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 (⌬atpFH ⌬adhE ⌬sucA) with mutations that eliminate acetate production [poxB::FRT (FLP recognition target) ⌬ackA] and fermentation products (⌬focA-pflB ⌬frdBC ⌬ldhA ⌬adhE). In mineral salts medium containing glucose as the sole carbon source, strain TC44(⌬focA-pflB ⌬frdBC ⌬ldhA ⌬atpFH ⌬adhE ⌬sucA poxB::FRT ⌬ackA) 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 ⅐h ؊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 biocatalyts (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.metabolic engineering ͉ glycolytic flux ͉ fermentation ͉ ATPase

The technology is available to produce fuel ethanol from renewable lignocellulosic biomass. The current challenge is to assemble the various process options into a commercial venture and begin the task of incremental improvement. Current process designs for lignocellulose are far more complex than grain to ethanol processes. This complexity results in part from the complexity of the substrate and the biological limitations of the catalyst. Our work at the University of Florida has focused primarily on the genetic engineering of Enteric bacteria using genes encoding Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase. These two genes have been assembled into a portable ethanol production cassette, the PET operon, and integrated into the chromosome of Escherichia coli B for use with hemicellulose-derived syrups. The resulting strain, KO11, produces ethanol efficiently from all hexose and pentose sugars present in the polymers of hemicellulose. By using the same approach, we integrated the PET operon into the chromosome of Klebsiella oxytoca to produce strain P2 for use in the simultaneous saccharification and fermentation (SSF) process for cellulose. Strain P2 has the native ability to ferment cellobiose and cellotriose, eliminating the need for one class of cellulase enzymes. Recently, the ability to produce and secrete high levels of endoglucanase has also been added to strain P2, further reducing the requirement for fungal cellulase. The general approach for the genetic engineering of new biocatalysts using the PET operon has been most successful with Enteric bacteria but was also extended to Gram positive bacteria, which have other useful traits for lignocellulose conversion. Many opportunities remain for further improvements in these biocatalysts as we proceed toward the development of single organisms that can be used for the efficient fermentation of both hemicellulosic and cellulosic substrates.

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 ϳ98% 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.

Microbial processes for commodity chemicals have focused on reduced products and anaerobic conditions where substrate loss to cell mass and CO2 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 (⌬atpFH), disrupted the cyclic function of the tricarboxylic acid pathway (⌬sucA), and eliminated native fermentation pathways (⌬focA-pflB ⌬frdBC ⌬ldhA ⌬adhE). 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 mol⅐min ؊1 ⅐mg ؊1 protein) was twice that of the parent. Higher flux was attributed to a deletion of membrane-coupling subunits in (F1F0)H ؉ -ATP synthase that inactivated ATP synthesis while retaining cytoplasmic F1-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. metabolic engineering ͉ glycolytic flux ͉ acetic acid ͉ fermentation ͉ ATPase

Recombinant His-tagged proteins expressed in Escherichia coli and purified by immobilized metal affinity chromatography (IMAC) are commonly coeluted with native E. coli proteins, especially if the recombinant protein is expressed at a low level. The E. coli contaminants display high affinity to divalent nickel or cobalt ions, mainly due to the presence of clustered histidine residues or biologically relevant metal binding sites. To improve the final purity of expressed His-tagged protein, we engineered E. coli BL21(DE3) expression strains in which the most recurring contaminants are either expressed with an alternative tag or mutated to decrease their affinity to divalent cations. The current study presents the design, engineering, and characterization of two E. coli BL21(DE3) derivatives, NiCo21(DE3) and NiCo22(DE3), which express the endogenous proteins SlyD, Can, ArnA, and (optionally) AceE fused at their C terminus to a chitin binding domain (CBD) and the protein GlmS, with six surface histidines replaced by alanines. We show that each E. coli CBD-tagged protein remains active and can be efficiently eliminated from an IMAC elution fraction using a chitin column flowthrough step, while the modification of GlmS results in loss of affinity for nickel-containing resin. The "NiCo" strains uniquely complement existing methods for improving the purity of recombinant His-tagged protein.Over the past 25 years, several techniques and tools have been developed to express and purify recombinant proteins for protein structure-function studies, for the development of new drugs, or simply for the manufacture of enzymes. The most frequently used method for isolating recombinant protein from a cell lysate in a single purification step is immobilized metal ion affinity chromatography (IMAC). In the simplest application of this method, the target protein is tagged with a polyhistidine sequence (typically 6ϫHis), which mediates chelation to immobilized divalent metal ions such as nickel or cobalt. Other studies have demonstrated that peptides with nonconsecutive histidines are also capable of chelation to immobilized divalent metal ions (5) (U.S. patent 7,176,298 [41] and U.S. patent application 2006/0030007 A1).Escherichia coli is the most commonly used host for highyield expression of recombinant protein, usually by exploiting the high promoter specificity and transcriptional activity of bacteriophage T7 RNA polymerase. However, several E. coli host proteins also contain nonconsecutive histidine residues exposed to the surface of their ternary structure. In addition, metal binding motifs often mediate binding to nickel-and/or cobalt-containing purification resins. Such host proteins are routinely copurified during IMAC procedures and are therefore referred to as "contaminants." Several metal binding proteins that behave as IMAC contaminants have been identified in recent years. For example, Bolanos-Garcia et al. reviewed this issue in detail by classifying the E. coli metal binding proteins according to their affinity for Ni...