Biomedical subjects

K T Shanmugam

Publications and source records attributed to K T Shanmugam.

At least 19 recordsLinked to original sources

PubMed2007

Development of plasmid vector and electroporation condition for gene transfer in sporogenic lactic acid bacterium, Bacillus coagulans.

Bacillus coagulans is a sporogenic lactic acid bacterium that ferments glucose and xylose, major components of plant biomass, a potential feedstock for cellulosic ethanol. The temperature and pH for optimum rate of growth of B. coagulans (50 to 55 degrees C, pH 5.0) are very similar to that of commercially developed fungal cellulases (50 degrees C; pH 4.8). Due to this match, simultaneous saccharification and fermentation (SSF) of cellulose to products by B. coagulans is expected to require less cellulase than needed if the SSF is conducted at a sub-optimal temperature, such as 30 degrees C, the optimum for yeast, the main biocatalyst used by the ethanol industry. To fully exploit B. coagulans as a platform organism, we have developed an electroporation method to transfer plasmid DNA into this genetically recalcitrant bacterium. We also constructed a B. coagulans/E. coli shuttle vector, plasmid pMSR10 that contains the rep region from a native plasmid (pMSR0) present in B. coagulans strain P4-102B. The native plasmid, pMSR0 (6823bp), has 9 ORFs, and replicates by rolling-circle mode of replication. Plasmid pNW33N, developed for Geobacillus stearothermophilus, was also transformed into this host and stably maintained while several other Bacillus/Escherichia coli shuttle vector plasmids were not transformed into B. coagulans. The transformation efficiency of B. coagulans strain P4-102B using the plasmids pNW33N or pMSR10 was about 1.5x10(16) per mole of DNA. The availability of shuttle vectors and an electroporation method is expected to aid in genetic and metabolic engineering of B. coagulans.

Amino Acid Sequence↗

PubMed2006

Low salt medium for lactate and ethanol production by recombinant Escherichia coli B.

Individual nutrient salts were experimentally varied to determine the minimum requirements for efficient L (+)-lactate production by recombinant strains of Escherichia coli B. Based on these results, AM1 medium was formulated with low levels of alkali metals (4.5 mM and total salts (4.2 g l(-1)). This medium was equally effective for ethanol production from xylose and lactate production from glucose with average productivities of 18-19 mmol l(-1) h(-1) for both (initial 48 h of fermentation).

Bioreactors↗

PubMed2006

Methylglyoxal bypass identified as source of chiral contamination in l(+) and d(-)-lactate fermentations by recombinant Escherichia coli.

Two new strains of Escherichia coli B were engineered for the production of lactate with no detectable chiral impurity. All chiral impurities were eliminated by deleting the synthase gene (msgA) that converts dihydroxyacetone-phosphate to methylglyoxal, a precursor for both L: (+)- and D: (-)-lactate. Strain TG113 contains only native genes and produced optically pure D: (-)-lactate. Strain TG108 contains the ldhL gene from Pediococcus acidilactici and produced only L: (+)-lactate. In mineral salts medium containing 1 mM betaine, both strains produced over 115 g (1.3 mol) lactate from 12% (w/v) glucose, >95% theoretical yield.

Biotechnology↗

PubMed2006

Fermentation of 12% (w/v) glucose to 1.2 M lactate by Escherichia coli strain SZ194 using mineral salts medium.

A non-recombinant mutant of Escherichia coli B, strain SZ194, was developed that produces over 1 M D-lactate from glucose (or sucrose) in 72 h using mineral salts medium supplemented with 1 mM: betaine in simple anaerobic fermentations. Rates and yields were highest at pH 7.5. Yields approached the theoretical maximum with only trace amounts of co-products. Chiral purity of D-lactate was estimated to be 95%. Specific and volumetric productivities for SZ194 in mineral salts medium (pH 7.5) with betaine were equivalent to those in Luria broth.

Base Sequence↗

PubMed2006

Betaine tripled the volumetric productivity of D(-)-lactate by Escherichia coli strain SZ132 in mineral salts medium.

Osmotic stress restricts glycolytic flux, growth (rate and yield), D-lactate productivity, and D-lactate tolerance in Escherichia coli B strain SZ132 during batch fermentation in mineral salts medium with 10% (w/v) sugar. Addition of 1 mM: betaine, a non-metabolized protective osmolyte, doubled cell yield, increased specific productivity of D-lactate and glycolytic flux by 50%, and tripled volumetric productivity (from 8.6 to 25.7 mmol l(-1) h(-1); 0.8 to 2.3 g l(-1) h(-1)). Glycolytic flux and specific productivity in mineral salts medium with betaine exceeded that in Luria broth, substantially eliminating the need for complex nutrients during D-lactate production. In mineral salts medium supplemented with betaine, SZ132 produced approximately 1 mol D-lactate (90 g) per 100 g sugar (glucose or sucrose).

Betaine↗

PubMed2006

Isolation and characterization of acid-tolerant, thermophilic bacteria for effective fermentation of biomass-derived sugars to lactic acid.

Biomass-derived sugars, such as glucose, xylose, and other minor sugars, can be readily fermented to fuel ethanol and commodity chemicals by the appropriate microbes. Due to the differences in the optimum conditions for the activity of the fungal cellulases that are required for depolymerization of cellulose to fermentable sugars and the growth and fermentation characteristics of the current industrial microbes, simultaneous saccharification and fermentation (SSF) of cellulose is envisioned at conditions that are not optimal for the fungal cellulase activity, leading to a higher-than-required cost of cellulase in SSF. We have isolated bacterial strains that grew and fermented both glucose and xylose, major components of cellulose and hemicellulose, respectively, to l(+)-lactic acid at 50 degrees C and pH 5.0, conditions that are also optimal for fungal cellulase activity. Xylose was metabolized by these new isolates through the pentose-phosphate pathway. As expected for the metabolism of xylose by the pentose-phosphate pathway, [(13)C]lactate accounted for more than 90% of the total (13)C-labeled products from [(13)C]xylose. Based on fatty acid profile and 16S rRNA sequence, these isolates cluster with Bacillus coagulans, although the B. coagulans type strain, ATCC 7050, failed to utilize xylose as a carbon source. These new B. coagulans isolates have the potential to reduce the cost of SSF by minimizing the amount of fungal cellulases, a significant cost component in the use of biomass as a renewable resource, for the production of fuels and chemicals.

Bacillus↗

PubMed2005

Global gene expression analysis revealed an unsuspected deo operon under the control of molybdate sensor, ModE protein, in Escherichia coli.

ModE protein, a molybdate sensor/regulator, controls the transcription of genes coding for molybdate uptake (mod), molybdopterin synthesis (moa), molybdoenzymes nitrate reductase (nap) and dimethylsulfoxide reductase (dms), as well as fermentative dihydrogen production (fdhF and hyc) and respiratory nitrate reductase (narXL) in Escherichia coli. The catalytic product of a second protein, MoeA, is also required for molybdate-dependent positive regulation of hyc and nar operons. To explore the potential role of ModE and MoeA in the regulation of other E. coli genes, the global gene expression profile of a wild type and a modE, moeA double mutant grown in glucose-minimal medium under anaerobic conditions were compared. Expression of 67 genes was affected by the modE and moeA mutations (P value <0.01). Of these, 17 differed by at least 2-fold or higher. Fourteen genes were expressed at a higher level in the mutant (2.4- to 23.9-fold) (notably, mod-molybdate transport, deo-nucleoside catabolism and opp-oligopeptide transport operons) and dmsA and yli operon were expressed at a higher level in the wild type parent (2.6- to 5.7-fold). One of the unexpected findings was repression of the deo operon by ModE. This was confirmed by quantitative RT-PCR and by the analysis of a deoC-lacZ fusion. The deo promoter/operator region contains a putative ModE-consensus sequence centered at -35 in which the adenines are replaced by guanines (TGTGT-N7-TGTGT). The ModE protein did bind to the deo upstream DNA and shifted its electrophoretic mobility. Bioinformatics analysis of the E. coli genome for ModE-consensus motif (TATAT-N7-TAYAT) identified 21 additional genes/operons including the moa as potential targets for Mo-control. The physiological role of many of the genes identified solely by bioinformatics (19/21) is unknown. Expression levels of these genes were similar in the parent and the isogenic modE, moeA mutant when cultured anaerobically in glucose-minimal medium. This study identified additional targets, such as deo and opp, for the Mo-dependent control in E. coli.

Escherichia coli↗

PubMed2005

Fermentation of 10% (w/v) sugar to D: (-)-lactate by engineered Escherichia coli B.

Derivatives of ethanologenic Escherichia coli K011 were constructed for D: (-)-lactate production by deleting genes encoding competing pathways followed by metabolic evolution, a growth-based selection for mutants with improved performance. Resulting strains, SZ132 and SZ186, contain native genes for sucrose utilization. No foreign genes are present in SZ186. Strain SZ132 also contains a chromosomally integrated endoglucanase gene (Erwinia chrysanthemi celY). Strain SZ132 produced over 1 mol lactate per liter of complex medium containing 10% (w/v) sugar (fermentation times of 48 h for glucose, 120 h for sucrose). Both strains produced 667-700 mmol lactate per liter of mineral salts medium. Yields for metabolized sugar ranged from 88% to 95% in both media.

Carboxylic Acids↗

PubMed2004

Engineering Escherichia coli for efficient conversion of glucose to pyruvate.

Escherichia coli TC44, a derivative of W3110, was engineered for the production of pyruvate from glucose by combining mutations to minimize ATP yield, cell growth, and CO2 production (DeltaatpFH DeltaadhE DeltasucA) with mutations that eliminate acetate production [poxB::FRT (FLP recognition target) DeltaackA] and fermentation products (DeltafocA-pflB DeltafrdBC DeltaldhA DeltaadhE). In mineral salts medium containing glucose as the sole carbon source, strain TC44(DeltafocA-pflB DeltafrdBC DeltaldhA DeltaatpFH DeltaadhE DeltasucA poxB::FRT DeltaackA) converted glucose to pyruvate with a yield of 0.75 g of pyruvate per g of glucose (77.9% of theoretical yield; 1.2 g of pyruvate liters(-1).h(-1)). A maximum of 749 mM pyruvate was produced with excess glucose. Glycolytic flux was >50% faster for TC44 producing pyruvate than for the wild-type W3110 during fully aerobic metabolism. The tolerance of E. coli to such drastic changes in metabolic flow and energy production implies considerable elasticity in permitted pool sizes for key metabolic intermediates such as pyruvate and acetyl-CoA. In strain TC44, pyruvate yield, pyruvate titer, and the rate of pyruvate production in mineral salts medium were equivalent or better than previously reported for other biocatalysts (yeast and bacteria) requiring complex vitamin feeding strategies and complex nutrients. TC44 offers the potential to improve the economics of pyruvate production by reducing the costs of materials, product purification, and waste disposal.

Escherichia coli↗

PubMed2004

Production of D(-)-lactate from sucrose and molasses.

Escherichia coli W3110 derivatives, strains SZ63 and SZ85, were previously engineered to produce optically pure D(-) and L(+)-lactate from hexose and pentose sugars. To expand the substrate range, a cluster of sucrose genes (cscR' cscA cscKB) was cloned and characterized from E. coli KO11. The resulting plasmid was functionally expressed in SZ63 but was unstable in SZ85. Over 500 mM D(-)-lactate was produced from sucrose and from molasses by SZ63(pLOI3501).

Biotechnology↗

PubMed2004

Fermentation of sugar cane bagasse hemicellulose hydrolysate to L(+)-lactic acid by a thermotolerant acidophilic Bacillus sp.

Sugar cane bagasse hemicellulose, hydrolyzed by dilute H2SO4, supplemented with mineral salts and 0.5% corn steep liquor, was fermented to L(+)-lactic acid using a newly isolated strain of Bacillus sp. In batch fermentations at 50 degrees C and pH 5, over 5.5% (w/v) L(+)-lactic acid was produced (89% theoretical yield; 0.9 g lactate per g sugar) with an optical purity of 99.5%.

Bacillus↗

PubMed2004

Lack of protective osmolytes limits final cell density and volumetric productivity of ethanologenic Escherichia coli KO11 during xylose fermentation.

Limited cell growth and the resulting low volumetric productivity of ethanologenic Escherichia coli KO11 in mineral salts medium containing xylose have been attributed to inadequate partitioning of carbon skeletons into the synthesis of glutamate and other products derived from the citrate arm of the anaerobic tricarboxylic acid pathway. The results of nuclear magnetic resonance investigations of intracellular osmolytes under different growth conditions coupled with those of studies using genetically modified strains have confirmed and extended this hypothesis. During anaerobic growth in mineral salts medium containing 9% xylose (600 mM) and 1% corn steep liquor, proline was the only abundant osmolyte (71.9 nmol x ml(-1) optical density at 550 nm [OD(550)] unit(-1)), and growth was limited. Under aerobic conditions in the same medium, twice the cell mass was produced, and cells contained a mixture of osmolytes: glutamate (17.0 nmol x ml(-1) OD(550) unit(-1)), trehalose (9.9 nmol x ml(-1) OD(550) unit(-1)), and betaine (19.8 nmol x ml(-1) OD(550) unit(-1)). Two independent genetic modifications of E. coli KO11 (functional expression of Bacillus subtilis citZ encoding NADH-insensitive citrate synthase; deletion of ackA encoding acetate kinase) and the addition of a metabolite, such as glutamate (11 mM) or acetate (24 mM), as a supplement each increased the intracellular glutamate pool during fermentation, doubled cell growth, and increased volumetric productivity. This apparent requirement for a larger glutamate pool for increased growth and volumetric productivity was completely eliminated by the addition of a protective osmolyte (2 mM betaine or 0.25 mM dimethylsulfoniopropionate), consistent with adaptation to osmotic stress rather than relief of a specific biosynthetic requirement.

Betaine↗

PubMed2004

Expression and regulation of a silent operon, hyf, coding for hydrogenase 4 isoenzyme in Escherichia coli.

On the basis of hyf-lacZ fusion studies, the hyf operon of Escherichia coli, noted for encoding the fourth hydrogenase isoenzyme (HYD4), is not expressed at a significant level in a wild-type strain. However, mutant FhlA proteins (constitutive activators of the hyc-encoded hydrogenase 3 isoenzyme) activated hyf-lacZ. HyfR, an FhlA homolog encoded by the hyfR gene present at the end of the hyf operon, also activated transcription of hyf-lacZ but did so only when hyfR was expressed from a heterologous promoter. The HYD4 isoenzyme did not substitute for HYD3 in H(2) production. Optimum expression of hyf-lacZ required the presence of cyclic AMP receptor protein-cyclic AMP complex and anaerobic conditions when HyfR was the activator.

Aerobiosis↗

PubMed2004

Pyruvate formate lyase and acetate kinase are essential for anaerobic growth of Escherichia coli on xylose.

During anaerobic growth of bacteria, organic intermediates of metabolism, such as pyruvate or its derivatives, serve as electron acceptors to maintain the overall redox balance. Under these conditions, the ATP needed for cell growth is derived from substrate-level phosphorylation. In Escherichia coli, conversion of glucose to pyruvate yields 2 net ATPs, while metabolism of a pentose, such as xylose, to pyruvate only yields 0.67 net ATP per xylose due to the need for one (each) ATP for xylose transport and xylulose phosphorylation. During fermentative growth, E. coli produces equimolar amounts of acetate and ethanol from two pyruvates, and these reactions generate one additional ATP from two pyruvates (one hexose equivalent) while still maintaining the overall redox balance. Conversion of xylose to acetate and ethanol increases the net ATP yield from 0.67 to 1.5 per xylose. An E. coli pfl mutant lacking pyruvate formate lyase cannot convert pyruvate to acetyl coenzyme A, the required precursor for acetate and ethanol production, and could not produce this additional ATP. E. coli pfl mutants failed to grow under anaerobic conditions in xylose minimal medium without any negative effect on their survival or aerobic growth. An ackA mutant, lacking the ability to generate ATP from acetyl phosphate, also failed to grow in xylose minimal medium under anaerobic conditions, confirming the need for the ATP produced by acetate kinase for anaerobic growth on xylose. Since arabinose transport by AraE, the low-affinity, high-capacity, arabinose/H+ symport, conserves the ATP expended in pentose transport by the ABC transporter, both pfl and ackA mutants grew anaerobically with arabinose. AraE-based xylose transport, achieved after constitutively expressing araE, also supported the growth of the pfl mutant in xylose minimal medium. These results suggest that a net ATP yield of 0.67 per pentose is only enough to provide for maintenance energy but not enough to support growth of E. coli in minimal medium. Thus, pyruvate formate lyase and acetate kinase are essential for anaerobic growth of E. coli on xylose due to energetic constraints.

Acetate Kinase↗

PubMed2003

Engineering the metabolism of Escherichia coli W3110 for the conversion of sugar to redox-neutral and oxidized products: homoacetate production.

Microbial processes for commodity chemicals have focused on reduced products and anaerobic conditions where substrate loss to cell mass and CO(2) are minimal and product yields are high. To facilitate expansion into more oxidized chemicals, Escherichia coli W3110 was genetically engineered for acetate production by using an approach that combines attributes of fermentative and oxidative metabolism (rapid growth, external electron acceptor) into a single biocatalyst. The resulting strain (TC36) converted 333 mM glucose into 572 mM acetate, a product of equivalent oxidation state, in 18 h. With excess glucose, a maximum of 878 mM acetate was produced. Strain TC36 was constructed by sequentially assembling deletions that inactivated oxidative phosphorylation (deltaatpFH), disrupted the cyclic function of the tricarboxylic acid pathway (deltasucA), and eliminated native fermentation pathways (deltafocA-pflB deltafrdBC deltaldhA deltaadhE). These mutations minimized the loss of substrate carbon and the oxygen requirement for redox balance. Although TC36 produces only four ATPs per glucose, this strain grows well in mineral salts medium and has no auxotrophic requirement. Glycolytic flux in TC36 (0.3 micromol.min(-1).mg(-1) protein) was twice that of the parent. Higher flux was attributed to a deletion of membrane-coupling subunits in (F(1)F(0))H(+)-ATP synthase that inactivated ATP synthesis while retaining cytoplasmic F(1)-ATPase activity. The effectiveness of this deletion in stimulating flux provides further evidence for the importance of ATP supply and demand in the regulation of central metabolism. Derivatives of TC36 may prove useful for the commercial production of a variety of commodity chemicals.

Acetates↗

PubMed2003

Production of optically pure D-lactic acid in mineral salts medium by metabolically engineered Escherichia coli W3110.

The resistance of polylactide to biodegradation and the physical properties of this polymer can be controlled by adjusting the ratio of L-lactic acid to D-lactic acid. Although the largest demand is for the L enantiomer, substantial amounts of both enantiomers are required for bioplastics. We constructed derivatives of Escherichia coli W3110 (prototrophic) as new biocatalysts for the production of D-lactic acid. These strains (SZ40, SZ58, and SZ63) require only mineral salts as nutrients and lack all plasmids and antibiotic resistance genes used during construction. D-Lactic acid production by these new strains approached the theoretical maximum yield of two molecules per glucose molecule. The chemical purity of this D-lactic acid was approximately 98% with respect to soluble organic compounds. The optical purity exceeded 99%. Competing pathways were eliminated by chromosomal inactivation of genes encoding fumarate reductase (frdABCD), alcohol/aldehyde dehydrogenase (adhE), and pyruvate formate lyase (pflB). The cell yield and lactate productivity were increased by a further mutation in the acetate kinase gene (ackA). Similar improvements could be achieved by addition of 10 mM acetate or by an initial period of aeration. All three approaches reduced the time required to complete the fermentation of 5% glucose. The use of mineral salts medium, the lack of antibiotic resistance genes or plasmids, the high yield of D-lactate, and the high product purity should reduce costs associated with nutrients, purification, containment, biological oxygen demand, and waste treatment.

Acetates↗

PubMed2003

Functional replacement of the Escherichia coli D-(-)-lactate dehydrogenase gene (ldhA) with the L-(+)-lactate dehydrogenase gene (ldhL) from Pediococcus acidilactici.

The microbial production of L-(+)-lactic acid is rapidly expanding to allow increased production of polylactic acid (PLA), a renewable, biodegradable plastic. The physical properties of PLA can be tailored for specific applications by controlling the ratio of L-(+) and D-(-) isomers. For most uses of PLA, the L-(+) isomer is more abundant. As an approach to reduce costs associated with biocatalysis (complex nutrients, antibiotics, aeration, product purification, and waste disposal), a recombinant derivative of Escherichia coli W3110 was developed that contains five chromosomal deletions (focA-pflB frdBC adhE ackA ldhA). This strain was constructed from a D-(-)-lactic acid-producing strain, SZ63 (focA-pflB frdBC adhE ackA), by replacing part of the chromosomal ldhA coding region with Pediococcus acidilactici ldhL encoding an L-lactate dehydrogenase. Although the initial strain (SZ79) grew and fermented poorly, a mutant (SZ85) was readily isolated by selecting for improved growth. SZ85 exhibited a 30-fold increase in L-lactate dehydrogenase activity in comparison to SZ79, functionally replacing the native D-lactate dehydrogenase activity. Sequencing revealed mutations in the upstream, coding, and terminator regions of ldhL in SZ85, which are presumed to be responsible for increased L-lactate dehydrogenase activity. SZ85 produced L-lactic acid in M9 mineral salts medium containing glucose or xylose with a yield of 93 to 95%, a purity of 98% (based on total fermentation products), and an optical purity greater than 99%. Unlike other recombinant biocatalysts for L-lactic acid, SZ85 remained prototrophic and is devoid of plasmids and antibiotic resistance genes.

Culture Media↗

PubMed2002

Genetic changes to optimize carbon partitioning between ethanol and biosynthesis in ethanologenic Escherichia coli.

The production of ethanol from xylose by ethanologenic Escherichia coli strain KO11 was improved by adding various medium supplements (acetate, pyruvate, and acetaldehyde) that prolonged the growth phase by increasing cell yield and volumetric productivity (approximately twofold). Although added pyruvate and acetaldehyde were rapidly metabolized, the benefit of these additives continued throughout fermentation. Both additives increased the levels of extracellular acetate through different mechanisms. Since acetate can be reversibly converted to acetyl coenzyme A (acetyl-CoA) by acetate kinase and phosphotransacetylase, the increase in cell yield caused by each of the three supplements is proposed to result from an increase in the pool of acetyl-CoA. A similar benefit was obtained by inactivation of acetate kinase (ackA), reducing the production of acetate (and ATP) and sparing acetyl-CoA for biosynthetic needs. Inactivation of native E. coli alcohol-aldehyde dehydrogenase (adhE), which uses acetyl-CoA as an electron acceptor, had no beneficial effect on growth, which was consistent with a minor role for this enzyme during ethanol production. Growth of KO11 on xylose appears to be limited by the partitioning of carbon skeletons into biosynthesis rather than the level of ATP. Changes in acetyl-CoA production and consumption provide a useful approach to modulate carbon partitioning. Together, these results demonstrate that xylose fermentation to ethanol can be improved in KO11 by redirecting small amounts of pyruvate away from fermentation products and into biosynthesis. Though negligible with respect to ethanol yield, these small changes in carbon partitioning reduced the time required to complete the fermentation of 9.1% xylose in 1% corn steep liquor medium from over 96 h to less than 72 h.

Acetic Acid↗