Preparation, Characterization, and Optimization of an In Vitro C₃₀Carotenoid Pathway

Abstract

The ispA gene encoding farnesyl pyrophosphate (FPP) synthase from Escherichia coli and the crtM gene encoding 4,4-diapophytoene (DAP) synthase from Staphylococcus aureus were overexpressed and purified for use in vitro. Steady-state kinetics for FPP synthase and DAP synthase, individually and in sequence, were determined under optimized reaction conditions. For the two-step reaction, the DAP product was unstable in aqueous buffer; however, in situ extraction using an aqueous-organic two-phase system resulted in a 100% conversion of isopentenyl pyrophosphate and dimethylallyl pyrophosphate into DAP. This aqueous-organic two-phase system is the first demonstration of an in vitro carotenoid synthesis pathway performed with in situ extraction, which enables quantitative conversions. This approach, if extended to a wide range of isoprenoidbased pathways, could lead to the synthesis of novel carotenoids and their derivatives.Carotenoids are naturally occurring pigments found in a wide variety of plants and microorganisms (22,23). Recent studies indicate that these isoprenoid-based natural products possess biologically active properties (21, 24), thereby making them of interest to the medicinal chemist. Most carotenoids belong to the C 30 and C 40 classes, being distinguished by the number of iterative steps of isopentenyl pyrophosphate (IPP) condensation to add C 5 isoprene units. The initial synthetic step is the condensation of IPP with ␥,␥-dimethylallyl pyrophosphate (DMAPP) catalyzed by farnesyl pyrophosphate (FPP) synthase (ispA) to give geranyl pyrophosphate (GPP) and, sequentially, FPP (9). This C 15 compound serves as a central node for the synthesis of sterols (29), farnesylated proteins (4), hemes (20), sesquiterpenes (6), and dolichols (5), as well as the crucial precursor for carotenoids.C 30 carotenoids are present in the nonphotosynthetic bacteria, such as Streptococcus faecium, Staphylococcus aureus, and Methylobacterium rhodinum, and in the photosynthetic Heliobacterium species (16,25,27,28). Only the genes encoding 4,4Ј-diapophytoene (DAP) synthase (crtM) and 4,4Ј-diapophytoene desaturase (crtN) from S. aureus have been cloned and functionally expressed in Escherichia coli, resulting in the yellow 4,4Ј-diaponeurosporene from FPP (30) (Fig. 1). The first committed step in C 30 carotenoid biosynthesis is the condensation of two molecules of FPP catalyzed by DAP synthase to form the colorless carotenoid 4,4Ј-diapophytoene.Until now, only a few reports have discussed the formation of DAP through a reconstituted C 30 carotenoid metabolic pathway in vitro (13, 17), and none have provided a full kinetic analysis or reaction optimization of the in vitro pathway. Nevertheless, such a study would be valuable in order to design novel carotenoids and carotenoid-based hybrid natural products. Therefore, in the current work, we set out to optimize the C 30 carotenoid pathway reconstructed in vitro for the synthesis of DAP from IPP and DMAPP. Furthermore, we elucidated the kinetic behavior of both the FPP synthase a...

Use of signal sequences as an in situ removable sequence element to stimulate protein synthesis in cell-free extracts

“…As demonstrated by Ku et al . (31), the in vitro reproduction of a metabolic pathway can be ideally used to further understand and optimize the production of metabolites.…”

Cyclization mechanism of phomopsene synthase: mass spectrometry based analysis of various site-specifically labeled terpenes

“…In order to perform the chemoenzymatic synthesis of various [ 2 H]-GGPPs, we prepared IPP isomerase (IDI), 15 GPP synthase (IspA-S81F), 16 FPP synthase (IspA) 17 and PaPS 7 according to literature procedures. Although the PT domain of PaPS was able to convert dimethylallyl diphosphate (DMAPP) and GPP into GGPP, the reaction was too sluggish.…”

“…Protein expression and purification was performed according to the literature procedure. [15][16][17] Enzyme synthesis of deuterium labeled-2…”

Carotenoid cleavage in chromoplasts of white and yellow‐fleshed peach varieties

“…), chromoplast suspension in the assay should be regarded as a two‐phase system, in which the rate of substrate partitioning into membranes influences the rate of its breakdown . Indeed, non‐linear kinetics have been reported in vitro in the case of some enzymes involved in carotenoid biosynthesis . Consistently, a low non‐linear decrease of absorbance was evident also in heat‐inactivated controls that may be due, at least in part, to substrate solubilization in the membrane (Fig.…”