AUTHOR=Bengelsdorf Frank R. , Poehlein Anja , Linder Sonja , Erz Catarina , Hummel Tim , Hoffmeister Sabrina , Daniel Rolf , Dürre Peter 

TITLE=Industrial Acetogenic Biocatalysts: A Comparative Metabolic and Genomic Analysis

JOURNAL=Frontiers in Microbiology

VOLUME=7

YEAR=2016

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2016.01036

DOI=10.3389/fmicb.2016.01036

ISSN=1664-302X

ABSTRACT=<p>Synthesis gas (syngas) fermentation by anaerobic acetogenic bacteria employing the Wood–Ljungdahl pathway is a bioprocess for production of biofuels and biocommodities. The major fermentation products of the most relevant biocatalytic strains (<italic>Clostridium ljungdahlii, C. autoethanogenum, C. ragsdalei</italic>, and <italic>C. coskatii</italic>) are acetic acid and ethanol. A comparative metabolic and genomic analysis using the mentioned biocatalysts might offer targets for metabolic engineering and thus improve the production of compounds apart from ethanol. Autotrophic growth and product formation of the four wild type (WT) strains were compared in uncontrolled batch experiments. The genomes of <italic>C. ragsdalei</italic> and <italic>C. coskatii</italic> were sequenced and the genome sequences of all four biocatalytic strains analyzed in comparative manner. Growth and product spectra (acetate, ethanol, 2,3-butanediol) of <italic>C. autoethanogenum, C. ljungdahlii</italic>, and <italic>C. ragsdalei</italic> were rather similar. In contrast, <italic>C. coskatii</italic> produced significantly less ethanol and its genome sequence lacks two genes encoding aldehyde:ferredoxin oxidoreductases (AOR). Comparative genome sequence analysis of the four WT strains revealed high average nucleotide identity (ANI) of <italic>C. ljungdahlii</italic> and <italic>C. autoethanogenum</italic> (99.3%) and <italic>C. coskatii</italic> (98.3%). In contrast, <italic>C. ljungdahlii</italic> WT and <italic>C. ragsdalei</italic> WT showed an ANI-based similarity of only 95.8%. Additionally, recombinant <italic>C. ljungdahlii</italic> strains were constructed that harbor an artificial <underline>a</underline>cetone <underline>s</underline>ynthesis <underline>o</underline>peron (ASO) consisting of the following genes: <italic>adc, ctfA, ctfB</italic>, and <italic>thlA</italic> (encoding acetoacetate decarboxylase, acetoacetyl-CoA:acetate/butyrate:CoA-transferase subunits A and B, and thiolase) under the control of <italic>thlA</italic> promoter (P<italic><sub>thlA</sub></italic>) from <italic>C. acetobutylicum</italic> or native <italic>pta-ack</italic> promoter (P<italic><sub>pta-ack</sub></italic>) from <italic>C. ljungdahlii</italic>. Respective recombinant strains produced 2-propanol rather than acetone, due to the presence of a NADPH-dependent primary-secondary alcohol dehydrogenase that converts acetone to 2-propanol. Furthermore, the ClosTron<sup>TM</sup> system was used to construct an <italic>adhE1</italic> integration mutant. These results provide extensive insights into genetic features of industrially relevant bacterial biocatalysts and expand the toolbox for metabolic engineering of acetogenic bacteria able to ferment syngas.</p>