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J Ind Microbiol Biotechnol (2013) 40:749758 DOI 10.1007/s10295-013-1279-1
METABOLIC ENGINEERING AND SYNTHETIC BIOLOGY
Gene replacement and elimination using kRed- and FLP-based tool to re-direct carbon ux in acetogen biocatalystduring continuous CO2/H2 blend fermentation
Michael Tyurin
Received: 23 February 2013 / Accepted: 23 April 2013 / Published online: 7 May 2013 Society for Industrial Microbiology and Biotechnology 2013
Abstract A time- and cost-efcient two-step gene elimination procedure was used for acetogen Clostridium sp. MT1834 capable of fermenting CO2/H2 blend to 245 mM acetate (p \ 0.005). The rst 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 CO2/H2 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 rst 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 CO2/H2 blend fermentation (p \ 0.005) with cell duplication time almost nearing that of the wild strain.
Keywords Acetogens Continuous fermentation Gene
elimination Gene replacement Mevalonate Formate
Introduction
Energy production from fossil fuels and manufacture of food and chemicals are associated with multi-step oxidation of organic carbon of carbohydrates to inorganic carbon of carbon dioxide (CO2) with oxygen as the end electron acceptor. An economic approach to use vent gas of [100 MW power plants utilizing integrated coal gasication combined cycle (IGCC) (waste gas 100 % CO2) for
biocatalysis to carbon of carbohydrates gains strong interest. The CO2 may be directly biocatalyzed to chemicals and food components without employing the photosyn-thesis part of the global carbon cycle. Bacteria are known for the highest ratio of cell surface area-to-cell volume [8] with cell...
