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Final Report: Environmentally Benign Synthesis of Resorcinol from Glucose

EPA Grant Number: R826116
Title: Environmentally Benign Synthesis of Resorcinol from Glucose
Investigators: Frost, John W.
Institution: Michigan State University
EPA Project Officer: Richards, April
Project Period: November 1, 1997 through October 31, 2000
Project Amount: $337,202
RFA: Technology for a Sustainable Environment (1997)
Research Category: Pollution Prevention/Sustainable Development



Resorcinol is a widely used chemical-building block globally produced at volumes of 3.0?3.5 x 107 kg/yr. Demand for resorcinol ranges from its formulation with formaldehyde to produce wood adhesives and tackifiers in the manufacture of steel tires to its use in the synthesis of fine chemicals ranging from UV blockers to the active ingredient of SucretsTM. New, environmentally benign routes were proposed for the synthesis of resorcinol that share the common feature of employing microbial biocatalysis and use of glucose as a starting material.

Current resorcinol manufacture (Scheme 1) employs benzene as a starting material. Benzene is carcinogenic and volatile and is obtained from nonrenewable petroleum feedstocks. All of resorcinol's carbon atoms in the proposed syntheses would be derived from the carbon atoms of glucose. In addition to being nontoxic and nonvolatile, glucose can be derived from the renewable starch and cellulose content of plants cultivated on an enormous scale in the United States. Resorcinol's oxygen atoms also would be derived from the oxygen atoms of glucose. Use of a starting material that already contains what will become the two oxygen atoms of resorcinol avoids high-temperature fusion with caustic lye, high-salt effluent streams, and explosive intermediates that are associated with chemically forcing oxygen atoms onto the aromatic ring of benzene.

Scheme 1

(a) biocatalysis; (b) chemical catalysis; (c) SO3, Na2SO4, 150?C; (d) 2-propene,
HZSM-12; (e) NaOH, 350?C; (f) O2, NaOH, 90?100?C

By taking advantage of the diversity of aromatic-biosynthesizing pathways and enzymes found in nature, the spectrum of chemicals synthesized by environmentally benign routes from renewable feedstocks can be greatly expanded. Proposed research was distinguished by the fact that polyketide biosynthesis or fatty acid biosynthesis would be the pathway chosen for converting glucose into resorcinol. Previous efforts to replace benzene with glucose as a starting material in chemical manufacture have employed the common pathway of aromatic amino acid biosynthesis (the Shikimate pathway) as the biocatalytic route for synthesis of key intermediates for the final chemical product.

Research objectives for the project included the following: (1) construct a triacetic acid lactone (TAL)-synthesizing biocatalyst by genetic alteration of fatty acid biosynthesis in Escherichia (E.) coli; (2) construct TAL- and 3-hydroxy-5-ketohexanoic acid (HKH)-synthesizing biocatalysts by alteration of the gene encoding 6-methylsalicylic acid synthase expressed in Streptomyces coelicolor; and (3) elaborate the reaction steps, catalysts, and associated reaction conditions necessary for abiotic, chemical conversion of TAL and HKH into resorcinol.

Summary/Accomplishments (Outputs/Outcomes):

To focus our efforts on developing a biocatalyst either for TAL biosynthesis or HKH biosynthesis, we chose to turn our attention initially to elaborating the chemical steps for converting TAL and HKH into resorcinol. Our original plan to convert TAL into resorcinol relied on phloroglucinol as a synthetic intermediate. Phloroglucinol (4) is an important chemical-building block that is no longer produced in the United States due to the danger of the manufacturing process. Trinitrotoluene (TNT), which is ultimately prepared from benzene, is an explosive intermediate used in phloroglucinol synthesis. Conversion of TAL into phloroglucinol also would broaden the utility of a TAL-synthesizing biocatalyst. Precedent in the literature indicated that NaBH4 could be used stoichiometrically for reduction of phloroglucinol to resorcinol (Scheme 2), and we were interested in exploring catalytic reactions to carry out this same transformation.

An intramolecular Claisen condensation of TAL was expected to afford phloroglucinol in a single step. Reaction of TAL with either Mg(OCH3)2 or KOH in methanol failed to afford phloroglucinol. In a second approach (Scheme 2), the hydroxyl group of TAL was first protected as a methyl ether, which was expected to be stable to subsequent nucleophilic attack. Several sets of conditions were explored to access the methyl ether of TAL (2). Refluxing TAL in methanol in the presence of Dowex-50 afforded 2 in yields of approximately 43 percent. Alternatively, reaction of TAL with dimethyl sulfate in refluxing acetone in the presence of Na2CO3 provided 2 in 85 percent yield after recrystallization. To avoid the toxicity of dimethyl sulfate, TAL also was reacted in neat trimethyl phosphate in the presence of K2CO3, which afforded 2 in 79 percent yield.

Scheme 2

(a) MeOH, Dowex-50, , 43%; (b) (CH3O)2SO2, Na2CO3, acetone, , 85%; (c) (CH3O)3PO, K2CO3, 140?C, 79%; (d) Na, MeOH, 185?C, 87%; (e) 12 N HCl, rt., 56%; (f) NaBH4; (g) (i) H2, 5% Rh/Al2O3, NaOH, H2O; (ii) 0.5 M H2SO4, , 82%

Conversion of 2 to the monomethyl ether of phloroglucinol 3 required proton abstraction followed by ring opening and subsequent ring closure. Addition of 2 to a solution of Na in methanol was followed by distillation of the excess methanol. The resulting residue was heated to 185?C. The residue was then dissolved in water, and the solution was adjusted to pH 2 and subsequently extracted with ethyl acetate. Kugel-Rohr distillation of the resulting product afforded 3 in 87 percent yield.

Deprotection of the monomethyl ether 3 to afford phloroglucinol 4 was more difficult than originally expected. Reaction of 3 in 12 N HCl at room temperature afforded 4 in 56 percent yield. Altering the concentration of HCl or the reaction temperature resulted in either incomplete reaction or dimerization of the phloroglucinol. Lewis acid-mediated dealkylation failed to improve the yield of 4.

A variety of catalysts were examined for reduction of phloroglucinol 4 to resorcinol 5, including palladium, platinum, and Raney nickel, and each was unsuccessful. Under basic conditions, phloroglucinol was reduced in the presence of 5 percent rhodium on alumina and 50 psi of hydrogen pressure to produce the sodium salt of dihydrophloroglucinol, which was subsequently dehydrated in 0.5 M sulfuric acid to afford resorcinol in 80 percent overall yield. Further exploration of the reaction conditions revealed that under the same reaction conditions, 3 could be converted into resorcinol in 82 percent yield, eliminating the need for phloroglucinol intermediacy.

Having established conditions for high-yielding conversion of TAL into resorcinol, attention was next focused on creation of an organism capable of biosynthesizing TAL from glucose. One approach for TAL biosynthesis was to utilize 6-methylsalicylic acid synthase (MSAS), an enzyme found in a number of organisms, including Penicillium (P.) patulum. MSAS is a multifunctional enzyme that catalyzes at least 11 separate reactions (Scheme 3) that result in conversion of acetyl-CoA and two molecules of malonyl-CoA into 6-methylsalicylic acid. These reactions are carried out by catalytic active sites possessing ketosynthase, ketoreductase, and dehydrase functions. It already had been established that TAL is a byproduct formed by MSAS, even in the presence of NADPH concentrations that are optimal for formation of 6-methylsalicylic acid. In the absence of NADPH, TAL is the exclusive product synthesized by MSAS. Altering MSAS by inactivation of the ketoreductase site was expected to result in formation of TAL.

The P. patulum gene encoding MSAS already had been cloned and expressed in Saccharomyces (S.) cerevisiae. Functional expression of the protein was verified by the formation of 6-methylsalicylic acid. A ketoreductase-deficient MSAS also had been reported by Khosla. Site-directed mutagenesis of the gene led to three amino acid substitutions in the region of the protein involved in NADPH binding. The gene encoding the mutated protein was generously provided by Khosla.

(a) ketosynthase; (b) ketoreductase; (c) dehydrase

Plasmids pMR228 encoding the mutated MSAS gene and pKOS12-128a encoding an essential phosphopantetheinyl transferase gene were cotransformed into S. cerevisiae InvSc1. InvSc1/pMR228/pKOS12-128a was grown under a variety of culture conditions ranging from small shake flask reactions to bench-top fermentations. Several variations were made in the fermentation medium, although yeast extract was added in all cases due to slow growth of the organism. Under all conditions examined, TAL was never detected in the culture medium. Possible explanations for the inability of the organism to synthesize TAL include poor expression of MSAS and lack of functionality of the protein. Although it also is possible that the ketoreductase activity of MSAS was still functional, the absence of 6-methylsalicylic acid formation tempers that argument. Exploitation of MSAS as a viable means of TAL biosynthesis will require additional experimentation, including possible expression of the enzyme in E. coli.

Effort next focused on exploiting enzymes normally involved in fatty acid synthesis for TAL biosynthesis (Scheme 4). TAL already has been reported to be produced by purified fatty acid synthases isolated from baker's yeast, pigeon liver, and E. coli. Although TAL accumulation during fatty acid biosynthesis is not significant under physiological conditions, these reports establish the direction to take in constructing a TAL-synthesizing microbe. An inadequate in vitro supply of reducing equivalents in the form of NADPH was the common feature for each report of TAL synthesized by purified fatty acid synthase. Our goal then was to reduce the availability of NADPH to fatty acid biosynthesis in E. coli. This would be accomplished by reducing or eliminating the ability of the ketoreductase to bind NADPH.

(a) ketosynthase; (b) ketoreducatase; (c) thiolase; (d) dehydrase; (e) enoylreductase

In one variation on this theme, E. coli strain YZ166 was obtained from Cronan in which the native fabG gene encoding the ketoreductase was not transcribed. Because mutation of fabG appears to be lethal to the organism, the S. typhimurium fabG gene was included in YZ166 on a plasmid and was transcribed from the regulatable araBAD promoter. Addition of L-arabinose to the medium activates expression from the araBAD promoter, while addition of glucose prohibits expression from the promoter. Our plan was to grow YZ166 to stationary phase in the presence of L-arabinose and to then inactivate expression of fabG by addition of glucose. Under all conditions examined, no TAL was detected in the culture medium. One possible explanation for the failure to accumulate TAL is a long cellular half-life for the ketoreductase. Even if glucose prevents continued expression of fabG, ketoreductase protein already in the cell may be sufficient to negate TAL formation.

A second strategy relied on the native E. coli fatty acid machinery to biosynthesize fatty acids necessary for growth, while a second fatty acid synthase from Brevibacterium (B.) ammoniagenes would be exploited for TAL biosynthesis. Unlike the E. coli type II fatty acid synthases in which each of the catalytic enzymes is a separate protein, the type I fatty acid synthase from B. ammoniagenes is a single, multifunctional protein that catalyzes all of the needed steps for fatty acid synthesis. Importantly, functional expression of the B. ammoniagenes fasB gene in E. coli requires coexpression of the B. ammoniagenes PPT1 gene, which encodes a protein necessary for conversion of the nonfunctional apo-protein into the functional holo-protein.

It was first necessary to confirm that the B. ammoniagenes FAS-B protein could catalyze TAL formation. Plasmid pGM44 encoding fasB and PPT1 was obtained from Schweizer. E. coli DH5a/pGM44 was grown to stationary phase, and the cells were lysed. Following precipitation with ammonium sulfate and ultracentrifugation, the partially purified cellular extract was incubated with [14C]-acetyl-CoA and malonyl-CoA. The reaction was extracted with ethyl acetate, and products were separated by thin layer chromatography. Reactions containing FAS-B lysate indicated that TAL was formed both in the presence and absence of added NADPH. Reactions that contained lysate of similarly treated DH5 failed to result in formation of TAL.

To utilize FAS-B for TAL formation, the binding site for the NADPH cofactor used by the ketoreductase was mutated. Site-specific mutagenesis was used to generate three variant genes of fasB that would lead to proteins encoding one, three, and four amino acid changes in the putative NADPH binding site of the protein. Strains expressing the fasB genes encoding three or four amino acid changes grew extremely poorly relative to strains expressing wild-type FAS-B or the single mutant protein. SDS-PAGE analysis of cellular extracts revealed that cells expressing FAS-B with multiple mutations failed to synthesize a protein of the appropriate molecular weight, while cells expressing the single mutation did produce a protein of the expected size of FAS-B. Cells expressing the single mutation were grown under a variety of conditions, including shake flasks and bench-top fermentations. In no case was TAL detected in the culture medium.

Further analysis indicated that expression of FAS-B was low compared to activity that had been reported by Schweitzer. To improve expression of the fasB locus, the gene was localized behind the strong T7 promoter. Bench-top fermentations using this construct were carried out. Enzyme assays of cells collected from the fermentation indicated that FAS-B activity was not measurable, calling into question the expression of the protein.

In summary, it remains unclear as to why TAL is not accumulating in the culture supernatant of any of the strains that have been developed. Because chemical experiments with TAL indicate that the molecule is stable to fermentation conditions, it is unlikely that TAL is being synthesized and then degrading prior to detection. Further work will have to focus on improving expression of the enzymes that have been targeted for TAL biosynthesis.

Supplemental Keywords:

green chemistry, metabolic engineering, biocatalysis, resorcinol, phloroglucinol, glucose, benzene, renewable, triacetic acid lactone., RFA, Scientific Discipline, Toxics, Sustainable Industry/Business, cleaner production/pollution prevention, Environmental Chemistry, Sustainable Environment, HAPS, Technology for Sustainable Environment, Economics and Business, triacetic acid lactone, cleaner production, waste minimization, waste reduction, environmentally conscious manufacturing, microbial biocatalysis, green process systems, UV blockers, Benzene (including benzene from gasoline), pollution prevention, biosynthesis, alternative chemical synthesis, environmentally-friendly chemical synthesis, green chemistry

Relevant Websites:

http://www.cem.msu.edu/~orgweb/brochure/frost.htmlExit EPA icon

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