AUTHOR=Nogle Robert , Nagaraju Shilpa , Utturkar Sagar M. , Giannone Richard J. , Reynoso Vinicio , Leang Ching , Hettich Robert L. , Mitchell Wayne P. , Simpson Sean D. , Jewett Michael C. , Köpke Michael , Brown Steven D. 

TITLE=Clostridium autoethanogenum isopropanol production via native plasmid pCA replicon

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=10

YEAR=2022

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2022.932363

DOI=10.3389/fbioe.2022.932363

ISSN=2296-4185

ABSTRACT=<p><italic>Clostridium autoethanogenum</italic> is a model gas-fermenting acetogen for commercial ethanol production. It is also a platform organism being developed for the carbon-negative production of acetone and isopropanol by gas fermentation. We have assembled a 5.5 kb pCA plasmid for type strain DSM10061 (JA1-1) using three genome sequence datasets. pCA is predicted to encode seven open-reading frames and estimated to be a low-copy number plasmid present at approximately 12 copies per chromosome. RNA-seq analyses indicate that pCA genes are transcribed at low levels and two proteins, CAETHG_05090 (putative replication protein) and CAETHG_05115 (hypothetical, a possible Mob protein), were detected at low levels during batch gas fermentations. Thiolase (<italic>thlA</italic>), CoA-transferase (<italic>ctfAB</italic>), and acetoacetate decarboxylase (<italic>adc</italic>) genes were introduced into a vector for isopropanol production in <italic>C. autoethanogenum</italic> using the native plasmid origin of replication. The availability of the pCA sequence will facilitate studies into its physiological role and could form the basis for genetic tool optimization.</p>