A synthetic biochemistry system for the in vitro production of isoprene from glycolysis intermediates

Abstract

The high yields required for the economical production of chemicals and fuels using microbes can be difficult to achieve due to the complexities of cellular metabolism. An alternative to performing biochemical transformations in microbes is to build biochemical pathways in vitro, an approach we call synthetic biochemistry. Here we test whether the full mevalonate pathway can be reconstituted in vitro and used to produce the commodity chemical isoprene. We construct an in vitro synthetic biochemical pathway that uses the carbon and ATP produced from the glycolysis intermediate phosphoenolpyruvate to run the mevalonate pathway. The system involves 12 enzymes to perform the complex transformation, while providing and balancing the ATP, NADPH, and acetyl-CoA cofactors. The optimized system produces isoprene from phosphoenolpyruvate in ∼100% molar yield. Thus, by inserting the isoprene pathway into previously developed glycolysis modules it may be possible to produce isoprene and other acetyl-CoA derived isoprenoids from glucose in vitro.

Keywords: biofuel; commodity chemicals; green chemistry; in vitro synthesis; isoprenoids; metabolic engineering.

Figure 1

Schematic illustration of the in vitro production of isoprene from glycolysis intermediates. Steps that recycle CoA, ADP, and NADPH are shown in blue, red, and purple, respectively. Enzymes involved in the recycle of ADP and ATP are highlighted with a star. The enzymes used in this study are numbered as in Table1.

Figure 2

Cofactor specifity of GsPDH and LlNoxE. A: Reduction of NAD⁺ or NADP⁺ by GsPDH in the presence of pyruvate and coenzyme A. B: Oxidation of NADH or NADPH by LlNoxE. Reactions for A and B were performed in 50 mM Tris-Cl pH 8.0 containing 2 mM MgCl₂ at 37°C and monitored at 340 nm.

Figure 3

Analysis of the mevalonate pathway in vitro. A: The top portion of the mevalonate pathway (EfTHL-HMGR, EfHMGS, ScHMGR-t) was assayed alone (acetyl-CoA) or in combination with GsPDH/LlNoxE (pyruvate) or GsPyk/GsPDH/LlNoxE (PEP) by monitoring the reduction of HMG-CoA by HMGR. Controls lacking PDH or EfHMGS are shown as gray lines. B: The bottom portion of the mevalonate pathway (ScMVK, SsPMVK, ScMDC) was assayed by limiting mevalonate (0.3 mM). The extent of reaction was monitored by the disappearance of NADH using an ATP regeneration assay consisting of 0.1 mM ADP, 1.5 mM PEP, 5 mM MgCl₂, 10 mM KCl, and PK/LDH (Sigma). Addition of each kinase in succession allows the reaction to consume more ATP.

Figure 4

Isoprene production via the in vitro mevalonate pathway. The mevalonate pathway was reconstituted in vitro alone or in combination with GsPDH/LlNoxE or GsPyk/GsPDH/LlNoxE as described in the text and in Table2. NADPH was added to 2 mM from the beginning (gray bars) or regenerated from 0.2 mM NADP+ (white bars) using 6 mM Glucose-6-phosphate and 1 unit of G6PDH. Reactions were incubated for 16 h at 32°C. The % conversion was based on the number of nmol isoprene that should be produced from 1.5 mM Acetyl-CoA, Pyruvate, or PEP in 200 μL reactions. Values represent the average of two independent experiments.

Figure 5

Time course of isoprene production from PEP in vitro. Time course of isoprene production from 1.5 mM PEP. The percent conversion of PEP to isoprene at each time point is shown. The data represent an average of three independent trials. The reaction contained 0.2 mM CoA, 0.2 mM NAD⁺, 0.2 mM NADP⁺, 0.5 mM ADP, 5 mM MgCl₂, 10 mm KCl, 0.05 mM thiamine pyrophosphate, 0.1 mM AMP, and 6 mM G6P in 100 mM tris-Cl, pH 8.0 and was incubated at 32°C. The amount of each enzyme added is listed in Table2.