O. V. Berezina

Bacterial acetone and butanol production by industrial fermentation in the Soviet Union: use of hydrolyzed agricultural waste for biorefinery

Clostridial acetone–butanol fermentation from renewable carbohydrates used to be the largest biotechnological process second only to yeast ethanol fermentation and the largest process ever run under sterile conditions. With the rising prices for mineral oil, it has now the economical and technological potential to replace petrochemistry for the production of fuels from renewable resources. Various methods for using non-food biomass such as cellulose and hemicellulose in agricultural products and wastes have been developed at laboratory scale. To our knowledge, the AB plants in Russia were the only full-scale industrial plants which used hydrolyzates of lignocellosic waste for butanol fermentation. These plants were further developed into the 1980s, and the process was finally run in a continual mode different from plants in Western countries. A biorefinery concept for the use of all by-products has been elaborated and was partially put into practice. The experience gained in the Soviet Union forms a promising basis for the development of modern large-scale processes to replace a considerable fraction of the current chemical production of fuel for our future needs on a sustainable basis.

Isolation of a new butanol-producing Clostridium strain: High level of hemicellulosic activity and structure of solventogenesis genes of a new Clostridium saccharobutylicum isolate ☆

New isolates of solventogenic bacteria exhibited high hemicellulolytic activity. They produced butanol and acetone with high selectivity for butanol (about 80% of butanol from the total solvent yield). Their 16S rDNA sequence was 99% identical to that of Clostridium saccharobutylicum. The genes responsible for the last steps of solventogenesis and encoding crotonase, butyryl-CoA dehydrogenase, electron-transport protein subunits A and B, 3-hydroxybutyryl-CoA dehydrogenase, alcohol dehydrogenase, CoA-transferase (subunits A and B), acetoacetate decarboxylase, and aldehyde dehydrogenase were identified in the new C. saccharobutylicum strain Ox29 and cloned into Escherichia coli. The genes for crotonase, butyryl-CoA dehydrogenase, electron-transport protein subunits A and B, and 3-hydroxybutyryl-CoA dehydrogenase composed the bcs-operon. A monocistronic operon containing the alcohol dehydrogenase gene was located downstream of the bcs-operon. Genes for aldehyde dehydrogenase, CoA-transferase (subunits A and B), and acetoacetate decarboxylase composed the sol-operon. The gene sequences and the gene order within the sol- and bcs-operons of C. saccharobutylicum Ox29 were most similar to those of Clostridium beijerinckii. The activity of some of the bcs-operon genes, expressed in heterologous E. coli, was determined.

Extracellular glycosyl hydrolase activity of the Clostridium strains producing acetone, butanol, and ethanol

Production of acetone, butanol, ethanol, acetic acid, and butyric acid by three strains of anaerobic bacteria, which we identified as Clostridium acetobutylicum, was studied. The yield of acetone and alcohols in 6% wheat flour medium amounted to 12.7–15 g/l with butanol constituting 51.0–55.6%. Activities of these strains towards xylan, β-glucan, carboxymethylcellulose, and crystalline and amorphous celluloses were studied. C. acetobutylicum 6, C. acetobutylicum 7, and C. acetobutylicum VKPM B-4786 produced larger amounts of acetone and alcohols and displayed higher cellulase and hemicellulase activities than the type strain C. acetobutylicum ATCC 824 in lab-scale butch cultures. It was demonstrated that starch in the medium could be partially substituted with plant biomass.

Microbial producers of butanol

This review is written due to an increased interest in the production of energy carriers and basic substrates of the chemical industry from renewable natural resources. In this review, the microbiological aspects of biobutanol production are reflected and the microbial producers of butanol (both natural, i.e., members of the Clostridium genus, and recombinant), obtained by genetic modification of Clostridia and other microorganisms, are characterised.

Thermoanaerobacter ethanolicus Gene Cluster Containing the α- and β-Galactosidase Genes melA and lacA and Properties of Recombinant LacA

The nucleotide sequence of a 4936-bp genomic DNA fragment from the thermophilic bacterium Thermoanaerobacter ethanolicus has been determined. The fragment contains three open reading frames (ORFs). The product of the incomplete first ORF is highly homologous to α-galactosidases (melibiases). The second ORF corresponds to the lacA gene for a thermostable β-galactosidase. The product of the third ORF is similar to α-D-mannosidases. A putative ρ-independent terminator is located immediately downstream of the lacA stop codon, suggesting a cotranscription of the α- and β-galactosidase genes. The deduced molecular weight of LacA is 86 kDa. LacA belongs to glycosylhydrolase family 2 (GH2). Native recombinant LacA is a dimer and shows the highest activity at pH 5.7–6.0 and 75–80°C. LacA is most active with lactose (480 units per mg protein, Km = 30 mM); the activities with pNP-Gal and oNP-Gal are 330 and 420 units per mg protein, respectively. Immobilized on aldehyde silochrome, LacA is even more thermostable and retains its high activity.

Cloning and characterisation of a large metagenomic DNA fragment containing glycosyl-hydrolase genes

The problem of searching for and characterizing enzymes produced by uncultured microorganisms is presently settled by creating metagenomic libraries. A 6000-clone library with the average size of inserts of about 15 kb has been constructed based on total DNA isolated from cow rumen microorganisms. As a result of library screening on plates with different substrates, a clone was selected that efficiently hydrolysed lichenan and carboxymethylcellulose. The clone contained the recombinant plasmid pBlue-13 bearing a 12071 bp-long metagenomic fragment carrying ten open reading frames, two of which were identified as glycosyl hydrolase genes. No homology of the metagenomic DNA with any sequences known microorganism genomes was revealed. The amino acid sequence deduced from frame 4 was denoted of Xyl3A, and bears resemblance with β-xylosidases of glycosyl hydrolase family 3. Frame 6 encodes polypeptide Cel5A homologous to cellulases of glycosyl hydorlase family 5. The amino acid sequences deducted from on seven out of ten open reading frames were homologous to proteins of microorganisms belonging to the Bacteroides sp. family and the bacteria inhabiting mammalian intestines.

A Cluster of Thermotoga neapolitana Genes Involved in the Degradation of Starch and Maltodextrins: the Molecular Structure of the Locus

A 5451-bp genome fragment of the hyperthermophilic anaerobic eubacterium Thermotoga neapolitana has been cloned and sequenced. The fragment contains one truncated and three complete open reading frames highly homologous to the starch/maltodextrin utilization gene cluster from T. maritima whose genome sequence is known. The incomplete product of the first frame is highly homologous to MalG, the Escherichia coli protein of starch and maltodextrin transport. The product of the second frame, AglB, is highly homologous to cyclomaltodextrinase with the α-glucosidase activity TMG belonging to family 13 of glycosyl hydrolases (GH13). The product of the third frame, AglA, is homologous to the T. maritima cofactor-dependent α-glucosidase from the GH4 family. The two enzymes form a separate branch on the phylogenetic tree of the family. The AglA and AglB proteins supplement each other in substrate specificity and can ensure complete hydrolysis to glucose of cyclic and linear maltodextrins, the intermediate products of starch degradation. The product of the fourth reading frame has sequence similarity with the riboflavin-specific deaminase RibD from T. maritima. The homologous locus of this bacterium, between the aglA and ribD genes, has five open reading frames missing in T. neapolitana. The nucleotide sequences of two frames are homologous to transposase genes. The deletion size is 2.9 kb.

Expression of the Genes celA and xylA isolated from a fragment of metagenomic DNA in Escherichia coli

The glycosyl hydrolase genes cel5A and xyl3A previously isolated by us within a fragment of DNA from the methagenomic library of cow rumen microflora DNA were subcloned and expressed in E. coli. The recombinant proteins Cel5A and Xyl3A were purified and characterized. Cellulase Cel5A belongs to family 5 of glycosyl hydrolases and is a single-module 38.2 kDa enzyme that hydrolyses the 1,4-glycoside bonds of soluble cellulose substrates and amorphous cellulose, which displays its maximal activity (31200 u/mg) on lichenan, a soluble substrate with mixed (beta-1,3-1,4) bonds. The end product of the amorphous cellulose hydrolysis is cellobiose. Cel5A is inactive toward the crystal forms of cellulose. The enzyme is an endoglucanase capable of exohydrolysis. The molecular mass of beta-xylosidase Xyl3A belonging to family 3 of glycosyl hydrolases is 83.7 kDa. The enzyme is active on only xylooligosaccharides with the maximal activity shown on xylobiose and the end product of the reaction is xylose. No activity on xylane has yet been observed. Recombinant Cel5A and Xyl3A are stable over a wide range of pH and temperature and their maximal activity is observed at pH 6.5 and at 55°C.

Comparative Analysis of the Recombinant α-Glucosidases from the Thermotoga neapolitana and Thermotoga maritima Maltodextrin Utilization Gene Clusters

Abstract The hyperthermophilic bacterium Thermotoga maritima contains an amylolytic gene cluster with two adjacent α-glucosidase genes, aglB and aglA. We have now identified a similar pair of α-glucosidase genes on a 5,451 bp fragment of T. neapolitana genomic DNA. Like in T. maritima, aglA of T. neapolitana is located downstream of aglB. The deduced AglB primary structure allows its assignment to glycoside hydrolase family 13 (GHF13), whereas AglA belongs to GHF4. The aglB gene of T. neapolitana and the corresponding gene from T. maritima were expressed in E. coli, and the recombinant enzymes were characterized. Both enzymes hydrolyzed cyclomaltodextrins and linear maltooligosaccharides to yield glucose and maltose. Evidence from the hydrolysis of non-natural oligosaccharides and the pseudo-tetrasaccharide acarbose suggests that linear malto-oligosaccharides are progressively degraded by T. neapolitana and T. maritima AglB from the reducing end, which is highly uncommon for α-glucosidases. AglB, in contrast to the cofactor-dependent (NAD+, Mn2+) α-glucosidase AglA, does not cleave maltose. The recent elucidation of the crystal structure of T. miritima AglA indicates that AglA and AglB employ different catalytic mechanisms for glycosidic bond cleavage. Possible reasons for the presence of two α-glucosidase genes in the same amylolytic gene cluster of Thermotoga species are discussed.

Characteristics of fungal strains producing thermostable xyloglucanases from the Russian National Collection of industrial microorganisms

Fungal strains possessing xyloglucanase activity have been selected from the microorganisms degrading plant biomass deposited in the Russian National Collection of Industrial Microorganisms (VKPM). The thermophilic strains Sporotrichum thermophile VKPM F-972, Myceliophthora thermophila VKPM F-244, and Sporotrichum pruinosum VKPM F-235 produced extracellular xyloglucanases under conditions of submerged cultivation with optimum temperature of 60°C and pH of 5.0. The enzymes retained 88–100 and 79–84% of the initial activity after 1-h incubation at 50° and 60°C, respectively. Thus, strains S. thermophile VKPM F-972, M. thermophila VKPM F-244, and S. pruinosum VKPM F-235 may be used as sources of genes for construction of highly active producers of thermostable xyloglucanases.