AUTHOR=Omorotionmwan Bunmi B. , Wang Hengzheng , Baker Jonathan P. , Gizynski Krzysztof , Yoo Minyeong , Akaluka Cynthia , Zhang Ying , Minton Nigel P. 

TITLE=Chromosomal engineering of inducible isopropanol- butanol-ethanol production in Clostridium acetobutylicum

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=11

YEAR=2023

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2023.1218099

DOI=10.3389/fbioe.2023.1218099

ISSN=2296-4185

ABSTRACT=<p>The use of environmentally damaging petrochemical feedstocks can be displaced by fermentation processes based on engineered microbial chassis that recycle biomass-derived carbon into chemicals and fuels. The stable retention of introduced genes, designed to extend product range and/or increase productivity, is essential. Accordingly, we have created multiply marked auxotrophic strains of <italic>Clostridium acetobutylicum</italic> that provide distinct loci (<italic>pyrE</italic>, <italic>argH</italic>, <italic>purD</italic>, <italic>pheA</italic>) at which heterologous genes can be rapidly integrated using allele-coupled exchange (ACE). For each locus, ACE-mediated insertion is conveniently selected on the basis of the restoration of prototrophy on minimal media. The <italic>Clostridioides difficile</italic> gene (<italic>tcdR</italic>) encoding an orthogonal sigma factor (TcdR) was integrated at the <italic>pyrE</italic> locus under the control of the lactose-inducible, <italic>bgaR::</italic>P<sub><italic>bgaL</italic></sub> promoter to allow the simultaneous control of genes/operons inserted at other disparate loci (<italic>purD</italic> and <italic>pheA</italic>) that had been placed under the control of the P<sub><italic>tcdB</italic></sub> promoter. In control experiments, dose-dependent expression of a <italic>catP</italic> reporter gene was observed with increasing lactose concentration. At the highest doses tested (10 mM) the level of expression was over 10-fold higher than if <italic>catP</italic> was placed directly under the control of <italic>bgaR::</italic>P<sub><italic>bgaL</italic></sub> and over 2-fold greater than achieved using the strong P<sub><italic>fdx</italic></sub> promoter of the <italic>Clostridium sporogenes</italic> ferredoxin gene. The utility of the system was demonstrated in the production of isopropanol by the <italic>C. acetobutylicum</italic> strain carrying an integrated copy of <italic>tcdR</italic> following the insertion of a synthetic acetone operon (<italic>ctfA/B, adc</italic>) at the <italic>purD</italic> locus and a gene (<italic>sadh</italic>) encoding a secondary dehydrogenase at <italic>pheA</italic>. Lactose induction (10 mM) resulted in the production of 4.4 g/L isopropanol and 19.8 g/L Isopropanol-Butanol-Ethanol mixture.</p>