Biochemical characterization of acyl activating enzymes for side chain moieties of Taxol and its analogs.

Taxol (paclitaxel) is a very widely used anticancer drug, but its commercial sources mainly consist of stripped bark or suspension cultures of members of the plant genus Taxus. Taxol accumulates as part of a complex mixture of chemical analogs, termed taxoids, which complicates its production in pure form, highlighting the need for metabolic engineering approaches for high-level Taxol production in cell cultures or microbial hosts. Here, we report on the characterization of acyl-activating enzymes (AAEs) that catalyze the formation of CoA esters of different organic acids relevant for the N-substitution of the 3-phenylisoserine side chain of taxoids. On the basis of similarities to AAE genes of known function from other organisms, we identified candidate genes in publicly available transcriptome data sets obtained with Taxus × media. We cloned 17 AAE genes, expressed them heterologously in Escherichia coli, purified the corresponding recombinant enzymes, and performed in vitro assays with 27 organic acids as potential substrates. We identified TmAAE1 and TmAAE5 as the most efficient enzymes for the activation of butyric acid (Taxol D side chain), TmAAE13 as the best candidate for generating a CoA ester of tiglic acid (Taxol B side chain), TmAAE3 and TmAAE13 as suitable for the activation of 4-methylbutyric acid (N-debenzoyl-N-(2-methylbutyryl)taxol side chain), TmAAE15 as a highly efficient candidate for hexanoic acid activation (Taxol C side chain), and TmAAE4 as suitable candidate for esterification of benzoic acid with CoA (Taxol side chain). This study lays important groundwork for metabolic engineering efforts aimed at improving Taxol production in cell cultures.

Coenzyme M biosynthesis in bacteria involves phosphate elimination by a functionally distinct member of the aspartase/fumarase superfamily.

For nearly 30 years, coenzyme M (CoM) was assumed to be present solely in methanogenic archaea. In the late 1990s, CoM was reported to play a role in bacterial propene metabolism, but no biosynthetic pathway for CoM has yet been identified in bacteria. Here, using bioinformatics and proteomic approaches in the metabolically versatile bacterium Xanthobacter autotrophicus Py2, we identified four putative CoM biosynthetic enzymes encoded by the xcbB1, C1, D1, and E1 genes. Only XcbB1 was homologous to a known CoM biosynthetic enzyme (ComA), indicating that CoM biosynthesis in bacteria involves enzymes different from those in archaea. We verified that the ComA homolog produces phosphosulfolactate from phosphoenolpyruvate (PEP), demonstrating that bacterial CoM biosynthesis is initiated similarly as the phosphoenolpyruvate-dependent methanogenic archaeal pathway. The bioinformatics analysis revealed that XcbC1 and D1 are members of the aspartase/fumarase superfamily (AFS) and that XcbE1 is a pyridoxal 5'-phosphate-containing enzyme with homology to d-cysteine desulfhydrases. Known AFS members catalyze ß-elimination reactions of succinyl-containing substrates, yielding fumarate as the common unsaturated elimination product. Unexpectedly, we found that XcbC1 catalyzes ß-elimination on phosphosulfolactate, yielding inorganic phosphate and a novel metabolite, sulfoacrylic acid. Phosphate-releasing ß-elimination reactions are unprecedented among the AFS, indicating that XcbC1 is an unusual phosphatase. Direct demonstration of phosphosulfolactate synthase activity for XcbB1 and phosphate ß-elimination activity for XcbC1 strengthened their hypothetical assignment to a CoM biosynthetic pathway and suggested functions also for XcbD1 and E1. Our results represent a critical first step toward elucidating the CoM pathway in bacteria.

Sample preparation for single cell transcriptomics: essential oil glands in Citrus fruit peel as an example.

Many plant natural products are synthesized in specialized cells and tissues. To learn more about metabolism in these cells, they have to be studied in isolation. Here, we describe a protocol for the isolation of epithelial cells that surround secretory cavities in Citrus fruit peel. Cells isolated using laser microdissection are suitable for RNA isolation and downstream transcriptome analyses.

Kinetic modeling of plant metabolism and its predictive power: peppermint essential oil biosynthesis as an example.

The integration of mathematical modeling with analytical experimentation in an iterative fashion is a powerful approach to advance our understanding of the architecture and regulation of metabolic networks. Ultimately, such knowledge is highly valuable to support efforts aimed at modulating flux through target pathways by molecular breeding and/or metabolic engineering. In this article we describe a kinetic mathematical model of peppermint essential oil biosynthesis, a pathway that has been studied extensively for more than two decades. Modeling assumptions and approximations are described in detail. We provide step-by-step instructions on how to run simulations of dynamic changes in pathway metabolites concentrations.

Improving peppermint essential oil yield and composition by metabolic engineering.

Peppermint (Mentha × piperita L.) was transformed with various gene constructs to evaluate the utility of metabolic engineering for improving essential oil yield and composition. Oil yield increases were achieved by overexpressing genes involved in the supply of precursors through the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. Two-gene combinations to enhance both oil yield and composition in a single transgenic line were assessed as well. The most promising results were obtained by transforming plants expressing an antisense version of (+)-menthofuran synthase, which is critical for adjusting the levels of specific undesirable oil constituents, with a construct for the overexpression of the MEP pathway gene 1-deoxy-D-xylulose 5-phosphate reductoisomerase (up to 61% oil yield increase over wild-type controls with low levels of the undesirable side-product (+)-menthofuran and its intermediate (+)-pulegone). Elite transgenic lines were advanced to multiyear field trials, which demonstrated consistent oil yield increases of up to 78% over wild-type controls and desirable effects on oil composition under commercial growth conditions. The transgenic expression of a gene encoding (+)-limonene synthase was used to accumulate elevated levels of (+)-limonene, which allows oil derived from transgenic plants to be recognized during the processing of commercial formulations containing peppermint oil. Our study illustrates the utility of metabolic engineering for the sustainable agricultural production of high quality essential oils at a competitive cost.