Gene replacement and elimination using λRed- and FLP-based tool to re-direct carbon flux in acetogen biocatalyst during continuous CO/H blend fermentation.

A time- and cost-efficient two-step gene elimination procedure was used for acetogen Clostridium sp. MT1834 capable of fermenting CO/H blend to 245 mM acetate ( p < 0.005). The first step rendered the targeted gene replacement without affecting the total genome size. We replaced the acetate pta- ack cluster with synthetic bi-functional acetaldehyde-alcohol dehydrogenase ( al- adh). Replacement of pta- ack with al- adh rendered initiation of 243 mM ethanol accumulation at the expense of acetate production during CO/H blend continuous fermentation ( p < 0.005). At the second step, al- adh was eliminated to reduce the genome size. Resulting recombinants accumulated 25 mM mevalonate in fermentation broth ( p < 0.005). Cell duplication time for recombinants with reduced genome size decreased by 9.5 % compared to Clostridium sp. MT1834 strain under the same fermentation conditions suggesting better cell energy pool management in the absence of the ack- pta gene cluster in the engineered biocatalyst. If the first gene elimination step was used alone for spo0A gene replacement with two copies of synthetic formate dehydrogenase in recombinants with a shortened genome, mevalonate production was replaced with 76.5 mM formate production in a single step continuous CO/H blend fermentation ( p < 0.005) with cell duplication time almost nearing that of the wild strain. [ABSTRACT FROM AUTHOR]