Engineering cofactor and transport mechanisms in Saccharomyces cerevisiae for enhanced acetyl-CoA and polyketide biosynthesis.

Synthesis of polyketides at high titer and yield is important for producing pharmaceuticals and biorenewable chemical precursors. In this work, we engineered cofactor and transport pathways in Saccharomyces cerevisiae to increase acetyl-CoA, an important polyketide building block. The highly regulated yeast pyruvate dehydrogenase bypass pathway was supplemented by overexpressing a modified Escherichia coli pyruvate dehydrogenase complex (PDHm) that accepts NADP(+) for acetyl-CoA production. After 24h of cultivation, a 3.7-fold increase in NADPH/NADP(+) ratio was observed relative to the base strain, and a 2.2-fold increase relative to introduction of the native E. coli PDH. Both E. coli pathways increased acetyl-CoA levels approximately 2-fold relative to the yeast base strain. Combining PDHm with a ZWF1 deletion to block the major yeast NADPH biosynthesis pathway resulted in a 12-fold NADPH boost and a 2.2-fold increase in acetyl-CoA. At 48h, only this coupled approach showed increased acetyl-CoA levels, 3.0-fold higher than that of the base strain. The impact on polyketide synthesis was evaluated in a S. cerevisiae strain expressing the Gerbera hybrida 2-pyrone synthase (2-PS) for the production of the polyketide triacetic acid lactone (TAL). Titers of TAL relative to the base strain improved only 30% with the native E. coli PDH, but 3.0-fold with PDHm and 4.4-fold with PDHm in the Δzwf1 strain. Carbon was further routed toward TAL production by reducing mitochondrial transport of pyruvate and acetyl-CoA; deletions in genes POR2, MPC2, PDA1, or YAT2 each increased titer 2-3-fold over the base strain (up to 0.8g/L), and in combination to 1.4g/L. Combining the two approaches (NADPH-generating acetyl-CoA pathway plus reduced metabolite flux into the mitochondria) resulted in a final TAL titer of 1.6g/L, a 6.4-fold increase over the non-engineered yeast strain, and 35% of theoretical yield (0.16g/g glucose), the highest reported to date. These biological driving forces present new avenues for improving high-yield production of acetyl-CoA derived compounds.

Procedures for the isolation and quantification of the intermediates of the mevalonic acid pathway.

Procedures for the isolation and analysis of all 11 intermediates of the mevalonic acid pathway from acetyl-CoA through geranylgeranyl pyrophosphate were developed. Both acid-labile and base-labile metabolites are simultaneously extracted with good recoveries into 7 M urea at neutral pH and low temperature to minimize hydrolytic and degradative enzyme losses, and the extract is partially purified by adsorption and desorption in high yield from an anion-exchange membrane. With the use of internal standards, the pathway intermediates are subsequently separated and quantified by reversed-phase ion-pair HPLC with on-line radiodetection. Permeable secretory cells specialized for monoterpene biosynthesis were isolated from peppermint (Mentha x piperita) leaves and employed as a model system to test the analytical protocols by examining the incorporation of [14C]pyruvate, [14C]mevalonate, and [3H]isopentenyl pyrophosphate as precursors. This simple new method should be readily adaptable to a wide range of cell and tissue types that can be administered basic metabolic precursors, and should allow measurements of both flux and steady-state levels of the intermediates of the mevalonic acid pathway.

Controlled potential enzymology of methyl transfer reactions involved in acetyl-CoA synthesis by CO dehydrogenase and the corrinoid/iron-sulfur protein from Clostridium thermoaceticum.

Many anaerobic bacteria fix CO2 via the Wood pathway of acetyl-CoA synthesis. Carbon monoxide dehydrogenase (CODH), also called acetyl-CoA synthase, accepts the methyl group from the methylated corrinoid/iron-sulfur protein (C/Fe-SP), binds a carbonyl group from CO, CO2, or the carboxyl of pyruvate, and binds coenzyme A. Then CODH catalyzes the synthesis of acetyl-CoA from these enzyme-bound groups. Here, we have characterized the methyl transfer steps involved in acetyl-CoA synthesis. We have studied the reactions leading to methylation of CODH by methyl iodide and shown an absolute requirement of the C/Fe-SP in this reaction. In addition, we have discovered and partly characterized two previously unknown exchange reactions catalyzed by CODH: between the methylated C/Fe-SP and methylated CODH and between methylated CODH and the methyl moiety of acetyl-CoA. We have performed these two exchange reactions, methylation of the C/Fe-SP, and methylation of CODH at controlled potentials. The rates of all these reactions except the exchange between methylated C/Fe-SP and methylated CODH are accelerated (from 1 to 2 orders of magnitude) when run at low potentials. Our results provide strong evidence for a nucleophilic redox-active metal center on CODH as the initial acceptor of the methyl group from the methylated C/Fe-SP. This metal center also is proposed to be involved in the cleavage of acetyl-CoA in the reverse reaction.

Synthesis of radiolabeled acetyl-coenzyme A from sodium acetate.

The synthesis of high specific radioactivity [14C]-acetyl-Coenzyme A from [14C]sodium acetate, 2,6-dichlorobenzoic acid, 1,1'-carbonyldiimidazole, and CoA is reported. Starting with 1 mumol of [14C]sodium acetate, this method yields pure [14C]acetyl-CoA in yields approaching 40%. Chromatography on a reversed-phase ODS column was used to separate acetyl-CoA from Coenzyme A and side products. The acetylating agent is apparently a reaction intermediate, acetylimidazole.

Radioisotopic assay of picomolar amounts of coenzyme A.

A two-step method of determining reduced coenzyme A (CoASH) concentrations in tissue or cell extracts is described. In the first step, CoASH is reacted with acetylphosphate in a reaction catalyzed by phosphotransacetylase to yield acetyl-CoA. Acetyl-CoA is then condensed with [14C]oxaloacetate by citrate synthase to give [14C]citrate. This method allows the measurement of 10-200 pmol of CoASH. By omitting the phosphotransacetylase step, measurement of the same amount of acetyl-CoA is possible.

Preparation of bromo[1-14C]acetyl-coenzyme A as an affinity label for acetyl-coenzyme A binding sites.

Bromo[1-14C]acetyl-CoA has been prepared from CoASH and the N-hydroxysuccinimide ester of bromo[1-14C]acetic acid, and unlabeled bromoacetyl-CoA by reaction of CoASH with bromoacetyl bromide. The products were purified by high-pressure liquid chromatography. Purified bromoacetyl-CoA was characterized, and found to be a potent alkylating agent with a substantial stability in aqueous solution: it decomposed at 30 degrees C and pH 6.6 and 8.0 with halftimes of 3.3 and 2.5 h, respectively. The major breakdown products were CoASH and CoAS X CO X CH2 X SCoA. Bromo[1-14C]acetyl-CoA has been used to affinity label the acetyl-CoA binding site of 3-hydroxy-3-methylglutaryl-CoA synthase from ox liver. It was found to irreversibly inhibit the enzyme activity and bind covalently with a stoichiometry for complete inhibition of about 0.8 mol/mol enzyme dimer.