Methanol is an attractive substrate for biological production of chemicals and fuels. Engineering methylotrophic Escherichia coli as a platform organism for converting methanol to metabolites is desirable. Prior efforts to engineer methylotrophic E. coli were limited by methanol dehydrogenases (Mdhs) with unfavorable enzyme kinetics. We engineered E. coli to utilize methanol using a superior NAD-dependent Mdh from Bacillus stearothermophilus and ribulose monophosphate (RuMP) pathway enzymes from B. methanolicus. Using C-labeling, we demonstrate this E. coli strain converts methanol into biomass components. For example, the key TCA cycle intermediates, succinate and malate, exhibit labeling up to 39%, while the lower glycolytic intermediate, 3-phosphoglycerate, up to 53%. Multiple carbons are labeled for each compound, demonstrating a cycling RuMP pathway for methanol assimilation to support growth. By incorporating the pathway to synthesize the flavanone naringenin, we demonstrate the first example of in vivo conversion of methanol into a specialty chemical in E. coli.

Synthetic methylotrophy is the development of non-native methylotrophs that can utilize methane and methanol as sole carbon and energy sources or as co-substrates with carbohydrates to produce metabolites as biofuels and chemicals. The availability of methane (from natural gas) and its oxidation product, methanol, has been increasing, while prices have been decreasing, thus rendering them as attractive fermentation substrates. As they are more reduced than most carbohydrates, methane and methanol, as co-substrates, can enhance the yields of biologically produced metabolites. Here we discuss synthetic biology and metabolic engineering strategies based on the native biology of aerobic methylotrophs for developing synthetic strains grown on methanol, with Escherichia coli as the prototype.

ABSTRACTVibrio parahaemolyticus, a marine bacterium, is the causative agent of gastroenteritis associated with the consumption of seafood. It contains a homologue of thetoxRSoperon that inV. choleraeis the key regulator of virulence gene expression. We examined a nonpolar mutation intoxRSto determine the role of these genes inV. parahaemolyticusRIMD2210633, an O3:K6 isolate, and showed that compared to the wild type, ΔtoxRSwas significantly more sensitive to acid, bile salts, and sodium dodecyl sulfate stresses. We demonstrated that ToxRS is a positive regulator ofompUexpression, and that the complementation of ΔtoxRSwithompUrestores stress tolerance. Furthermore, we showed that ToxRS also regulates type III secretion system genes in chromosome I via the regulation of theleuOhomologue VP0350. We examined the effect of ΔtoxRS in vivousing a new orogastric adult murine model of colonization. We demonstrated that streptomycin-treated adult C57BL/6 mice experienced prolonged intestinal colonization along the entire intestinal tract by the streptomycin-resistantV. parahaemolyticus. In contrast, no colonization occurred in non-streptomycin-treated mice. A competition assay between the ΔtoxRSand wild-typeV. parahaemolyticusstrains marked with the β-galactosidase genelacZdemonstrated that the ΔtoxRSstrain was defective in colonization compared to the wild-type strain. This defect was rescued by ectopically expressingompU. Thus, the defect in stress tolerance and colonization in ΔtoxRSis solely due to OmpU. To our knowledge, the orogastric adult murine model reported here is the first showing sustained intestinal colonization byV. parahaemolyticus.

Vibrio parahaemolyticus inhabits marine, brackish, and estuarine waters worldwide, where fluctuations in salinity pose a constant challenge to the osmotic stress response of the organism. Vibrio parahaemolyticus is a moderate halophile, having an absolute requirement for salt for survival, and is capable of growth at 1 to 9% NaCl. It is the leading cause of seafood-related bacterial gastroenteritis in the United States and much of Asia. We determined whether growth in differing NaCl concentrations alters the susceptibility of V. parahaemolyticus O3:K6 to other environmental stresses. Vibrio parahaemolyticus was grown at a 1% or 3% NaCl concentration, and the growth and survival of the organism were examined under acid or temperature stress conditions. Growth of V. parahaemolyticus in 3% NaCl versus that in 1% NaCl increased survival under both inorganic (HCl) and organic (acetic acid) acid conditions. In addition, at 42°C and ؊20°C, 1% NaCl had a detrimental effect on growth. The expression of lysine decarboxylase (encoded by cadA), the organism's main acid stress response system, was induced by both NaCl and acid conditions. To begin to address the mechanism of regulation of the stress response, we constructed a knockout mutation in rpoS, which encodes the alternative stress sigma factor, and in toxRS, a two-component regulator common to many Vibrio species. Both mutant strains had significantly reduced survival under acid stress conditions. The effect of V. parahaemolyticus growth in 1% or 3% NaCl was examined using a cytotoxicity assay, and we found that V. parahaemolyticus grown in 1% NaCl was significantly more toxic than that grown in 3% NaCl.Vibrio parahaemolyticus is a Gram-negative bacterium that inhabits coastal waters worldwide. Vibrio parahaemolyticus grows optimally in warmer waters and is most commonly isolated during the summer months, often in association with plankton, crustaceans, mollusks, and fish (16,17). During the winter months, the organism is typically scarce and usually is isolated from sediment samples (16). While V. parahaemolyticus has been shown to be the etiological agent of disease in several kinds of crustaceans and shellfish, it is most notably a pathogen of humans (17). Vibrio parahaemolyticus was first discovered in Japan during an outbreak of gastroenteritis in 1950 (12). It is the leading cause of seafood-related bacterial gastroenteritis in the United States and much of Asia (6,39). Infection is most frequently associated with the consumption of oysters harvested from warm waters, particularly along the U.S. Gulf Coast, where vibrios grow to high levels during the summer months (6, 7, 42). Newly released data from the CDC comparing the incidence rates of laboratory-confirmed infections by gastrointestinal pathogens in 1996 to 2008 revealed an increase of 47% for Vibrio infections, of which V. parahaemolyticus accounted for 55%, while rates for all other enteric pathogens decreased or remained the same (5). An outbreak of V. parahaemolyticus infections which caused rapid hos...

Methanol is an important feedstock derived from natural gas and can be chemically converted into commodity and specialty chemicals at high pressure and temperature. Although biological conversion of methanol can proceed at ambient conditions, there is a dearth of engineered microorganisms that use methanol to produce metabolites. In nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction. Thus, efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism. An emerging strategy to promote efficient substrate channeling is to spatially organize pathway enzymes in an engineered assembly to provide kinetic driving forces that promote carbon flux in a desirable direction. Here, we report a scaffoldless, self-assembly strategy to organize Mdh, Hps, and Phi into an engineered supramolecular enzyme complex using an SH3-ligand interaction pair, which enhances methanol conversion to fructose-6-phosphate (F6P). To increase methanol consumption, an "NADH Sink" was created using Escherichia coli lactate dehydrogenase as an NADH scavenger, thereby preventing reversible formaldehyde reduction. Combination of the two strategies improved in vitro F6P production by 97-fold compared with unassembled enzymes. The beneficial effect of supramolecular enzyme assembly was also realized in vivo as the engineered enzyme assembly improved whole-cell methanol consumption rate by ninefold. This approach will ultimately allow direct coupling of enhanced F6P synthesis with other metabolic engineering strategies for the production of many desired metabolites from methanol. methane | methylotophs | supramolcular | scaffold | substrate channeling

bVibrio parahaemolyticus is the leading cause of bacterial seafood-borne gastroenteritis worldwide, yet little is known about how this pathogen colonizes the human intestine. The alternative sigma factor RpoN/sigma-54 is a global regulator that controls flagellar synthesis, as well as a wide range of nonflagellar genes. We constructed an in-frame deletion mutation in rpoN (VP2670) in V. parahaemolyticus RIMD2210633, a clinical serogroup O3:K6 isolate, and examined the effects in vivo using a streptomycintreated mouse model of colonization. We confirmed that deletion of rpoN rendered V. parahaemolyticus nonmotile, and it caused reduced biofilm formation and an apparent defect in glutamine synthetase production. In in vivo competition assays between the rpoN mutant and a wild-type RIMD2210633 strain marked with the ␤-galactosidase gene lacZ (WBWlacZ), the mutant colonized significantly more proficiently. Intestinal persistence competition assays also demonstrated that the rpoN mutant had enhanced fitness and outcompeted WBWlacZ. Mutants defective in the polar flagellum biosynthesis FliAP sigma factor also outcompeted WBWlacZ but not to the same level as the rpoN mutant, which suggested that lack of motility is not the sole cause of the fitness effect. In an in vitro growth competition assay in mouse intestinal mucus, the rpoN mutant also outcompeted the wild type and exhibited faster doubling times when grown in mucus and on individual components of mucus. Genes in the pathways for the catabolism of mucus sugars also had significantly higher expression levels in a ⌬rpoN mutant than in the wild type. These data suggest that in V. parahaemolyticus, RpoN plays an important role in carbon utilization regulation, which may significantly affect host colonization. Vibrio parahaemolyticus is a Gram-negative bacterium ubiquitous in the marine and estuarine environments worldwide (1-4). Vibrio parahaemolyticus is also the leading cause of seafoodassociated bacterial gastroenteritis in the United States and Asia (5, 6), which usually stems from the consumption of raw or undercooked shellfish (7,8). Typically, infection by this organism leads to nausea, vomiting, fever, and a diarrhea distinct from that of the related Vibrio cholerae. Less commonly, infection by V. parahaemolyticus can cause wound infection and septicemia, leading to mortality in immunocompromised individuals (9-12).Much effort has gone into understanding the mechanisms that contribute to V. parahaemolyticus pathogenesis, with a particular focus on virulence factors produced by this bacterium. Strains that caused disease often possessed either the thermostable direct hemolysin (TDH) or the TDH-related hemolysin (TRH), while nonpathogenic strains typically lacked these two markers (13-15). Additionally, sequence analysis of RIMD2210633, an O3:K6 isolate (TDH ϩ TRH Ϫ ), revealed the presence of two type 3 secretion systems (T3SS), one on each chromosome (T3SS-1 and T3SS-2) (16, 17). T3SS-1 is common to both clinical and nonclinical strains of V. parahaemolyticu...

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Sialic acids (neuraminic acids) are a diverse family of 9 carbon (nonulosonic) α-keto acidic carbohydrates. The canonical sialic acid, 2-keto-3-deoxy-5-acetamido-D-glycero-D-galactononulosonic acid, also known as N-acetylneuraminic acid (Neu5Ac) is the backbone on which a large number of known modifications are made (6). The Neu5Ac structure is typified by a 6 carbon carboxylic acid ring structure with a glycerol tail, an acetamido at the C-5 position and hydroxyl groups present on C-4, C-7, C-8, and C-9. Modifications occur primarily on the hydroxyl groups, with O-acetylation being the most common alteration, and substitutions have been shown to occur after the completion of the core structure (18). Other modifications such as O-methylation, O-lactylation, and O-sulfation add to the diversity of this molecule in vivo. Two structurally similar sialic acids, N-glycolylneuraminic acid (Neu5Gc), which differs from Neu5Ac by the presence of a hydroxyl group on the N-5 acetyl moiety, and 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (KDN), a deaminated form of Neu5Ac also occur in nature and similar modifications are made to their core structure (6). These three main structures (Neu5Ac, Neu5Gc, and KDN) encompass the family of sialic acids due to their retention of the same stereochemical configuration of the 9-carbon backbone.