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Biotechnological Applications of Extremely Thermophilic Microbes

Microbial “extremophiles” thrive at temperatures up to 110°C, at pH <2 or >10, or in saturated saline.  Most belong to a newly discovered third domain of life, the Archaea, which diverges as much from Bacteria or Eukarya as the latter do from one another.  In collaboration with UCLA microbiologists and Professor Liao’s group, we are discovering new thermostable enzymes and metabolic pathways of extreme thermophiles.

Project Overview

This project will establish a functional proteomics approach involving coordinated use of high-throughput LC/MS-based enzyme assays, DNA microarrays, and gene cloning and expression for fast screening of enzyme activities and for identification of genes in hypothesized metabolic pathways. In particular, the research will focus on the aromatic biosynthesis pathway s of the extremely thermophilic sulfate-reducing archaeon, Archaeoglobus fulgidus.

The aromatic amino acid synthesis pathway has been engineered successfully for the synthesis of natural and unnatural chiral amino acids, which are important drug intermediates, as well as other industrially important aromatics, such as indigo. Intense interest therefore has developed in the enzymes of these metabolic pathways. A. fulgidus is representative of the third, most primitive domain of life, and the aromatic amino acid synthesis pathways have not been explored in these microorganisms despite the fact that they may offer a far more robust set of biosynthetic enzymes well suited both for in vivo and in vitro synthesis applications.

Recently, the entire genome of A. fulgidus was sequenced and a thorough study of open reading frames for sequences homologous to known enzymes was conducted. It is noteworthy that a number of enzymes involved in common aromatic amino acid synthesis routes were not identified on the genome. Our goal is to identify these new enzymes/pathways by a functional preteomics approach and to identify, isolate, sequence, clone and express (in E. coli) new enzymes from this microbe.

The isoprenoid tetraether lipid biosynthetic pathway of thermophilic organisms is not understood despite numerous previous studies.  Archaeoglobus fulgidus has previously uncharacterized membranes that are suspected to include diether and tetraether lipids that are more stable at high temperatures than the standard diester lipids of non-thermophilic organisms.  In addition, a variable number of cyclopentane groups also may appear in the core isoprenoid lipid structure, a feature that has been found in the lipids of several other thermophilic archaea.  After finding methods to consistently isolate, purify, and hydrolyze the lipids of this organism, the core lipid is characterized over a range of growth temperatures using LC-MS.  Preliminary data suggests that the ratio of tetraether lipid to diether lipid in Archaeoglobus fulgidus increases with the growth temperature of the organism.  LC-MS is being used in the development of assays for enzymes of isoprenoid lipid biosynthesis.

Students

Publications

This project is a collaboration between three labs: Monbouquette, Liao (UCLA Chemical Engineering), and Schroeder (UCLA Microbiology and Molecular Genetics).

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