Final Technical Report: Microbial Production of Isoprene

Fall, Ray

University of Colorado, Boulder, CO (United States)

关键词: Hydrocarbons;    Biofilms;    59 Basic Biological Sciences Isoprene;    Bioreactor;    Plant Roots;   

DOI  :  10.2172/814920
RP-ID  :  DOE/ER20274
RP-ID  :  FG03-97ER20274
RP-ID  :  814920

美国|英语

来源: UNT Digital Library

【 摘 要 】

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 the 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.