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Title: Microbial Production of Isoprene

Abstract

Isoprene is a volatile hydrocarbon of unknown function, produced by certain bacteria, plants and animals, sometimes in huge amounts—the Earth’s forests are estimated to emit >500 x 106 tons of isoprene per year. With funding from this program we explored the biochemistry and regulation of isoprene formation in the model bacterial system, Bacillus subtilis, with the goals of explaining the biological rationale for isoprene biogenesis and constructing an isoprene-overproducing microbial system. Although the role for isoprene formation in B. subtilis is still uncertain, our current model for regulation of this hydrocarbon’s synthesis is that isoprene production in B. subtilis is controlled by a combination of i) rapid regulation of isoprene synthase activity and ii) supply of the substrate for isoprene synthase, dimethyallyl diphosphate (DMAPP). This model parallels our current thinking about the control of isoprene formation in plant chloroplasts. In this reporting period we have been working to test part ii) of this model; this work has produced new results using genetic and analytical approaches. For examples, we have developed an analytical method to resolve DMAPP and its isomer, isopentenyl diphosphate, from each other in bacteria and plants. We have also shown that the IPP isomerase (type 2) of B.more » subtilis is not the source of “isoprene synthase” activity, and discovered that B. subtilis releases C5 isoprenoid alcohols to the medium, suggesting that isoprene plus other C5 isoprenoids may be common by-products of metabolism. In addition, we have continued to work on our discovery that wild type B. subtilis strains form prolific biofilms, are normal components of plant root microflora, and are testing the idea that B. subtilis growing in biofilms uses isoprene to induce plant root exudation.« less

Authors:
Publication Date:
Research Org.:
University of Colorado, Boulder
Sponsoring Org.:
USDOE - Office of Energy Research (ER)
OSTI Identifier:
910307
Report Number(s):
DOE/ER/15400-1
TRN: US200821%%333
DOE Contract Number:
FG02-03ER15400
Resource Type:
Technical Report
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ANIMALS; BACILLUS SUBTILIS; BACTERIA; BIOCHEMISTRY; BY-PRODUCTS; CHLOROPLASTS; GENETICS; HYDROCARBONS; ISOMERASES; ISOPRENE; METABOLISM; MICROORGANISMS; REGULATIONS; STRAINS; SUBSTRATES; SYNTHESIS; TESTING; Bacillus, isoprene, dimethylallyl diphosphate, plant roots

Citation Formats

Ray Fall. Microbial Production of Isoprene. United States: N. p., 2007. Web. doi:10.2172/910307.
Ray Fall. Microbial Production of Isoprene. United States. doi:10.2172/910307.
Ray Fall. Sun . "Microbial Production of Isoprene". United States. doi:10.2172/910307. https://www.osti.gov/servlets/purl/910307.
@article{osti_910307,
title = {Microbial Production of Isoprene},
author = {Ray Fall},
abstractNote = {Isoprene is a volatile hydrocarbon of unknown function, produced by certain bacteria, plants and animals, sometimes in huge amounts—the Earth’s forests are estimated to emit >500 x 106 tons of isoprene per year. With funding from this program we explored the biochemistry and regulation of isoprene formation in the model bacterial system, Bacillus subtilis, with the goals of explaining the biological rationale for isoprene biogenesis and constructing an isoprene-overproducing microbial system. Although the role for isoprene formation in B. subtilis is still uncertain, our current model for regulation of this hydrocarbon’s synthesis is that isoprene production in B. subtilis is controlled by a combination of i) rapid regulation of isoprene synthase activity and ii) supply of the substrate for isoprene synthase, dimethyallyl diphosphate (DMAPP). This model parallels our current thinking about the control of isoprene formation in plant chloroplasts. In this reporting period we have been working to test part ii) of this model; this work has produced new results using genetic and analytical approaches. For examples, we have developed an analytical method to resolve DMAPP and its isomer, isopentenyl diphosphate, from each other in bacteria and plants. We have also shown that the IPP isomerase (type 2) of B. subtilis is not the source of “isoprene synthase” activity, and discovered that B. subtilis releases C5 isoprenoid alcohols to the medium, suggesting that isoprene plus other C5 isoprenoids may be common by-products of metabolism. In addition, we have continued to work on our discovery that wild type B. subtilis strains form prolific biofilms, are normal components of plant root microflora, and are testing the idea that B. subtilis growing in biofilms uses isoprene to induce plant root exudation.},
doi = {10.2172/910307},
journal = {},
number = ,
volume = ,
place = {United States},
year = {Sun Jul 29 00:00:00 EDT 2007},
month = {Sun Jul 29 00:00:00 EDT 2007}
}

Technical Report:

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  • We have discovered that microorganisms produce and emit the hydrocarbon isoprene (2-methyl-1,3-butadiene), and have suggested that if isoprene-producing enzymes and their genes can be harnessed, useful hydrocarbon-producing systems might be constructed. The main goal of the proposed work is to establish the biochemical mechanism and regulation of isoprene formation in the bacterial system, Bacillus subtilis. Specific objectives of the proposed work are the following: (A) to characterize the physiological regulation of isoprene formation in B. subtilis; (B) to characterize mutations in B. subtilis 168 that suppress isoprene formation, clone these genes, and determine how isoprene and isoprenoid carbon flow aremore » regulated; and (C) to test ''overflow'' and ''signaling'' models for Bacillus isoprene formation. We are also pursuing the isolation and cloning of B. subtilis isoprene synthase, which we believe may be a regulatory enzyme.« less
  • OAK B135 We have discovered that bacteria produce and emit the hydrocarbon isoprene (2-methyl-1,3-butadiene), and have suggested that if isoprene-producing enzymes and their genes can be harnessed, useful hydrocarbon-producing systems might be constructed. The main goal of the proposed work was to establish the biochemical mechanism and regulation of isoprene formation in the model bacterial system, Bacillus subtilis. In this 3-year project we (a) characterized the physiological regulation of isoprene formation in B. subtilis and its relationship to isoprene formation in plant chloroplasts; (b) analyzed genetic controls on isoprene formation in B. subtilis; and (c) developed models to explain themore » biochemical rationale for isoprene formation. We are also pursued (d) new methods for continuous measurement of isoprene release in bioreactors, and (e) determined the presence of isoprene-forming Bacillus on plant roots and used B. subtilis as a biocontrol agent for protection of plant roots from plant pathogenic bacteria. We have made significant advances in several areas. These include: (1) establishing the enzymatic basis of isoprene formation in B. subtilis, and demonstrating throughout growth in a bioreactor that isoprene synthase activity rises and falls with each of three peaks of isoprene release (i.e. it appears to be a regulated enzyme). (2) We have explored genetic aspects of isoprene formation, using gene disruption methods to greatly alter the patterns of isoprene formation in bioreactors. Analysis of these mutants and alteration of cellular levels of dimethylallyl diphosphate (DMAPP), the substrate for isoprene synthase, has led to the formulation of two models to explain why isoprene is formed: an isoprenoid overflow model and a signaling model. We have obtained compelling evidence that isoprene releases in bioreactors result from metabolic overflow. However, we have yet to determine the pattern of isoprene formation when these bacteria are grown in a more natural state (e.g. as biofilms on surfaces). (3) We successfully used on-line mass spectrometry methods to measure release of volatiles, including isoprene, from bioreactors during growth of B. subtilis. This methodology, still in its infancy, may provide a new means to assess physiological processes during industrial growth of Bacillus species, and use isoprene formation as a barometer of carbon flow in these bacteria. (4) We also addressed the question: is Bacillus isoprene formation analogous to chloroplast processes? This research was initiated because of the continuing interest in the puzzle of isoprene formation in leaf chloroplasts. In pursuit of linkages between bacterial and plant isoprene formation, we used our DMAPP assay to demonstrate that leaves of the isoprene-emitter (cottonwood) show a diurnal cycle, peaking at mid-day in parallel with isoprene release. Thus it appears that in two different biological systems isoprene formation is highly regulated, and linked to isoprenoid carbon availability. (5) We developed a new method to detect Bacillus species in plant root samples, and demonstrated that plant roots are a rich source of biofilm-forming B. subtilis. Furthermore, using cultured Arabidopsis roots as a test system, we were able to demonstrate the formation of stable, viable Bacillus biofilms on the roots. Such roots were protected from killing by a root pathogenic Pseudomonas syringae strain. We have now formulated a mechanism to explain how such biocontrol by B. subtilis occurs, and future work will explore the role of isoprene in signaling between different rhizobacteria and plant roots.« less
  • Biogeochemical activity is an ongoing and dynamic process due to bacterial activity in the subsurface. Bacteria contribute significantly to biotransformation of metals and radionuclides. As basic science reveals more information about specific mechanisms of bacterial-metal reduction, an even greater contribution of bacteria to biogeochemical activities is realized. An understanding and application of the mechanisms of metal and radionuclide reduction offers tremendous potential for development into bioremedial processes and technologies. Most bacteria are capable of biogeochemical transformation as a result of meeting nutrient requirements. These assimilatory mechanisms for metals transformation include production of small molecules that serve as electron shuttles formore » metal reduction. This contribution to biogeochemistry is small however due to only trace requirements for minerals by bacteria. Dissimilatory metal reducing bacteria (DMRB) reduce oxidized metals and insoluble mineral oxides as a means for biological energy production during growth. These types of bacteria offer considerable potential for bioremediation of environments contaminated with toxic metals and radionuclides because of the relatively large amount of metal biotransformation they require for growth. One of the mechanisms employed by some DMRB for electron transfer to insoluble metal oxides is melanin production. The electrochemical properties of melanin provide this polymeric, humic-type compound with electron shuttling properties. Melanin, specifically, pyomelanin, increases the rate and degree of metal reduction in DMRB as a function of pyomelanin concentration. Due to its electron shuttling behavior, only low femtogram quantities per cell are required to significantly increase metal reduction capacity of DMRB. Melanin production is not limited to DMRB. In fact melanin is one of the most common pigments produced by biological systems. Numerous soil microorganisms produce melanin, contributing to about 2-4 percent of the humic fraction of soils. Our recent work has shown that melanin production by one species of bacteria could be used by other species for metal reduction. This melanin ''sharing'' is the area of focus for this project. In addition, melanin contributes to significant increases in metal reduction by both assimilatory and dissimilatory metal reducing bacteria. Stimulation of melanin production in the subsurface offers potential for accelerating metal reduction. Another focus of this project was to determine the potential of melanin production in portions of the Tims Branch watershed as it relates to metal and radionuclide immobilization in-situ.« less
  • The neutral lipids of nine species of methanogenic bacteria, two thermoacidophiles, two alkalinophiles and 20 algal samples were analyzed. The major components were C/sub 30/, C/sub 25/, and/or C/sub 20/ acyclic isoprenoid hydrocarbons with a continuous range of hydroisoprenoid homologues. The range or acyclic isoprenoids detected were from C/sub 14/ to C/sub 30/. The neutral lipid composition from these bacteria resembles the isoprenoid distribution isolated from ancient sediments and petroleum. Therefore, these findings may have major implications to biological and biogeochemical evolution. In this connection, samples and cores from ancient sediments and future fossil fuel source beds are being analyzedmore » for these neutral lipids as well as the more polar isopranyl glycerol-ether lipids. The derivation of fossil fuels and the biomass accumulations are the focal points of this phase of the study. Ancient and recent sediments, future source beds, and local esturaries are being enriched for microorganisms to establish a range and capability profile for hydrocarbon production. Only a relatively small percent of the microorganisms isolated demonstrated the ability to synthesize hydrocarbons; however, one particular algal isolate demonstrated that it can synthesize hydrocarbons while in a green physiological stage. Greater production is expected in the brown phase of growth. Hydrocarbon biosynthesis studies were conducted in an attempt to better understand the conditions required to maximize hydrocarbon production. The program involved physical and chemical parameters as well as assays of specifically labelled precusors with a cell free enzyme system to measure their conversions to hydrocarbons. The results have indicated a complex one enzyme system is involved in condensation and reduction of two fatty acids into hydrocarbons.« less