John W. Frost

Benzene‐Free Synthesis of Adipic Acid

Strains of Escherichia coli were constructed and evaluated that synthesized cis,cis‐muconic acid from d‐glucose under fed‐batch fermentor conditions. Chemical hydrogenation of the cis, cis‐muconic acid in the resulting fermentation broth has also been examined. Biocatalytic synthesis of adipic acid from glucose eliminates two environmental concerns characteristic of industrial adipic acid manufacture: use of carcinogenic benzene and benzene‐derived chemicals as feedstocks and generation of nitrous oxide as a byproduct of a nitric acid catalyzed oxidation. While alternative catalytic syntheses that eliminate the use of nitric acid have been developed, most continue to rely on petroleum‐derived benzene as the ultimate feedstock. In this study, E. coli WN1/pWN2.248 was developed that synthesized 36.8 g/L of cis, cis‐muconic acid in 22% (mol/mol) yield from glucose after 48 h of culturing under fed‐batch fermentor conditions. Optimization of microbial cis,cis‐muconic acid synthesis required expression of three enzymes not typically found in E. coli. Two copies of the Klebsiella pneumoniae aroZ gene encoding DHS dehydratase were inserted into the E. coli chromosome, while the K. pneumoniae aroY gene encoding PCA decarboxylase and the Acinetobacter calcoaceticus catA gene encoding catechol 1,2‐dioxygenase were expressed from an extrachromosomal plasmid. After fed‐batch culturing of WN1/pWN2.248 was complete, the cells were removed from the broth, which was treated with activated charcoal and subsequently filtered to remove soluble protein. Hydrogenation of the resulting solution with 10% Pt on carbon (5% mol/mol) at 3400 kPa of H2 pressure for 2.5 h at ambient temperature afforded a 97% (mol/mol) conversion of cis, cis‐muconic acid into adipic acid.

Structure of dehydroquinate synthase reveals an active site capable of multistep catalysis

Dehydroquinate synthase (DHQS) has long been regarded as a catalytic marvel because of its ability to perform several consecutive chemical reactions in one active site. There has been considerable debate as to whether DHQS is actively involved in all these steps,, or whether several steps occur spontaneously, making DHQS a spectator in its own mechanism. DHQS performs the second step in the shikimate pathway, which is required for the synthesis of aromatic compounds in bacteria, microbial eukaryotes and plants. This enzyme is a potential target for new antifungal and antibacterial drugs, as the shikimate pathway is absent from mammals and DHQS is required for pathogen virulence. Here we report the crystal structure of DHQS, which has several unexpected features, including a previously unobserved mode for NAD+-binding and an active-site organization that is surprisingly similar to that of alcohol dehydrogenase, in a new protein fold. The structure reveals interactions between the active site and a substrate-analogue inhibitor, which indicate how DHQS can perform multistep catalysis without the formation of unwanted by-products.

Phosphoenolpyruvate Availability and the Biosynthesis of Shikimic Acid

The impact of increased availability of phosphoenolpyruvate during shikimic acid biosynthesis has been examined in Escherichia coliK‐12 constructs carrying plasmid‐localized aroFFBR and tktAinserts encoding, respectively, feedback‐insensitive 3‐deoxy‐d‐arabino‐heptulosonic acid 7‐phosphate synthase and transketolase. Strategies for increasing the availability of phosphoenolpyruvate were based on amplified expression of E. coli ppsA‐encoded phosphoenolpyruvate synthase or heterologous expression of the Zymomonas mobilis glf‐encoded glucose facilitator. The highest titers and yields of shikimic acid biosynthesized from glucose in 1 L fermentor runs were achieved using E. coli SP1.lpts/pSC6.090B, which expressed both Z. mobilis glf‐encoded glucose facilitator protein and Z. mobilis glk‐encoded glucose kinase in a host deficient in the phosphoenolpyruvate:carbohydrate phosphotransferase system. At 10 L scale with yeast extract supplementation, E. coli SP1.lpts/pSC6.090B synthesized 87 g/L of shikimic acid in 36% (mol/mol) yield with a maximum productivity of 5.2 g/L/h for shikimic acid synthesized during the exponential phase of growth.

Altered Glucose Transport and Shikimate Pathway Product Yields in E.coli

Different glucose transport systems are examined for their impact on phosphoenolpyruvate availability as reflected by the yields of 3‐dehydroshikimic acid and byproducts 3‐deoxy‐d‐arabino‐heptulosonic acid, 3‐dehydroquinic acid, and gallic acid synthesized by Escherichia coli from glucose. 3‐Dehydroshikimic acid is an advanced shikimate pathway intermediate in the syntheses of a spectrum of commodity, pseudocommodity, and fine chemicals. All constructs carried plasmid aroFFBR and tktA inserts encoding, respectively, a feedback‐insensitive isozyme of 3‐deoxy‐d‐arabino‐heptulosonic acid 7‐phosphate synthase and transketolase. Reliance on the native E. coli phosphoenolpyruvate:carbohydrate phosphotransferase system for glucose transport led in 48 h to the synthesis of 3‐dehydroshikimic acid (49 g/L) and shikimate pathway byproducts in a total yield of 33% (mol/mol). Use of heterologously expressed Zymomonas mobilis glf‐encoded glucose facilitator and glk‐encoded glucokinase resulted in the synthesis in 48 h of 3‐dehydroshikimic acid (60 g/L) and shikimate pathway byproducts in a total yield of 41% (mol/mol). Recruitment of native E. coli galP‐encoded galactose permease for glucose transport required 60 h to synthesize 3‐dehydroshikimic acid (60 g/L) and shikimate pathway byproducts in a total yield of 43% (mol/mol). Direct comparison of the impact of altered glucose transport on the yields of shikimate pathway products synthesized by E. coli has been previously hampered by different experimental designs and culturing conditions. In this study, the same product and byproduct mixture synthesized by E. coli constructs derived from the same progenitor strain is used to compare strategies for increasing phosphoenolpyruvate availability. Constructs are cultured under the same set of fermentor‐controlled conditions.

Fed-batch fermentor synthesis of 3-dehydroshikimic acid using recombinant Escherichia coli

3-Dehydroshikimic acid (DHS), in addition to being a potent antioxidant, is the key hydroaromatic intermediate in the biocatalytic conversion of glucose into aromatic bioproducts and a variety of industrial chemicals. Microbial synthesis of DHS, like other intermediates in the common pathway of aromatic amino acid biosynthesis, has previously been examined only under shake flask conditions. In this account, synthesis of DHS using recombinant Escherichia coli constructs is examined in a fed-batch fermentor where glucose availability, oxygenation levels, and solution pH are controlled. DHS yields and titers are also determined by the activity of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthase. This enzymes expression levels, sensitivity to feedback inhibition, and the availability of its substrates, phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P), dictate its in vivo activity. By combining fed-batch fermentor control with amplified expression of a feedback-insensitive isozyme of DAHP synthase and amplified expression of transketolase, DHS titers of 69 g/L were synthesized in 30% yield (mol/mol) from D-glucose. Significant concentrations of 3-dehydroquinic acid (6.8 g/L) and gallic acid (6.6 g/L) were synthesized in addition to DHS. The pronounced impact of transketolase overexpression, which increases E4P availability, on DHS titers and yields indicates that PEP availability is not a limiting factor under the fed-batch fermentor conditions employed.

Modulation of Phosphoenolpyruvate Synthase Expression Increases Shikimate Pathway Product Yields in E. coli

Product yields in microbial synthesis are ultimately limited by the mechanism utilized for glucose transport. Altered expression of phosphoenolpyruvate synthase was examined as a method for circumventing these limits. Escherichia coli KL3/pJY1.216A was cultured under fed‐batch fermentor conditions where glucose was the only source of carbon for the formation of microbial biomass and the synthesis of product 3‐dehydroshikimic acid. Shikimate pathway byproducts 3‐deoxy‐d‐ arabino‐heptulosonic acid, 3‐dehydroquinic acid, and gallic acid were also generated. An optimal expression level of phosphoenolpyruvate synthase was identified, which did not correspond to the highest expression levels of this enzyme, where the total yield of 3‐dehydroshikimic acid and shikimate pathway byproducts synthesized from glucose was 51% (mol/mol). For comparison, the theoretical maximum yield is 43% (mol/mol) for synthesis of 3‐dehydroshikimic acid and shikimate pathway byproducts from glucose in lieu of amplified expression of phosphoenolpyruvate synthase.

Microbial synthesis of 3-dehydroshikimic acid: a comparative analysis of D-xylose, L-arabinose, and D-glucose carbon sources.

3‐Dehydroshikimic acid is a hydroaromatic precursor to chemicals ranging from l‐phenylalanine to adipic acid. The concentration and yield of 3‐dehydroshikimic acid microbially synthesized from various carbon sources has been examined under fed‐batch fermentor conditions. Examined carbon sources included d‐xylose, l‐arabinose, and d‐glucose. A mixture consisting of a 3:3:2 molar ratio of glucose/xylose/arabinose was also evaluated as a carbon source to model the composition of pentose streams potentially resulting from the hydrolysis of corn fiber. Escherichia coli KL3/pKL4.79B, which overexpresses feedback‐insensitive DAHP synthase, synthesizes higher concentrations and yields of 3‐dehydroshikimic acid when either xylose, arabinose, or the glucose/xylose/arabinose mixture is used as a carbon source relative to when glucose alone is used as a carbon source. E. coli KL3/pKL4.124A, which overexpresses transketolase and feedback‐insensitive DAHP synthase, synthesizes higher concentrations and yields of 3‐dehydroshikimic acid when the glucose/xylose/arabinose mixture is used as the carbon source relative to when either xylose or glucose is used as a carbon source. Observed high‐titer, high‐yielding synthesis of 3‐dehydroshikimic acid from the glucose/xylose/arabinose mixture carries significant ramifications relevant to the employment of corn fiber in the microbial synthesis of value‐added chemicals.

Aromatic inhibitors of dehydroquinate synthase: Synthesis, evaluation and implications for gallic acid biosynthesis

The role of the active site metal in determining binding to 3-dehydroquinate synthase has been examined. Protocatechuic acid, catechol, and derivatives of these aromatics were synthesized that shared the common element of an ortho dihydroxylated benzene ring. Inhibition constants were determined for each aromatic as well as the variation of this inhibition as a function of whether Co(+2) or Zn(+2) was the active site metal ion.

Myo-inositol-1-phosphate (MIP) synthase inhibition: in-vivo study in rats.

Summary.Lithium and valproate are the prototypic mood stabilizers and have diverse structures and targets. Both drugs influence inositol metabolism. Lithium inhibits IMPase and valproate inhibits MIP synthase. This study shows that MIP synthase inhibition does not replicate or augment the effects of lithium in the inositol sensitive pilocarpine-induced seizures model. This lack of effects may stem from the low contribution of de-novo synthesis to cellular inositol supply or to the inhibition of the de-novo synthesis by lithium itself.