Analysis of cellulose synthesis in a high-producing acetic acid bacterium Komagataeibacter hansenii.

Bacterial cellulose (BC) represents a renewable biomaterial with unique properties promising for biotechnology and biomedicine. Komagataeibacter hansenii ATCC 53,582 is a well-characterized high-yield producer of BC used in the industry. Its genome encodes three distinct cellulose synthases (CS), bcsAB1, bcsAB2, and bcsAB3, which together with genes for accessory proteins are organized in operons of different complexity. The genetic foundation of its high cellulose-producing phenotype was investigated by constructing chromosomal in-frame deletions of the CSs and of two predicted regulatory diguanylate cyclases (DGC), dgcA and dgcB. Proteomic characterization suggested that BcsAB1 was the decisive CS because of its high expression and its exclusive contribution to the formation of microcrystalline cellulose. BcsAB2 showed a lower expression level but contributes significantly to the tensile strength of BC and alters fiber diameter significantly as judged by scanning electron microscopy. Nevertheless, no distinct extracellular polymeric substance (EPS) from this operon was identified after static cultivation. Although transcription of bcsAB3 was observed, expression of the protein was below the detection limit of proteome analysis. Alike BcsAB2, deletion of BcsAB3 resulted in a visible reduction of the cellulose fiber diameter. The high abundance of BcsD and the accessory proteins CmcAx, CcpAx, and BglxA emphasizes their importance for the proper formation of the cellulosic network. Characterization of deletion mutants lacking the DGC genes dgcA and dgcB suggests a new regulatory mechanism of cellulose synthesis and cell motility in K. hansenii ATCC 53,582. Our findings form the basis for rational tailoring of the characteristics of BC. KEY POINTS: • BcsAB1 induces formation of microcrystalline cellulose fibers. • Modifications by BcsAB2 and BcsAB3 alter diameter of cellulose fibers. • Complex regulatory network of DGCs on cellulose pellicle formation and motility.

The Roles of the Various Cellulose Biosynthesis Operons in Komagataeibacter hansenii ATCC 23769.

Cellulose is the most abundant biopolymer on earth and offers versatile applicability in biotechnology. Bacterial cellulose, especially, is an attractive material because it represents pure microcrystalline cellulose. The cellulose synthase complex of acetic acid bacteria serves as a model for general studies on (bacterial) cellulose synthesis. The genome of Komagataeibacter hansenii ATCC 23769 encodes three cellulose synthase (CS) operons of different sizes and gene compositions. This implies the question of which role each of the three CS-encoding operons, bcsAB1, bcsAB2, and bcsAB3, plays in overall cellulose synthesis. Therefore, we constructed markerless deletions in K. hansenii ATCC 23769, yielding mutant strains that expressed only one of the three CSs. Apparently, BcsAB1 is the only CS that produces fibers of crystalline cellulose. The markerless deletion of bcsAB1 resulted in a nonfiber phenotype in scanning electron microscopy analysis. Expression of the other CSs resulted in a different, nonfibrous extracellular polymeric substance (nfEPS) structure wrapping the cells, which is proposed to contain acetylated cellulose. Transcription analysis revealed that all CSs were expressed continuously and that bcsAB2 showed a higher transcription level than bcsAB1. Moreover, we were able to link the expression of diguanylate cyclase B (dgcB) to cellulose production. IMPORTANCE Acetic acid bacteria form a massive biofilm called "mother of vinegar," which is built of cellulose fibers. Bacterial cellulose is an appealing biomaterial with manifold applications in biomedicine and biotechnology. Because most cellulose-producing acetic acid bacteria express several cellulose synthase operons, a deeper understanding of their contribution to the synthesis of modified forms of cellulose fibers within a natural biofilm is of special interest. For the first time, we were able to identify the contribution of each of the three cellulose synthases to cellulose formation in Komagataeibacter hansenii ATCC 23769 after a chromosomal clean deletion. Moreover, we were able to depict their roles in spatial composition of the biofilm. These findings might be applicable in the future for naturally modified biomaterials with novel properties.

L-Erythrulose production with a multideletion strain of Gluconobacter oxydans.

Many ketoses or organic acids can be produced by membrane-associated oxidation with Gluconobacter oxydans. In this study, the oxidation of meso-erythritol to L-erythrulose was investigated with the strain G. oxydans 621HΔupp BP.8, a multideletion strain lacking the genes for eight membrane-bound dehydrogenases. First batch biotransformations with growing cells showed re-consumption of L-erythrulose by G. oxydans 621HΔupp BP.8 in contrast to resting cells. The batch biotransformation with 2.8 g L-1 resting cells of G. oxydans 621HΔupp BP.8 in a DO-controlled stirred-tank bioreactor resulted in 242 g L-1 L-erythrulose with a product yield of 99% (w/w) and a space-time yield of 10 g L-1 h-1. Reaction engineering studies showed substrate excess inhibition as well as product inhibition of G. oxydans 621HΔupp BP.8 in batch biotransformations. In order to overcome substrate inhibition, a continuous membrane bioreactor with full cell retention was applied for meso-erythritol oxidation with resting cells of G. oxydans 621HΔupp BP.8. At a mean hydraulic residence time of 2 h, a space-time yield of 27 g L-1 h-1 L-erythrulose was achieved without changing the product yield of 99% (w/w) resulting in a cell-specific product yield of up to 4.4 gP gX-1 in the steady state. The product concentration (54 g L-1 L-erythrulose) was reduced in the continuous biotransformation process compared with the batch process to avoid product inhibition.

Markerless deletion of putative alanine dehydrogenase genes in Bacillus licheniformis using a codBA-based counterselection technique.

Bacillus licheniformis strains are used for the large-scale production of industrial exoenzymes from proteinaceous substrates, but details of the amino acid metabolism involved are largely unknown. In this study, two chromosomal genes putatively involved in amino acid metabolism of B. licheniformis were deleted to clarify their role. For this, a convenient counterselection system for markerless in-frame deletions was developed for B. licheniformis. A deletion plasmid containing up- and downstream DNA segments of the chromosomal deletion target was conjugated to B. licheniformis and integrated into the genome by homologous recombination. Thereafter, the counterselection was done by using a codBA cassette. The presence of cytosine deaminase and cytosine permease exerted a conditionally lethal phenotype on B. licheniformis cells in the presence of the cytosine analogue 5-fluorocytosine. Thereby clones were selected that lost the integrated vector sequence and the anticipated deletion target after a second recombination step. This method allows the construction of markerless mutants in Bacillus strains in iterative cycles. B. licheniformis MW3 derivatives lacking either one of the ORFs BL03009 or BL00190, encoding a putative alanine dehydrogenase and a similar putative enzyme, respectively, retained the ability to grow in minimal medium supplemented with alanine as the carbon source. In the double deletion mutant MW3 ΔBL03009 ΔBL00190, however, growth on alanine was completely abolished. These data indicate that the two encoded enzymes are paralogues fulfilling mutually replaceable functions in alanine utilization, and suggest that in B. licheniformis MW3 alanine utilization is initiated by direct oxidative transamination to pyruvate and ammonium.

Characterization of membrane-bound dehydrogenases of Gluconobacter oxydans 621H using a new system for their functional expression.

Acetic acid bacteria are used in biotechnology due to their ability to incompletely oxidize a great variety of carbohydrates, alcohols, and related compounds in a regio- and stereo-selective manner. These reactions are catalyzed by membrane-bound dehydrogenases (mDHs), often with a broad substrate spectrum. In this study, the promoters of six mDHs of Gluconobacter oxydans 621H were characterized. The constitutive promoter of the alcohol dehydrogenase and the glucose-repressed promoter of the inositol dehydrogenase were used to construct a shuttle vector system for the fully functional expression of mDHs in the multi-deletion strain G. oxydans BP.9 that lacks its mDHs. This system was used to express each mDH of G. oxydans 621H, in order to individually characterize the substrates, they oxidize. From 55 tested compounds, the alcohol dehydrogenase oxidized 30 substrates and the polyol dehydrogenase 25. The substrate spectrum of alcohol dehydrogenase overlapped largely with the aldehyde dehydrogenase and partially with polyol dehydrogenase. Thus, we were able to resolve the overlapping substrate spectra of the main mDHs of G. oxydans 621H. The described approach could also be used for the expression and detailed characterization of substrates used by mDHs from other acetic acid bacteria or a metagenome.

Expression of membrane-bound dehydrogenases from a mother of vinegar metagenome in Gluconobacter oxydans.

Acetic acid bacteria are well-known for their membrane-bound dehydrogenases rapidly oxidizing a variety of substrates in the periplasm. Since many acetic acid bacteria have not been successfully cultured in the laboratory yet, studying membrane-bound dehydrogenases directly from a metagenome of vinegar microbiota seems to be a promising way to identify novel variants of these enzymes. To this end, DNA from a mother of vinegar was isolated, sequenced, and screened for membrane-bound dehydrogenases using an in silico approach. Six metagenomic dehydrogenases were successfully expressed using an expression vector with native promoters in the acetic acid bacterium strain Gluconobacter oxydans BP.9, which is devoid of its major native membrane-bound dehydrogenases. Determining the substrates converted by these enzymes, using a whole-cell DCPIP assay, revealed one glucose dehydrogenase with an enlarged substrate spectrum additionally oxidizing aldoheptoses, D-ribose and aldotetroses, one polyol dehydrogenase with an extreme diminished spectrum but distinguishing cis and trans-1,2-cyclohexandiol and a completely new secondary alcohol dehydrogenase, which oxidizes secondary alcohols with a hydroxyl group at position 2, as long as no primary hydroxyl group is present. Three further dehydrogenases were found with substrate spectra similar to known dehydrogenases of G. oxydans 621H.

RACK1/Asc1p, a ribosomal node in cellular signaling.

RACK1/Asc1p and its essential orthologues in higher eukaryotes, such as RACK1 in metazoa, are involved in several distinct cellular signaling processes. The implications of a total deletion have never been assessed in a comprehensive manner. This study reveals the major cellular processes affected in a Saccharomyces cerevisiae Δasc1 deletion background via de novo proteome and transcriptome analysis, as well as subsequent phenotypical characterizations. The deletion of ASC1 reduces iron uptake and causes nitrosative stress, both known indicators for hypoxia, which manifests in a shift of energy metabolism from respiration to fermentation in the Δasc1 strain. Asc1p further impacts cellular metabolism through its regulative role in the MAP kinase signal transduction pathways of invasive/filamentous growth and cell wall integrity. In the Δasc1 mutant strain, aberrations from the expected cellular response, mediated by these pathways, can be observed and are linked to changes in protein abundances of pathway-targeted transcription factors. Evidence of the translational regulation of such transcription factors suggests that ribosomal Asc1p is involved in signal transduction pathways and controls the biosynthesis of the respective final transcriptional regulators.

Identification of ecotype-specific marker genes for categorization of beer-spoiling Lactobacillus brevis.

The tolerance to hop compounds, which is mainly associated with inhibition of bacterial growth in beer, is a multi-factorial trait. Any approaches to predict the physiological differences between beer-spoiling and non-spoiling strains on the basis of a single marker gene are limited. We identified ecotype-specific genes related to the ability to grow in Pilsner beer via comparative genome sequencing. The genome sequences of four different strains of Lactobacillus brevis were compared, including newly established genomes of two highly hop tolerant beer isolates, one strain isolated from faeces and one published genome of a silage isolate. Gene fragments exclusively occurring in beer-spoiling strains as well as sequences only occurring in non-spoiling strains were identified. Comparative genomic arrays were established and hybridized with a set of L. brevis strains, which are characterized by their ability to spoil beer. As result, a set of 33 and 4 oligonucleotide probes could be established specifically detecting beer-spoilers and non-spoilers, respectively. The detection of more than one of these marker sequences according to a genetic barcode enables scoring of L. brevis for their beer-spoiling potential and can thus assist in risk evaluation in brewing industry.

Coping with anoxia: a comprehensive proteomic and transcriptomic survey of denitrification.

Ralstonia eutropha H16 is a denitrifying microorganism able to use nitrate and nitrite as terminal electron acceptors under oxygen deprivation. To identify proteins showing an altered expression pattern in response to oxygen supply, R. eutropha cells grown aerobically and anaerobically were compared in a comprehensive proteome and transcriptome approach. Nearly 700 proteins involved in several processes including respiration, formation of cell appendages, and DNA and cofactor biosynthesis were found to be differentially expressed. A combination of 1D gel-LC and conventional 2D gel analysis of six consecutive sample points covering the entire denitrification sequence revealed a detailed view on the shifting abundance of the key proteins of denitrification. Denitrification- or anaerobiosis-induced alterations of the respiratory chain included a distinct expression pattern for multiple terminal oxidases. Alterations in the central carbon metabolism were restricted to a few key functions including the isoenzymes for aconitase and isocitrate dehydrogenase. Although R. eutropha is a strictly respiratory bacterium, the abundance of certain fermentation enzymes was increased. This work represents a comprehensive survey of denitrification on the proteomic and transcriptomic levels and provides unique insight into how R. eutropha adapts its metabolism to low oxygen conditions.

Identification of genes essential for anaerobic growth of Listeria monocytogenes.

The facultative anaerobic bacterium Listeria monocytogenes encounters microaerophilic or anaerobic conditions in various environments, e.g. in soil, in decaying plant material, in food products and in the host gut. To elucidate the adaptation of Listeria monocytogenes to variations in oxygen tension, global transcription analyses using DNA microarrays were performed. In total, 139 genes were found to be transcribed differently during aerobic and anaerobic growth; 111 genes were downregulated and 28 genes were upregulated anaerobically. The oxygen-dependent transcription of central metabolic genes is in agreement with results from earlier physiological studies. Of those genes more strongly expressed under lower oxygen tension, 20 were knocked out individually. Growth analysis of these knock out mutants did not indicate an essential function for the respective genes during anaerobiosis. However, even if not essential, transcriptional induction of several genes might optimize the bacterial fitness of Listeria monocytogenes in anaerobic niches, e.g. during colonization of the gut. For example, expression of the anaerobically upregulated gene lmo0355, encoding a fumarate reductase α chain, supported growth on 10 mM fumarate under anaerobic but not under aerobic growth conditions. Genes essential for anaerobic growth were identified by screening a mutant library. Eleven out of 1360 investigated mutants were sensitive to anaerobiosis. All 11 mutants were interrupted in the atp locus. These results were further confirmed by phenotypic analysis of respective in-frame deletion and complementation mutants, suggesting that the generation of a proton motive force via F1F0-ATPase is essential for anaerobic proliferation of Listeria monocytogenes.

Chemostat cultivation and transcriptional analyses of Clostridium acetobutylicum mutants with defects in the acid and acetone biosynthetic pathways.

Clostridium acetobutylicum is a model organism for the biotechnologically important acetone-butanol-ethanol (ABE) fermentation. With the objective to rationally develop strains with improved butanol production, detailed insights into the physiological and genetic mechanisms of solvent production are required. Therefore, pH-controlled phosphate-limited chemostat cultivation and DNA microarray technology were employed for an in-depth analysis of knockout mutants with defects in the central fermentative metabolism. The set of studied mutants included strains with inactivated phosphotransacetylase (pta), phosphotransbutyrylase (ptb), and acetoacetate decarboxylase (adc) encoding genes, as well as an adc/pta double knockout mutant. A comprehensive physiological characterization of the mutants was performed by continuous cultivation, allowing for a well-defined separation of acidogenic and solventogenic growth, combined with the advantage of the high reproducibility of steady-state conditions. The ptb-negative strain C. acetobutylicum ptb::int(87) exhibited the most striking metabolite profile: Sizable amounts of butanol (29 ± 1.3 mM) were already produced during acidogenic growth. The product patterns of the mutants as well as accompanying transcriptomic data are presented and discussed.

Alternative hosts for functional (meta)genome analysis.

Microorganisms are ubiquitous on earth, often forming complex microbial communities in numerous different habitats. Most of these organisms cannot be readily cultivated in the laboratory using standard media and growth conditions. However, it is possible to gain access to the vast genetic, enzymatic, and metabolic diversity present in these microbial communities using cultivation-independent approaches such as sequence- or function-based metagenomics. Function-based analysis is dependent on heterologous expression of metagenomic libraries in a genetically amenable cloning and expression host. To date, Escherichia coli is used in most cases; however, this has the drawback that many genes from heterologous genomes and complex metagenomes are expressed in E. coli either at very low levels or not at all. This review emphasizes the importance of establishing alternative microbial expression systems consisting of different genera and species as well as customized strains and vectors optimized for heterologous expression of membrane proteins, multigene clusters encoding protein complexes or entire metabolic pathways. The use of alternative host-vector systems will complement current metagenomic screening efforts and expand the yield of novel biocatalysts, metabolic pathways, and useful metabolites to be identified from environmental samples.

Metabolic engineering of Bacillus subtilis for growth on overflow metabolites.

BACKGROUND: The genome of the important industrial host Bacillus subtilis does not encode the glyoxylate shunt, which is necessary to utilize overflow metabolites, like acetate or acetoin, as carbon source. In this study, the operon encoding the isocitrate lyase (aceB) and malate synthase (aceA) from Bacillus licheniformis was transferred into the chromosome of B. subtilis. The resulting strain was examined in respect to growth characteristics and qualities as an expression host. RESULTS: Our results show that the modified B. subtilis strain is able to grow on the C2 compound acetate. A combined transcript, protein and metabolite analysis indicated a functional expression of the native glyoxylate shunt of B. lichenifomis in B. subtilis. This metabolically engineered strain revealed better growth behavior and an improved activity of an acetoin-controlled expression system. CONCLUSIONS: The glyoxylate shunt of B. licheniformis can be functionally transferred to B. subtilis. This novel strain offers improved properties for industrial applications, such as growth on additional carbon sources and a greater robustness towards excess glucose feeding.

Characterization of membrane-bound dehydrogenases from Gluconobacter oxydans 621H via whole-cell activity assays using multideletion strains.

Gluconobacter oxydans, like all acetic acid bacteria, has several membrane-bound dehydrogenases, which oxidize a multitude of alcohols and polyols in a stereo- and regio-selective manner. Many membrane-bound dehydrogenases have been purified from various acetic acid bacteria, but in most cases without reporting associated sequence information. We constructed clean deletions of all membrane-bound dehydrogenases in G. oxydans 621H and investigated the resulting changes in carbon utilization and physiology of the organism during growth on fructose, mannitol, and glucose. Furthermore, we studied the substrate oxidation spectra of a set of strains where the membrane-bound dehydrogenases were consecutively deleted using a newly developed whole-cell 2,6-dichlorophenolindophenol (DCPIP) activity assay in microtiter plates. This allowed a detailed and comprehensive in vivo characterization of each membrane-bound dehydrogenase in terms of substrate specificity. The assays revealed that general rules can be established for some of the enzymes and extended the known substrate spectra of some enzymes. It was also possible to assign proteins whose purification and characterization had been reported previously, to their corresponding genes. Our data demonstrate that there are less membrane-bound dehydrogenases in G. oxydans 621H than expected and that the deletion of all of them is not lethal for the organism.

Importance of codB for new codA-based markerless gene deletion in Gluconobacter strains.

For the detailed molecular analysis, genomic modification, and application of acetic acid bacteria such as Gluconobacter in biotechnological processes, a simple markerless deletion system is essential. The available methods have either low efficiencies or their applicability is restricted to strains containing an upp mutation. We now developed a method based on counterselection by cytosine deaminase, encoded by the codA gene from Escherichia coli, in the presence of the fluorinated pyrimidine analogue 5-fluorocytosine (FC). The codA-encoded enzyme converts nontoxic FC to toxic 5-fluorouracil, which is channeled into the metabolism by the uracil phosphoribosyltransferase, encoded by the chromosomal upp gene of Gluconobacter. We found that the presence of E. coli codB, encoding a cytosine permease, was needed for a high efficiency of gene deletion. The system is applicable in wild-type strains because no preceding deletions are required. Based on the fact that a codA gene is absent and an upp gene is present in almost all acetic acid bacteria sequenced so far, the method should also be applicable for other genera of the Acetobacteraceae.

Deletion of pyruvate decarboxylase by a new method for efficient markerless gene deletions in Gluconobacter oxydans.

Gluconobacter oxydans, a biotechnologically relevant species which incompletely oxidizes a large variety of carbohydrates, alcohols, and related compounds, contains a gene for pyruvate decarboxylase (PDC). This enzyme is found only in very few species of bacteria where it is normally involved in anaerobic ethanol formation via acetaldehyde. In order to clarify the role of PDC in the strictly oxidative metabolism of acetic acid bacteria, we developed a markerless in-frame deletion system for strain G. oxydans 621H which uses 5-fluorouracil together with a plasmid-encoded uracil phosphoribosyltransferase as counter selection method and used this technique to delete the PDC gene (GOX1081) of G. oxydans 621H. The PDC deletion mutant accumulated large amounts of pyruvate but almost no acetate during growth on D-mannitol, D-fructose or in the presence of L-lactate. This suggested that in G. oxydans acetate formation occurs by decarboxylation of pyruvate and subsequent oxidation of acetaldehyde to acetate. This observation and the efficiency of the markerless deletion system were confirmed by constructing deletion mutants of two acetaldehyde dehydrogenases (GOX1122 and GOX2018) and of the acetyl-CoA-synthetase (GOX0412). Acetate formation during growth of these mutants on mannitol did not differ significantly from the wild-type strain.

Clostridium ljungdahlii represents a microbial production platform based on syngas.

Clostridium ljungdahlii is an anaerobic homoacetogen, able to ferment sugars, other organic compounds, or CO(2)/H(2) and synthesis gas (CO/H(2)). The latter feature makes it an interesting microbe for the biotech industry, as important bulk chemicals and proteins can be produced at the expense of CO(2), thus combining industrial needs with sustained reduction of CO and CO(2) in the atmosphere. Sequencing the complete genome of C. ljungdahlii revealed that it comprises 4,630,065 bp and is one of the largest clostridial genomes known to date. Experimental data and in silico comparisons revealed a third mode of anaerobic homoacetogenic metabolism. Unlike other organisms such as Moorella thermoacetica or Acetobacterium woodii, neither cytochromes nor sodium ions are involved in energy generation. Instead, an Rnf system is present, by which proton translocation can be performed. An electroporation procedure has been developed to transform the organism with plasmids bearing heterologous genes for butanol production. Successful expression of these genes could be demonstrated, leading to formation of the biofuel. Thus, C. ljungdahlii can be used as a unique microbial production platform based on synthesis gas and carbon dioxide/hydrogen mixtures.

Membrane-bound sorbitol dehydrogenase is responsible for the unique oxidation of D-galactitol to L-xylo-3-hexulose and D-tagatose in Gluconobacter oxydans.

BACKGROUND: Gluconobacter oxydans, is used in biotechnology because of its ability to oxidize a wide variety of carbohydrates, alcohols, and polyols in a stereo- and regio-selective manner by membrane-bound dehydrogenases located in periplasmic space. These reactions obey the well-known Bertrand-Hudson's rule. In our previous study (BBA-General Subjects, 2021, 1865:129740), we discovered that Gluconobacter species, including G. oxydans and G. cerinus strain can regio-selectively oxidize the C-3 and C-5 hydroxyl groups of D-galactitol to rare sugars D-tagatose and L-xylo-3-hexulose, which represents an exception to Bertrand Hudson's rule. The enzyme catalyzing this reaction is located in periplasmic space or membrane-bound and is PQQ (pyrroloquinoline quinine) and Ca2+-dependent; we were encouraged to determine which type of enzyme(s) catalyze this unique reaction. METHODS: Enzyme was identified by complementation of multi-deletion strain of Gluconobacter oxydans 621H with all putative membrane-bound dehydrogenase genes. RESULTS AND CONCLUSIONS: In this study, we identified this gene encoding the membrane-bound PQQ-dependent dehydrogenase that catalyzes the unique galactitol oxidation reaction in its 3'-OH and 5'-OH. Complement experiments in multi-deletion G. oxydans BP.9 strains established that the enzyme mSLDH (encoded by GOX0855-0854, sldB-sldA) is responsible for galactitol's unique oxidation reaction. Additionally, we demonstrated that the small subunit SldB of mSLDH was membrane-bound and served as an anchor protein by fusing it to a red fluorescent protein (mRubby), and heterologously expressed in E. coli and the yeast Yarrowia lipolytica. The SldB subunit was required to maintain the holo-enzymatic activity that catalyzes the conversion of D-galactitol to L-xylo-3-hexulose and D-tagatose. The large subunit SldA encoded by GOX0854 was also characterized, and it was discovered that its 24 amino acids signal peptide is required for the dehydrogenation activity of the mSLDH protein. GENERAL SIGNIFICANCE: In this study, the main membrane-bound polyol dehydrogenase mSLDH in G. oxydans 621H was proved to catalyze the unique galactitol oxidation, which represents an exception to the Bertrand Hudson's rule, and broadens its substrate ranges of mSLDH. Further deciphering the explicit enzymatic mechanism will prove this theory.

Continuous Production of Ethanol, 1-Butanol and 1-Hexanol from CO with a Synthetic Co-Culture of Clostridia Applying a Cascade of Stirred-Tank Bioreactors.

Syngas fermentation with clostridial co-cultures is promising for the conversion of CO to alcohols. A CO sensitivity study with Clostridium kluyveri monocultures in batch operated stirred-tank bioreactors revealed total growth inhibition of C. kluyveri already at 100 mbar CO, but stable biomass concentrations and ongoing chain elongation at 800 mbar CO. On/off-gassing with CO indicated a reversible inhibition of C. kluyveri. A continuous supply of sulfide led to increased autotrophic growth and ethanol formation by Clostridium carboxidivorans even at unfavorable low CO concentrations. Based on these results, a continuously operated cascade of two stirred-tank reactors was established with a synthetic co-culture of both Clostridia. An amount of 100 mbar CO and additional sulfide supply enabled growth and chain elongation in the first bioreactor, whereas 800 mbar CO resulted in an efficient reduction of organic acids and de-novo synthesis of C2-C6 alcohols in the second reactor. High alcohol/acid ratios of 4.5-9.1 (w/w) were achieved in the steady state of the cascade process, and the space-time yields of the alcohols produced were improved by factors of 1.9-5.3 compared to a batch process. Further improvement of continuous production of medium chain alcohols from CO may be possible by applying less CO-sensitive chain-elongating bacteria in co-cultures.

Effect of iron limitation and fur gene inactivation on the transcriptional profile of the strict anaerobe Clostridium acetobutylicum.

Iron is a nutrient of critical importance for the strict anaerobe Clostridium acetobutylicum, as it is involved in numerous basic cellular functions and metabolic pathways. A gene encoding a putative ferric uptake regulator (Fur) has been identified in the genome of C. acetobutylicum. In this work, we inactivated the fur gene by using insertional mutagenesis. The resultant mutant showed a slow-growing phenotype and enhanced sensitivity to oxidative stress, but essentially no dramatic change in its fermentation pattern. A unique feature of its physiology was the overflowing production of riboflavin. To gain further insights into the role of the Fur protein and the mechanisms for establishment of iron balance in C. acetobutylicum, we characterized and compared the gene-expression profile of the fur mutant and the iron-limitation stimulon of the parental strain. Not surprisingly, a repertoire of iron-transport systems was upregulated in both microarray datasets, suggesting that they are regulated by Fur according to the availability of iron. In addition, iron limitation and inactivation of fur affected the expression of several genes involved in energy metabolism. Among them, two genes, encoding a lactate dehydrogenase and a flavodoxin, were highly induced. In order to support the function of the latter, the ribDBAH operon responsible for riboflavin biosynthesis was also upregulated significantly. Furthermore, the iron-starvation response of C. acetobutylicum involved transcriptional modifications that were not detected in the fur mutant, suggesting that there exist additional mechanisms for adaptation to low-iron environments. Collectively, these results demonstrate that the strict anaerobe C. acetobutylicum senses and responds to availability of iron on multiple levels using a sophisticated system, and that Fur plays an important role in this process.