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ARS Home » Northeast Area » Ithaca, New York » Robert W. Holley Center for Agriculture & Health » Plant, Soil and Nutrition Research » Research » Publications at this Location » Publication #350899

Title: Genome structure of bacillus cereus tsu1 and genes involved in cellulose degradation and poly-3-hydroxybutyrate synthesis

item LI, HUI - Tennessee State University
item ZHOU, SUPING - Tennessee State University
item JOHNSON, TERRANCE - Tennessee State University
item VERCRUYSSE, KOEN - Tennessee State University
item LIZHI, OUYANG - Tennessee State University
item RANGANATHAN, PARTHASARATHY - Tennessee State University
item PHAMBU, NSOKI - Tennessee State University
item ROPELEWSKI, ALEXANDER - Computational Diagnostics, Inc
item Thannhauser, Theodore - Ted

Submitted to: International Journal of Polymer Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/9/2017
Publication Date: 9/10/2017
Citation: Li, H., Zhou, S., Johnson, T., Vercruysse, K., Lizhi, O., Ranganathan, P., Phambu, N., Ropelewski, A., Thannhauser, T.W. 2017. Genome structure of bacillus cereus tsu1 and genes involved in cellulose degradation and poly-3-hydroxybutyrate synthesis. International Journal of Polymer Science.

Interpretive Summary: Since the 1960s, bioplastics have attracted widespread attention, as eco-friendly, biodegradable, and sustainable alternatives feed stocks for industrial applications. In this work, a recently isolated bacterial strain, Bacilus cereus tsu1, was found to contain genes encoding for the enzymes of the poly-hydroxybutyrate (PHB) biosynthesis pathway. Five of these genes (phaA, phaB, phaR, phaC, and phaP) were selected, cloned in E. coli and expressed. The recombinant proteins were isolated and their identities confirmed by mass spectrometry. The clone over expressing the phaC gene was found to accumulate quantities of PHB particles. PHB is a biodegradable plastic with advantageous characteristics that make it an attractive starting material for commercial applications involving packaging plastics, medical materials, chiral specificity, etc. Thus, the phaC clone may prove useful for industrial applications.

Technical Abstract: In previous work, we reported on the isolation and genome sequence analysis of Bacillus cereus strain tsu1 NCBI accession number JPYN00000000. The 36 scaffolds in the assembled tsu1 genome were all aligned with B. cereus B4264 genome with variations. Genes encoding for xylanase and cellulase and the cluster of genes in the poly-3-hydroxybutyrate (PHB) biosynthesis pathway were identified in tsu1 genome. The PHB accumulation in B. cereus tsu1 was initially identified using Sudan Black staining and then confirmed using high-performance liquid chromatography. Physical properties of these PHB extracts, when analyzed with Raman spectra and Fourier transform infrared spectroscopy, were found to be comparable to the standard compound. The five PHB genes in tsu1 (phaA, phaB, phaR, phaC, and phaP) were cloned and expressed with TOPO cloning, and the recombinant proteins were validated using peptide mapping of in-gel trypsin digestion followed by mass spectrometry analysis. The recombinant E. coli BL21 (DE3) (over)expressing phaC was found to accumulate PHB particles. The cellulolytic activity of tsu1 was detected using carboxymethylcellulose (CMC) plate Congo red assay and the shift towards low-molecular size forms of CMC revealed by gel permeation chromatography in CMC liquid culture and the identification of a cellulase in the secreted proteome.