Title: Heterologous complementation of a pyrF deletion in Caldicellulosiruptor hydrothermalisgenerates a new host for the analysis of biomass deconstruction

Abstract

Members of the thermophilic, anaerobic Gram-positive bacterial genus Caldicellulosiruptor grow optimally at 65 to 78°C and degrade lignocellulosic biomass without conventional pretreatment. Decomposition of complex cell wall polysaccharides is a major bottleneck in the conversion of plant biomass to biofuels and chemicals, and conventional biomass pretreatment includes exposure to high temperatures, acids, or bases as well as enzymatic digestion. Members of this genus contain a variety of glycosyl hydrolases, pectinases, and xylanases, but the contribution of these individual enzymes to biomass deconstruction is largely unknown. C. hydrothermalis is of special interest because it is the least cellulolytic of all the Caldicellulosiruptor species so far characterized, making it an ideal naïve system to study key cellulolytic enzymes from these bacteria. To develop methods for genetic manipulation of C. hydrothermalis, we selected a spontaneous deletion of pyrF, a gene in the pyrimidine biosynthetic pathway, resulting in a strain that was a uracil auxotroph resistant to 5-fluoroorotic acid (5-FOA). This strain allowed the selection of prototrophic transformants with either replicating or non-replicating plasmids containing the wild-type pyrF gene. Counter-selection of the pyrF wild-type allele on non-replicating vectors allowed the construction of chromosomal deletions. To eliminate integration of the non-replicating plasmid at the pyrFmore » locus in the C. hydrothermalis chromosome, we used the non-homologous Clostridium thermocellum wild-type pyrF allele to complement the C. hydrothermalis pyrF deletion. The autonomously replicating shuttle vector was maintained at 25 to 115 copies per chromosome. Deletion of the ChyI restriction enzyme in C. hydrothermalis increased the transformation efficiency by an order of magnitude and demonstrated the ability to construct deletions and insertions in the genome of this new host. The use of C. hydrothermalis as a host for homologous and heterologous expression of enzymes important for biomass deconstruction will enable the identification of enzymes that contribute to the special ability of these bacteria to degrade complex lignocellulosic substrates as well as facilitate the construction of strains to improve and extend their substrate utilization capabilities.« less

Authors:

Groom, Joseph ^[1]; Chung, Daehwan ^[1]; Young, Jenna ^[1]; Westpheling, Janet ^[1]

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+ Show Author Affiliations

1. University of Georgia, Athens, GA (United States); Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Publication Date:

Tue Sep 16 00:00:00 EDT 2014

Research Org.:

Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER); University of Georgia Research Foundation

OSTI Identifier:

1626959

Grant/Contract Number:  

AC05-00OR22725

Resource Type:

Accepted Manuscript

Journal Name:

Biotechnology for Biofuels

Additional Journal Information:

Journal Volume: 7; Journal Issue: 1; Journal ID: ISSN 1754-6834

Publisher:

BioMed Central

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; 09 BIOMASS FUELS; switchgrass; shuttle vector; glycosyl hydrolase; clostridium thermocellum; native plasmid