Adh4, an alcohol dehydrogenase controls alcohol formation within bacterial microcompartments in the acetogenic bacterium Acetobacterium woodii

Acetobacterium woodii utilizes the Wood-Ljungdahl pathway for reductive synthesis of acetate from carbon dioxide. However, A. woodii can also perform non-acetogenic growth on 1,2-propanediol (1,2-PD) where instead of acetate, equal amounts of propionate and propanol are produced as metabolic end products. Metabolism of 1,2-PD occurs via encapsulated metabolic enzymes within large proteinaceous bodies called bacterial microcompartments. While the genome of A. woodii harbours 11 genes encoding putative alcohol dehydrogenases, the BMC-encapsulated propanol-generating alcohol dehydrogenase remains unidentified. Here, we show that Adh4 of A. woodii is the alcohol dehydrogenase required for propanol/ethanol formation within these microcompartments. It catalyses the NADH-dependent reduction of propionaldehyde or acetaldehyde to propanol or ethanol and primarily functions to recycle NADH within the BMC. Removal of adh4 gene from the A. woodii genome resulted in slow growth on 1,2-PD and the mutant displayed reduced propanol and enhanced propionate formation as a metabolic end product. In sum, the data suggest that Adh4 is responsible for propanol formation within the BMC and is involved in redox balancing in the acetogen, A. woodii.     

Genetic analysis of the protein shell of the microcompartments involved in coenzyme B12-dependent 1,2-propanediol degradation by Salmonella

Hundreds of bacterial species use microcompartments (MCPs) to optimize metabolic pathways that have toxic or volatile intermediates. MCPs consist of a protein shell encapsulating specific metabolic enzymes. In Salmonella, an MCP is used for 1,2-propanediol utilization (Pdu MCP). The shell of this MCP is composed of eight different types of polypeptides, but their specific functions are uncertain. Here, we individually deleted the eight genes encoding the shell proteins of the Pdu MCP. The effects of each mutation on 1,2-PD degradation and MCP structure were determined by electron microscopy and growth studies. Deletion of the pduBB', pduJ, or pduN gene severely impaired MCP formation, and the observed defects were consistent with roles as facet, edge, or vertex protein, respectively. Metabolite measurements showed that pduA, pduBB', pduJ, or pduN deletion mutants accumulated propionaldehyde to toxic levels during 1,2-PD catabolism, indicating that the integrity of the shell was disrupted. Deletion of the pduK, pduT, or pduU gene did not substantially affect MCP structure or propionaldehyde accumulation, suggesting they are nonessential to MCP formation. However, the pduU or pduT deletion mutants grew more slowly than the wild type on 1,2-PD at saturating B(12), indicating that they are needed for maximal activity of the 1,2-PD degradative enzymes encased within the MCP shell. Considering recent crystallography studies, this suggests that PduT and PduU may mediate the transport of enzyme substrates/cofactors across the MCP shell. Interestingly, a pduK deletion caused MCP aggregation, suggesting a role in the spatial organization of MCP within the cytoplasm or perhaps in segregation at cell division.     

Microcompartments for B12-dependent 1,2-propanediol degradation provide protection from DNA and cellular damage by a reactive metabolic intermediate

Salmonella enterica grows on 1,2-propanediol (1,2-PD) in a coenzyme B(12)-dependent fashion. Prior studies showed that a bacterial microcompartment (MCP) is involved in this process and that an MCP-minus mutant undergoes a 20-h period of growth arrest during 1,2-PD degradation. It was previously proposed that growth arrest resulted from propionaldehyde toxicity, but no direct evidence was presented. Here, high-pressure liquid chromatography analyses of culture medium were used to show that the major products of aerobic 1,2-PD degradation are propionaldehyde, propionate, and 1-propanol. A MCP-minus mutant accumulated a level of propionaldehyde 10-fold higher than that of the wild type (1.6 mM compared to 15.7 mM), associating this compound with growth arrest. The addition of propionaldehyde to cultures of S. enterica caused growth arrest from 8 to 20 mM, but not at 4 mM, providing direct evidence for propionaldehyde toxicity. Studies also indicated that propionaldehyde was toxic due to the inhibition of respiratory processes, and the growth arrest ended when propionaldehyde was depleted primarily by conversion to propionate and 1-propanol and secondarily due to volatility. The Ames test was used to show that propionaldehyde is a mutagen and that mutation frequencies are increased in MCP-minus mutants during 1,2-PD degradation. We propose that a primary function of the MCPs involved in 1,2-PD degradation is the mitigation of toxicity and DNA damage by propionaldehyde.     

Metabolic engineering of 1,2-propanediol pathways in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

We analyzed 1,2-propanediol (1,2-PD) production in metabolically engineered Corynebacterium glutamicum. Wild-type C. glutamicum produced 93 μM 1,2-PD after 132 h incubation under aerobic conditions. No gene encoding the methylglyoxal synthase (MGS) which catalyzes the first step of 1,2-PD synthesis from the glycolytic pathway was detected on the C. glutamicum genome, but several genes annotated as encoding putative aldo-keto reductases (AKRs) were present. AKR functions as a methylglyoxal reductase in the 1,2-PD synthesis pathway. Expressing Escherichia coli mgs gene in C. glutamicum increased 1,2-PD yield 100-fold, suggesting that wild-type C. glutamicum carries the genes downstream of MGS in the 1,2-PD synthesis pathway. Furthermore, simultaneous overexpression of mgs and cgR_2242, one of the genes annotated as AKRs, enhanced 1,2-PD production to 24 mM. This work establishes that 1,2-PD synthesis by C. glutamicum, previously unknown, is possible.     

Comparative genomics and proteomics of Eubacterium maltosivorans: functional identification of trimethylamine methyltransferases and bacterial microcompartments in a human intestinal bacterium with a versatile lifestyle

Eubacterium maltosivorans YI^T is a human intestinal isolate capable of acetogenic, propionogenic and butyrogenic growth. Its 4.3-Mb genome sequence contains coding sequences for 4227 proteins, including 41 different methyltransferases. Comparative proteomics of strain YI^T showed the Wood–Ljungdahl pathway proteins to be actively produced during homoacetogenic growth on H₂ and CO₂ while butyrogenic growth on a mixture of lactate and acetate significantly upregulated the production of proteins encoded by the recently identified lctABCDEF cluster and accessory proteins. Growth on H₂ and CO₂ unexpectedly induced the production of two related trimethylamine methyltransferases. Moreover, a set of 16 different trimethylamine methyltransferases together with proteins for bacterial microcompartments were produced during growth and deamination of the quaternary amines, betaine, carnitine and choline. Growth of strain YI^T on 1,2-propanediol generated propionate with propanol and induced the formation of bacterial microcompartments that were also prominently visible in betaine-grown cells. The present study demonstrates that E. maltosivorans is highly versatile in converting low-energy fermentation end-products in the human gut into butyrate and propionate whilst being capable of preventing the formation of the undesired trimethylamine by converting betaine and other quaternary amines in bacterial microcompartments into acetate and butyrate.     

Employing bacterial microcompartment technology to engineer a shell-free enzyme-aggregate for enhanced 1,2-propanediol production in Escherichia coli

Bacterial microcompartments (BMCs) enhance the breakdown of metabolites such as 1,2-propanediol (1,2-PD) to propionic acid. The encapsulation of proteins within the BMC is mediated by the presence of targeting sequences. In an attempt to redesign the Pdu BMC into a 1,2-PD synthesising factory using glycerol as the starting material we added N-terminal targeting peptides to glycerol dehydrogenase, dihydroxyacetone kinase, methylglyoxal synthase and 1,2-propanediol oxidoreductase to allow their inclusion into an empty BMC. 1,2-PD producing strains containing the fused enzymes exhibit a 245% increase in product formation in comparison to un-tagged enzymes, irrespective of the presence of BMCs. Tagging of enzymes with targeting peptides results in the formation of dense protein aggregates within the cell that are shown by immuno-labelling to contain the vast majority of tagged proteins. It can therefore be concluded that these protein inclusions are metabolically active and facilitate the significant increase in product formation.     

Characterization of 1,3-propanediol oxidoreductase (DhaT) from <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> J2B

1,3-propanediol oxidoreductase (DhaT) of Klebsiella pneumoniae converts 3-hydroxypropionaldehyde (3-HPA) to 1,3-propanediol (1,3-PD) during microbial production of 1,3-PD from glycerol. In this study, DhaT from newly isolated K. pneumoniae J2B was cloned, expressed, purified, and studied for its kinetic properties. It showed, on its physiological substrate 3-HPA, higher activity than similar aldehydes such as acetaldehyde, propionaldehyde and butyraldehyde. The turnover numbers (k _cat , 1/s) were estimated as 59.4 for the forward reaction (3-HPA to 1,3-PD at pH 7.0) and 10.0 for the reverse reaction (1,3-PD to 3-HPA at pH 9.0). The Michaelis constants (K _m , mM) were 0.77 (for 3-HPA) and 0.03 (for NADH) for the forward reaction (at pH 7.0), and 7.44 (for 1,3-PD) and 0.23 (for NAD⁺) for the reverse reaction (at pH 9.0). Between these forward and reverse reactions, the optimum temperature and pH were significantly different (37°C and 7.0 vs. 55°C and 9.0, respectively). These results indicate that, under physiological conditions, DhaT mostly catalyzes the forward reaction. The enzyme was seriously inhibited by heavy metal ions such as Ag⁺ and Hg²⁺. DhaT was highly unstable when incubated with its own substrate 3-HPA, indicating the necessity of enhancing its stability for improved 1,3-PD production from glycerol.     

Construction and Characterization of a 1,3-Propanediol Operon

The genes for the production of 1,3-propanediol (1,3-PD) in Klebsiella pneumoniae, dhaB, which encodes glycerol dehydratase, and dhaT, which encodes 1,3-PD oxidoreductase, are naturally under the control of two different promoters and are transcribed in different directions. These genes were reconfigured into an operon containing dhaB followed by dhaT under the control of a single promoter. The operon contains unique restriction sites to facilitate replacement of the promoter and other modifications. In a fed-batch cofermentation of glycerol and glucose, Escherichia coli containing the operon consumed 9.3 g of glycerol per liter and produced 6.3 g of 1,3-PD per liter. The fermentation had two distinct phases. In the first phase, significant cell growth occurred and the products were mainly 1,3-PD and acetate. In the second phase, very little growth occurred and the main products were 1,3-PD and pyruvate. The first enzyme in the 1,3-PD pathway, glycerol dehydratase, requires coenzyme B₁₂, which must be provided in E. coli fermentations. However, the amount of coenzyme B₁₂ needed was quite small, with 10 nM sufficient for good 1,3-PD production in batch cofermentations. 1,3-PD is a useful intermediate in the production of polyesters. The 1,3-PD operon was designed so that it can be readily modified for expression in other prokaryotic hosts; therefore, it is useful for metabolic engineering of 1,3-PD pathways from glycerol and other substrates such as glucose.     

The effect of carbon sources and lactate dehydrogenase deletion on 1,2-propanediol production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

In previous studies, we showed that cofactor manipulations can potentially be used as a tool in metabolic engineering. In this study, sugars similar to glucose, that can feed into glycolysis and pyruvate production, but with different oxidation states, were used as substrates. This provided a simple way of testing the effect of manipulating the NADH/NAD+ ratio or the availability of NADH on the metabolic patterns of Escherichia coli under anaerobic conditions and on the production of 1,2-propanediol (1,2-PD), which requires NADH for its synthesis. Production of 1,2-PD was achieved by overexpressing the two enzymes methylglyoxal synthase from Clostridium acetobutylicum and glycerol dehydrogenase from E. coli. In addition, the effect of eliminating a pathway competing for NADH by using a ldh ^– strain (without lactate dehydrogenase activity) on the production of 1,2-PD was investigated. The oxidation state of the carbon source significantly affected the yield of metabolites, such as ethanol, acetate and lactate. However, feeding a more reduced carbon source did not increase the yield of 1,2-PD. The production of 1,2-PD with glucose as the carbon source was improved by the incorporation of a ldh ^– mutation. The results of these experiments indicate that our current 1,2-PD production system is not limited by NADH, but rather by the pathways following the formation of methylglyoxal. Electronic Publication     

Microbial production of 1,3-propanediol

1,3-Propanediol (1,3-PD) production by fermentation of glycerol was described in 1881 but little attention was paid to this microbial route for over a century. Glycerol conversion to 1,3-PD can be carried out by Clostridia as well as Enterobacteriaceae. The main intermediate of the oxidative pathway is pyruvate, the further utilization of which produces CO₂, H₂, acetate, butyrate, ethanol, butanol and 2,3-butanediol. In addition, lactate and succinate are generated. The yield of 1,3-PD per glycerol is determined by the availability of NADH₂, which is mainly affected by the product distribution (of the oxidative pathway) and depends first of all on the microorganism used but also on the process conditions (type of fermentation, substrate excess, various inhibitions). In the past decade, research to produce 1,3-PD microbially was considerably expanded as the diol can be used for various polycondensates. In particular, polyesters with useful properties can be manufactured. A prerequisite for making a “green” polyester is a more cost-effective production of 1,3-PD, which, in practical terms, can only be achieved by using an alternative substrate, such as glucose instead of glycerol. Therefore, great efforts are now being made to combine the pathway from glucose to glycerol successfully with the bacterial route from glycerol to 1,3-PD. Thus, 1,3-PD may become the first bulk chemical produced by a genetically engineered microorganism. Received: 12 January 1999 / Received revision: 9 March 1999 / Accepted: 14 March 1999     

Development of a Saccharomyces cerevisiae strain for the production of 1,2-propanediol by gene manipulation

The main goal of this research was to achieve a more efficient production of 1,2-propanediol (1,2-PD) using mutated Saccharomyces cerevisiae. 1,2-PD cannot be produced by wild type S. cerevisiae. To develop a S. cerevisiae mutant that could produce 1,2-PD, the mgs gene of E. coli-K12 MG1655 and the dhaD gene of Citrobacter freundii were inserted into yeast expression vectors such as pESC-URA and pESC-TRP and transformed into the wild type of S. cerevisiae. As a result, the batch fermentation of S. cerevisiae YPH500, harboring an mgs gene inserted pJES27 vector, resulted in a yield of 0.17 g/L. On the other hand, the methylglyoxal synthase of the recombinant S. cerevisiae YPH500, harboring a dhaD gene inserted pJES29 vector, was inactive and produced no detectable amount of 1,2-PD. Therefore, in order to achieve a maximum yield of 1,2-PD, we selected the pESC-TRP vector that is able to co-express the dhaD gene with the pJES27 vector. By inserting the dhaD gene into the pESC-TRP vector, the pJES30 vector was constructed. The maximal yield of 1,2-PD achieved in a 1% galactose batch fermentation by pJES27 and pJES30 harboring S. cerevisiae was 0.45 g/L.     

The formation of unhydrated propionaldehyde by dioldehydrase.

When dl-1,2-propanediol is converted to propionaldehyde by dioldehydrase, an enzyme which requires B₁₂-coenzyme, the product is unhydrated propionaldehyde. Its formation was demonstrated by measuring the rate of reduction of the enzymically formed aldehyde by NADH, catalyzed by yeast alcohol dehydrogenase.     

Characterization of the PduS Cobalamin Reductase of Salmonella enterica and Its Role in the Pdu Microcompartment

Salmonella enterica degrades 1,2-propanediol (1,2-PD) in a coenzyme B₁₂ (adenosylcobalamin, AdoCbl)-dependent fashion. Salmonella obtains AdoCbl by assimilation of complex precursors, such as vitamin B₁₂ and hydroxocobalamin. Assimilation of these compounds requires reduction of their central cobalt atom from Co³⁺ to Co²⁺ to Co⁺, followed by adenosylation to AdoCbl. In this work, the His₆-tagged PduS cobalamin reductase from S. enterica was produced at high levels in Escherichia coli, purified, and characterized. The anaerobically purified enzyme reduced cob(III)alamin to cob(II)alamin at a rate of 42.3 ± 3.2 μmol min⁻¹ mg⁻¹, and it reduced cob(II)alamin to cob(I)alamin at a rate of 54.5 ± 4.2 nmol min⁻¹ mg⁻¹ protein. The apparent K_m values of PduS-His₆ were 10.1 ± 0.7 μM for NADH and 67.5 ± 8.2 μM for hydroxocobalamin in cob(III)alamin reduction. The apparent K_m values for cob(II)alamin reduction were 27.5 ± 2.4 μM with NADH as the substrate and 72.4 ± 9.5 μM with cob(II)alamin as the substrate. High-performance liquid chromatography (HPLC) and mass spectrometry (MS) indicated that each monomer of PduS contained one molecule of noncovalently bound flavin mononucleotide (FMN). Genetic studies showed that a pduS deletion decreased the growth rate of Salmonella on 1,2-PD, supporting a role in cobalamin reduction in vivo. Further studies demonstrated that the PduS protein is a component of the Pdu microcompartments (MCPs) used for 1,2-PD degradation and that it interacts with the PduO adenosyltransferase, which catalyzes the terminal step of AdoCbl synthesis. These studies further characterize PduS, an unusual MCP-associated cobalamin reductase, and, in conjunction with prior results, indicate that the Pdu MCP encapsulates a complete cobalamin assimilation system.Coenzyme B₁₂ (adenosylcobalamin, AdoCbl) is an indispensable cofactor for a variety of enzymes that are widely distributed among microbes and higher animals (2, 55). Organisms obtain AdoCbl by de novo synthesis or by assimilation of complex precursors, such as vitamin B₁₂ (cyanocobalamin, CN-Cbl) and hydroxocobalamin (OH-Cbl), which can be enzymatically converted to AdoCbl. De novo synthesis occurs only in prokaryotes, but the assimilation of complex precursors is more widespread, taking place in many microbes and in higher animals (56). A model for the assimilation of CN-Cbl and OH-Cbl to AdoCbl, based on work done in a number of laboratories, is shown in Fig. ​Fig.1.1. CN-Cbl is first reductively decyanated to cob(II)alamin (22, 30, 68). Next, cob(II)alamin is reduced to cob(I)alamin, and ATP:cob(I)alamin adenosyltransferase (ATR) transfers a 5′ deoxyadenosyl group from ATP to cob(I)alamin to form AdoCbl (10, 11, 28, 29, 35, 63, 64, 72). Studies indicate that prior to reduction cob(II)alamin binds the ATR and undergoes a transition to the 4-coordinate base-off conformer (41, 48, 59, 61, 62). Transition to the 4-coordinate state raises the midpoint potential of the cob(II)alamin/cob(I)alamin couple by about 250 mV, facilitating reduction (60). OH-Cbl assimilation occurs by a similar pathway except that the first step is reduction of OH-Cbl to cob(II)alamin by cobalamin reductase or by the reducing environment of the cell (19, 69).Open in a separate windowFIG. 1.Cobalamin assimilation and recycling pathway. Many organisms are able to take up CN-Cbl and OH-Cbl and convert them to the active coenzyme form, AdoCbl. This process involves reduction of the central cobalt atom of the corrin ring followed by addition of a 5′ deoxyadenosyl (Ado) group via a carbon-cobalt bond. The Ado group is unstable in vivo, and AdoCbl breaks down to form OH-Cbl. Consequently, cobalamin recycling is required for AdoCbl-dependent processes, and recycling uses the same pathway that functions in the assimilation of cobalamin from the environment. PPPi, triphosphate.The pathway used for the assimilation of OH-Cbl and CN-Cbl is also used for intracellular cobalamin recycling. During catalysis the adenosyl group of AdoCbl is periodically lost due to by-reactions and is usually replaced by a hydroxyl group, resulting in the formation of an inactive OH-Cbl enzyme complex (66). Cobalamin recycling begins with a reactivase that converts the inactive OH-Cbl-enzyme complex to OH-Cbl and apoenzyme (43, 44). Next, the process described in Fig. ​Fig.11 converts OH-Cbl to AdoCbl, which spontaneously associates with apoenzyme to form active holoenzyme (43, 44, 66). In the organisms that have been studied, cobalamin recycling is essential, and genetic defects in this process block AdoCbl-dependent metabolism (3, 16, 29).Salmonella enterica degrades 1,2-propanediol (1,2-PD) via an AdoCbl-dependent pathway (27). 1,2-PD is a major product of the anaerobic degradation of common plant sugars rhamnose and fucose and is thought to be an important carbon and energy source in natural environments (38, 46). Twenty-four genes for 1,2-PD utilization (pdu) are found in a contiguous cluster (pocR, pduF, and pduABB′CDEGHJKLMNOPQSTUVWX) (7, 27). This locus encodes enzymes for the degradation of 1,2-PD and cobalamin recycling, as well as proteins for the formation of a bacterial microcompartment (MCP) (7). Bacterial MCPs are simple proteinaceous organelles used by diverse bacteria to optimize metabolic pathways that have toxic or volatile intermediates (6, 13, 14, 71). They are polyhedral in shape, 100 to 150 nm in cross section (about the size of a large virus), and consist of a protein shell that encapsulates sequentially acting metabolic enzymes. Sequence analyses indicate that MCPs are produced by 20 to 25% of all bacteria and function in seven or more different metabolic processes (14). The function of the Pdu MCP is to confine the propionaldehyde formed in the first step of 1,2-PD degradation in order to mitigate its toxicity and prevent DNA damage (7, 23, 24, 51). Prior studies indicate that 1,2-PD traverses the protein shell and enters the lumen of the Pdu MCP, where it is converted to propionaldehyde and then to propionyl-coenzyme A (CoA) by AdoCbl-dependent diol dehydratase (DDH; PduCDE) and propionaldehyde dehydrogenase (PduP) (8, 33). Propionyl-CoA then exits the MCP into the cytoplasm, where it is converted to 1-propanol or propionate or enters central metabolism via the methylcitrate pathway (25, 47). The shell of the Pdu MCP is thought to limit the diffusion of propionaldehyde in order to protect cytoplasmic components from toxicity. The Pdu MCP was purified, and 14 major polypeptide components were identified (PduABB′CDEGHJKOPTU), all of which are encoded by the pdu locus (23). PduABB′JKTU are confirmed or putative shell proteins (23, 24, 51). PduCDE and PduP catalyze the first 2 steps of 1,2-PD degradation as described above (7, 8, 23, 33). The PduO and PduGH enzymes are used for cobalamin recycling. PduO is an adenosyltransferase (29), and PduGH is a homolog of the Klebsiella DDH reactivase, which mediates the removal of OH-Cbl from an inactive OH-Cbl-DDH complex (43, 44). However, a reductase which is also required for cobalamin recycling was not previously identified as a component of the Pdu MCP (23). This raises the question of how cobalamin is recycled for the AdoCbl-dependent DDH that resides within the Pdu MCP.Prior studies indicated that the PduS enzyme (which is encoded by the pdu locus) is a cobalamin reductase (52). Very recently PduS was purified from S. enterica and shown to be a flavoprotein that can mediate the reduction of 4-coordinate cob(II)alamin bound to ATR but was not further characterized (40). In this study, PduS from S. enterica is purified and more extensively characterized, including identification of its cofactor requirements and kinetic properties. In addition, we show that PduS is a component of the Pdu MCP. This finding in conjunction with prior work indicates that, in addition to 1,2-PD degradative enzymes, the Pdu MCP encapsulates a complete cobalamin recycling system.     

LplR,a Repressor Belonging to the TetR Family,Regulates Expression of the l-Pantoyl Lactone Dehydrogenase Gene in Rhodococcus erythropolis

The l-pantoyl lactone (l-PL) dehydrogenase (LPLDH) gene (lpldh) has been cloned from Rhodococcus erythropolis AKU2103, and addition of 1,2-propanediol (1,2-PD) was shown to be required for lpldh expression in this strain. In this study, based on an exploration of the nucleotide sequence around lpldh, a TetR-like regulator gene, which we designated lplR, was found upstream of lpldh, and three putative open reading frames existed between the two genes. Disruption of lplR led to 22.8 times higher lpldh expression, even without 1,2-PD induction, than that in wild-type R. erythropolis AKU2103 without 1,2-PD addition. Introduction of a multicopy vector carrying lplR (multi-lplR) into the wild-type and ΔlplR strains led to no detectable LPLDH activity even in the presence of 1,2-PD. The results of an electrophoretic mobility shift assay revealed that purified LplR bound to a 6-bp inverted-repeat sequence located in the promoter/operator region of the operon containing lpldh. These results indicated that LplR is a negative regulator in lpldh expression. Based on the clarification of the expression mechanism of lpldh, recombinant cells showing high LPLDH activity were constructed and used as a catalyst for the conversion of l-PL to ketopantoyl lactone. Finally, a promising production process of d-PL from dl-PL was constructed.     

Bacterial metabolism of propane-1,2-diol

The pathway of propane-1,2-diol metabolism by a species of Flavobacterium able to grow on the diol as the sole source of carbon was influenced by the degree of aeration of the growth medium. Under strongly aerobic conditions the diol was exclusively catabolised to lactaldehyde by an initial diol oxidase, subsequently metabolised to pyruvate and then oxidised to CO₂ by the tricarboxylic acid cyle. Under microaerophilic conditions some propane-1,2-diol was catabolised by the oxidase-initiated pathway, but some diol was alternatively catabolised by an inducible diol dehydrase to propionaldehyde and subsequently reduced to n-propanol as an end product of metabolism.     

Genetic and biocatalytic basis of formate dependent growth of Escherichia coli strains evolved in continuous culture

The reductive glycine pathway was described as the most energetically favorable synthetic route of aerobic formate assimilation. Here we report the successful implementation of formatotrophy in Escherichia coli by means of a stepwise adaptive evolution strategy. Medium swap and turbidostat regimes of continuous culture were applied to force the channeling of carbon flux through the synthetic pathway to pyruvate establishing growth on formate and CO₂ as sole carbon sources. Labeling with ¹³C-formate proved the assimilation of the C1 substrate via the pathway metabolites. Genetic analysis of intermediate isolates revealed a mutational path followed throughout the adaptation process. Mutations were detected affecting the copy number (gene ftfL) or the coding sequence (genes folD and lpd) of genes which specify enzymes implicated in the three steps forming glycine from formate and CO₂, the central metabolite of the synthetic pathway. The mutation R191S present in methylene-tetrahydrofolate dehydrogenase/cyclohydrolase (FolD) abolishes the inhibition of cyclohydrolase activity by the substrate formyl-tetrahydrofolate. The mutation R273H in lipoamide dehydrogenase (Lpd) alters substrate affinities as well as kinetics at physiological substrate concentrations likely favoring a reactional shift towards lipoamide reduction. In addition, genetic reconstructions proved the necessity of all three mutations for formate assimilation by the adapted cells. The largely unpredictable nature of these changes demonstrates the usefulness of the evolutionary approach enabling the selection of adaptive mutations crucial for pathway engineering of biotechnological model organisms.     

Isolation of microorganisms able to produce 1,3-propanediol and optimization of medium constituents for Klebsiella pneumoniae AJ4

Microbial fermentation under anaerobic and microaerobic conditions has been used for the production of 1,3-propanediol (1,3-PD), a monomer used to produce polymers such as polytrimethylene terephthalate. In this study, we screened microorganisms using the high throughput screening method and isolated the Klebsiella pneumoniae AJ4 strain, which is able to produce 1,3-PD under aerobic conditions. To obtain the maximum 1,3-PD concentration from glycerol, the response surface methodology based on a central composite design was chosen to show the statistical significance of the effects of glycerol, peptone, and (NH₄)₂SO₄ on 1,3-PD production by K. pneumoniae AJ4. The optimal culture medium factors for achieving maximum concentrations of 1,3-PD included glycerol, 108.5 g/L; peptone, 2.72 g/L; and (NH₄)₂SO_4, 4.38 g/L. Under this optimum condition, the maximum concentration of 1,3-PD, 54.76 g/L, was predicted. A concentration of about 52.59 g/L 1,3-PD was obtained using the optimized medium during 26-h batch fermentation, a finding that agreed well with the predicted value.     

Metabolic engineering of a 1,2-propanediol pathway in Escherichia coli

1,2-Propanediol (1,2-PD) is a major commodity chemical that is currently derived from propylene, a nonrenewable resource. A goal of our research is to develop fermentation routes to 1,2-PD from renewable resources. Here we report the production of enantiomerically pure R-1,2-PD from glucose in Escherichia coli expressing NADH-linked glycerol dehydrogenase genes (E. coli gldA or Klebsiella pneumoniae dhaD). We also show that E. coli overexpressing the E. coli methylglyoxal synthase gene (mgs) produced 1,2-PD. The expression of either glycerol dehydrogenase or methylglyoxal synthase resulted in the anaerobic production of approximately 0.25 g of 1,2-PD per liter. R-1,2-PD production was further improved to 0.7 g of 1,2-PD per liter when methylglyoxal synthase and glycerol dehydrogenase (gldA) were coexpressed. In vitro studies indicated that the route to R-1,2-PD involved the reduction of methylglyoxal to R-lactaldehyde by the recombinant glycerol dehydrogenase and the reduction of R-lactaldehyde to R-1, 2-PD by a native E. coli activity. We expect that R-1,2-PD production can be significantly improved through further metabolic and bioprocess engineering.