Discovery of novel geranylgeranyl reductases and characterization of their substrate promiscuity

Abstract

BackgroundGeranylgeranyl reductase (GGR) is a flavin-containing redox enzyme that hydrogenates a variety of unactivated polyprenyl substrates, which are further processed mostly for lipid biosynthesis in archaea or chlorophyll biosynthesis in plants. To date, only a few GGR genes have been confirmed to reduce polyprenyl substrates in vitro or in vivo.ResultsIn this work, we aimed to expand the confirmed GGR activity space by searching for novel genes that function under amenable conditions for microbial mesophilic growth in conventional hosts such as Escherichia coli or Saccharomyces cerevisiae. 31 putative GGRs were selected to test for potential reductase activity in vitro on farnesyl pyrophosphate, geranylgeranyl pyrophosphate, farnesol (FOH), and geranylgeraniol (GGOH). We report the discovery of several novel GGRs exhibiting significant activity toward various polyprenyl substrates under mild conditions (i.e., pH 7.4, T = 37 °C), including the discovery of a novel bacterial GGR isolated from Streptomyces coelicolor. In addition, we uncover new mechanistic insights within several GGR variants, including GGR-mediated phosphatase activity toward polyprenyl pyrophosphates and the first demonstration of completely hydrogenated GGOH and FOH substrates.ConclusionThese collective results enhance the potential for metabolic engineers to manufacture a variety of isoprenoid-based biofuels, polymers, and chemical feedstocks in common microbial hosts such as E. coli or S. cerevisiae.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1342-2) contains supplementary material, which is available to authorized users.

An automated workflow to screen alkene reductases using high-throughput thin layer chromatography

“…Collectively, the screen revealed six product bands in the TLC pro les, including an additional faint side product (R f = 0.26 ± 0.02) observed in many of the mutants contained in A-D wells ( Figure 6 and Supplementary Table S1). Notably, this side product could be the diepoxy adduct resulting from the regioisomer of H 2 -FOH in which the middle prenyl group is reduced instead of the terminal one as proposed in the previous GGR characterization study 1 . Retention factor values for non-enzymatic side products (R f (s2) = 0.51 ± 0.04 and R f (s3) = 0.60 ± 0.04) were shifted slightly downward in the A-D plate (R f (s2) = 0.46 ± 0.03 and R f (s3) = 0.52 ± 0.04).…”

“…Alkene hydrogenation is an important process in petroleum re ning, yet one that suffers pitfalls including catalyst poisoning, mass transport limitations, heat generation, and collective nancial barriers associated with hydrogen, storage, and catalysts. For these reasons, performing alkene hydrogenation in vivo using engineered microbes has emerged as a potential alternative capable of overcoming these barriers 1 . Moreover, as synthetic biology tools and high-throughput screening (HTS) technologies become more affordable and accessible, microbes like Escherichia coli or Saccharomyces cerevisiae are increasingly utilized to biosynthesize commercially important molecules at industrial scales [2][3][4][5][6][7] .…”

“…Its native function is believed to asymmetrically reduce all eight prenyl units within 2,3-di-O-geranylgeranylglyceryl phosphate, a precursor for archaeal lipid membrane biosynthesis 20 . SaGGR has been shown to reduce a variety of C 15 -C 20 isoprenoid-based intermediates synthesized from either the mevalonate or DXP pathways 1…”

An Automated Workflow to Screen Alkene Reductases Using High-throughput Thin Layer Chromatography

“…Notably, this side product could be the diepoxy adduct resulting from the regioisomer of H 2 -FOH in which the middle prenyl group is reduced instead of the terminal one as proposed in the previous GGR characterization study 1 . Retention factor values for non-enzymatic side products (R f (s2) = 0.51 ± 0.04 and R f (s3) = 0.60 ± 0.04) were shifted slightly downward in the A-D plate (R f (s2) = 0.46 ± 0.03 and R f (s3) = 0.52 ± 0.04).…”

A Review on Bio-Fuels and its Manufacturing

“…One example is, bio-ethanol produced from biomass. All these raw materials may be converted into bio-fuels via paths with enzymes and/or microorganisms, including microorganisms that were developed for this purpose [15].…”