrees C) enter a transient adaptation period before exponential growth at the new temperature. The de novo synthesis of some proteins coincides with the adaptation period.

Cloning and characterization of a glutamine transport operon of Bacillus stearothermophilus NUB36: effect of temperature on regulation of transcription.

We cloned and sequenced a fragment of the Bacillus stearothermophilus NUB36 chromosome that contains two open reading frames (ORFs) whose products were detected only in cells of cultures grown in complex medium at high temperature. The nucleotide sequence of the two ORFs exhibited significant identity to the sequence of the glnQ and glnH loci of the glutamine transport system in enteric bacteria. In addition, growth response to glutamine, sensitivity to the toxic glutamine analog gamma-L-glutamylhydrazide, and glutamine transport assays with parental strain NUB3621 and mutant strain NUB36500, in which the ORF1 coding segment in the chromosome was interrupted with the cat gene, demonstrated that glnQ and glnH encode proteins that are active in the glutamine transport system in B. stearothermophilus. The inferred promoter for the glnQH operon exhibited a low homology to the -35 and -10 regions of the consensus promoter sequences of Bacillus subtilis and Escherichia coli genes. In addition, the inferred promoter for the glnQH operon also exhibited a low homology with the consensus promoter sequence deduced from the sequences of the promoters of nine different genes from B. stearothermophilus. Transcription of the glnQH operon was activated in a nitrogen-rich medium at high temperature and inhibited under the same conditions at low temperature. Transcription of the glnQH operon was partially activated in a nitrogen-poor medium at low temperature. The region upstream from glnQ contains sequences that have a low homology with the nitrogen regulator I-binding sequences and the nitrogen-regulated promoters of enteric bacteria. The effect of temperature on the regulation of the glnQH operon is discussed.

Genetic map of the Bacillus stearothermophilus NUB36 chromosome.

A circular genetic map of Bacillus stearothermophilus NUB36 was constructed by transduction with bacteriophage TP-42C and protoplast fusion. Sixty-four genes were tentatively assigned a cognate Bacillus subtilis gene based on growth response to intermediates or end products of metabolism, cross-feeding, accumulation of intermediates, or their relative order in a linkage group. Although the relative position of many genes on the Bacillus stearothermophilus and Bacillus subtilis genetic map appears to be similar, some differences were detected. The tentative order of the genes in the Bacillus stearothermophilus aro region is aspB-aroBAFEC-tyrA-hisH-(trp), whereas it is aspB-aroE-tyrA-hisH-(trp)-aroHBF in Bacillus subtilis. The aroA, aroC, and aroG genes in Bacillus subtilis are located in another region. The tentative order of genes in the trp operon of Bacillus stearothermophilus is trpFCDABE, whereas it is trpABFCDE in Bacillus subtilis.

Protoplast transformation of Bacillus stearothermophilus NUB36 by plasmid DNA.

An efficient protoplast transformation system was established for Bacillus stearothermophilus NUB3621 using thermophilic plasmid pTHT15 Tcr (4.5 kb) and mesophilic plasmid pLW05 Cmr (3 kb), a spontaneous deletion derivative of pPL401 Cmr Kmr. The efficiency of transformation of NUB3621 with pLW05 and pTHT15 was 2 x 10(7) to 4 x 10(8) transformants per micrograms DNA. The transformation frequency (transformants per regenerant) was 0.5 to 1.0. Chloramphenicol-resistant and tetracycline-resistant transformants were obtained when competent cells of Bacillus subtilis were transformed with pLW05 [2.5 x 10(5) transformants (microgram DNA)-1] and pTHT15 [1.8 x 10(5) transformants (micrograms DNA)-1], respectively. Thus, these plasmids are shuttle vectors for mesophilic and thermophilic bacilli. Plasmid pLW05 Cmr was not stably maintained in cultures growing at temperatures between 50 and 65 degrees C but the thermostable chloramphenicol acetyltransferase was active in vivo at temperatures up to 70 degrees C. In contrast, thermophilic plasmid pTHT15 Tcr was stable in cultures growing at temperatures up to 60 degrees C but the tetracycline resistance protein was relatively thermolabile at higher temperatures. The estimated copy number of pLW05 in cells of NUB3621 growing at 50, 60, and 65 degrees C was 69, 18, and 1 per chromosome equivalent, respectively. The estimated copy number of pTHT15 in cells of NUB3621 growing at 50 or 60 degrees C was about 41 to 45 per chromosome equivalent and 12 in cells growing at 65 degrees C.

Transduction in Bacillus stearothermophilus.

Temperate and virulent bacteriophages isolated from soil were shown to carry out generalized transduction of Bacillus stearothermophilus NUB36. A transducing frequency of 1 X 10(-5) to 7 X 10(-4) was obtained for temperate phages TP-42 and TP-56. The transducing frequency for virulent phage TP-68 was two to three orders of magnitude lower. Cotransfer analysis with the three phages showed that hom-1 is linked to thr-1 and that gly-1 is linked to his-1.

Genetic analysis of Bacillus stearothermophilus by protoplast fusion.

Efficient and reliable protoplasting, regeneration, and fusion techniques were established for the prototrophic strain Bacillus stearothermophilus NUB36. Auxotrophic mutants were isolated, and protoplast fusion was used to construct isogenic mutant strains and for chromosomal mapping. Markers were mapped using two-, three-, and four-factor crosses. The order of the markers was hom-1-thr-1-his-1-(gly-1 or gly-2)-pur-1-pur-2. These markers may be analogous to hom, thrA, hisA, glyC, and purA markers on the Bacillus subtilis chromosome. No analogous pur-1 marker has been reported in B. subtilis. The relative order of three of the markers (hom-1-thr-1-gly-1) was independently confirmed by transduction.

Isolation of a Bacillus stearothermophilus mutant exhibiting increased thermostability in its restriction endonuclease.

A procedure was developed for the selection of spontaneous mutants of Bacillus stearothermophilus NUB31 that are more efficient than the wild type in the restriction of phage at elevated temperatures. Inactivation studies revealed that two mutants contained a more thermostable restriction enzyme and one mutant contained three times more enzyme than the wild type. The restriction endonucleases from the wild type and one of the mutants were purified to apparent homogeneity. The mutant enzyme was more thermostable than the wild-type enzyme. The subunit molecular weight, amino acid composition, N-terminal and C-terminal amino acid residues, tryptic peptide map, and catalytic properties of the two enzymes were determined. The two enzymes have similar catalytic properties, but the molecular size of the mutant enzyme is approximately 6 to 7 kilodaltons larger than that of the wild-type enzyme. The mutant enzyme contains 54 additional amino acid residues, of which 26 to 28 are aspartate/asparagine, 8 to 15 are glutamate/glutamine, and 8 to 9 are tyrosine residues. The two enzymes contained similar amounts of the other amino acids, identical N-terminal residues, and different C-terminal residues. Tryptic peptide analyses revealed a high degree of homology between the two enzymes. The increased thermostability observed in the mutant enzyme appears to have been achieved by a mutation that resulted in the addition of amino acid residues to the wild-type enzyme. A number of mechanisms are discussed that could account for the observed difference between the mutant and wild-type enzymes.

Molecular weight and amino acid composition of a thermostable lytic endopeptidase.

A thermostable lytic endopeptidase from Bacillus stearothermophilus 1503-4R was purified 14,500-fold, with a 34% recovery of lytic activity. The enzyme is a basic protein (pI, 9.7) with a molecular weight of 15,100 and is composed of approximately 129 amino acid residues.

Deoxyribonucleic acid modification methylase from Bacillus stearothermophilus.

A modification methylase was isolated from Bacillus stearothermophilus 1503-4R (Bst 1503I) and purified to homogeneity. The enzyme is an acidic protein and composed of a subunit with a molecular weight of 105 000, and only the tetrameric form was detected in solution. The methylase exhibited maximal activity between 54 and 61 degrees C and between pH 8.1 and 9.3. In contrast to Bst 1503I endonuclease [Catterall, J.F., & Welker, N. E. (1977) J. Bacteriol. 129, 1110-1120], the methylase is completely inactivated when exposed to temperatures near the optimal growth temperature (63-67 degrees C). The methylase was also inactivated when exposed to temperatures below the minimal growth temperature (48-53 degrees C). The thermostability of the methylase is significantly enhanced by Na+, K+, or NH4+. Membrane-bound methylase is resistant to heat inactivation at temperatures near the maximum growth temperature (73-75 degrees C). The methylase functions as a tetramer. The initial rates of methyl transfer are first order in methylase concentration, and the enzyme obeys Michaelis-Menten kinetics with respect to DNA but not to S-adenosyl-L-methionine.

Isolation and properties of a thermostable restriction endonuclease (ENDO R-Bst1503).

A restriction endonuclease was isolated from Bacillus stearothermophilus1503-4R (Bst1503) and purified to homogeneity. The enzyme required Mg2+ ion as a cofactor. Bst1503 exhibited maximal activity between pH 7.5 and 8.0, between 60 and 65 degrees C, and with about 0.2 mM Mg2+. Bst1503 was not inactivated after exposure at 55 or 65 degrees C for up to 10 h. After 2 h of incubation at 70 degrees C, Bst1503 was inactivated by 65%. Bst1503 was rapidly inactivated at 75 degrees C. A single protein-staining band having a molecular weight of 46,000 was observed when Bst1503 was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was found to exist in two active forms, the predominating form with an S value of 8.3 (180,000) and the second form with an S value of 5.4 (96,000). No conversion between the 8.3S and 5.4S forms was observed after storage. Bst1503 recognized six sites in TP-1C deoxyribonucleic acid (DNA), one site in pSC101 and simian virus 40 DNAs, and three sites in lambdavir DNA. Bst1503 and BamHI were determined to be isoschizomers. The effect of temperatures on the activity and stability of BamHI was determined.

Characterization of a bacteriophage-induced, host-specific lytic enzyme from lysates of Bacillus stearothermophilus infected with bacteriophage TP-8.

Phage TP-8 lysates of Bacillus stearothermophilus 4S or 4S(8) contain lytic activity exhibiting two pH optima, one at pH 6.5 and the other at pH 7.5. Using a variety of fractionation procedures, the two lytic activities could not be separated. At pH 7.5 the lytic enzyme is an endopeptidase which hydrolyzes the l-alanyl-d-glutamyl linkage in the peptide subunits of the cell wall peptidoglycan and at pH 6.5 it exhibits N-acetylmuramidase activity. Endopeptidase activity is inhibited by NaCl and neither lytic activity was significantly affected by divalent cations or ethylenediaminetetraacetic acid. Crude lysates contain 2.5 to 3.0 times more endopeptidase activity than N-acetylmuramidase activity. The ratio of the two lytic activities (endopeptidase/N-acetylmuramidase) changes to 1.3 to 1.7 during the course of purification, to 1.0 after isoelectric focusing, and 3.9 and 6.00 after exposure for 2 h at 60 and 65 C, respectively. We conclude that the two lytic activities may be associated with a single protein or a lytic enzyme complex composed of two enzymes. Lytic activity at pH 7.5 is more effective in solubilizing cells or cell walls than the lytic activity at pH 6.5. LiCl extracts of 4S and 4S(8) cells contain lytic activity exhibiting endopeptidase activity at pH 7.5 and N-acetylmuramidase activity at pH 6.5. Lytic activity in these LiCl extracts also has a number of other properties in common with those in lysates of phage TP-8. We proposed that the lytic enzyme(s) are not coded for by the phage genome but are part of the host autolytic system.