title: Gene replacement and elimination using λRed- and FLP-based tool to re-direct carbon flux in acetogen biocatalyst during continuous CO₂/H₂ blend fermentation.
abstract: A time- and cost-efficient 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 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 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 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 CO₂/H₂ blend fermentation (p < 0.005) with cell duplication time almost nearing that of the wild strain.
authors: Syngas Biofuels Energy, Inc., P.O. Box 300819, Houston, TX 77230, USA. michael@syngasbiofuelsenergy.com
authors: Michael
authors: M
authors: Tyurin
mesh_terms: D000079:Acetaldehyde / Q000378:metabolism; D019342:Acetic Acid / Q000378:metabolism; D000426:Alcohol Dehydrogenase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D019149:Bioreactors; D057486:Carbon Cycle / Q000235:genetics*; D002245:Carbon Dioxide / Q000378:metabolism* / Q000494:pharmacology; D049109:Cell Proliferation; D003013:Clostridium / Q000166:cytology / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D000431:Ethanol / Q000378:metabolism; D005285:Fermentation / Q000187:drug effects; D005560:Formate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D005561:Formates / Q000378:metabolism; D017353:Gene Deletion; D005798:Genes, Bacterial / Q000235:genetics; D016680:Genome, Bacterial / Q000235:genetics; D006859:Hydrogen / Q000378:metabolism* / Q000494:pharmacology; D006864:Hydrogenase / Q000235:genetics / Q000378:metabolism; D008798:Mevalonic Acid / Q000378:metabolism; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism; D016254:Mutagenesis, Insertional / Q000379:methods*; D045522:Recombinases / Q000235:genetics / Q000378:metabolism
chemical_list: D005561:Formates; D009097:Multienzyme Complexes; D045522:Recombinases; C030544:formic acid; D002245:Carbon Dioxide; D000431:Ethanol; D006859:Hydrogen; D000426:Alcohol Dehydrogenase; D006864:Hydrogenase; D005560:Formate Dehydrogenases; C054017:formate hydrogenlyase; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D000079:Acetaldehyde; D019342:Acetic Acid; D008798:Mevalonic Acid
references: Metab Eng. 2008 Nov;10(6):295-304
references: 18655844
references: PLoS One. 2012;7(5):e38171
references: 22675445
references: Appl Microbiol Biotechnol. 2012 Feb;93(4):1377-87
references: 22234536
references: Microb Cell Fact. 2009 Jan 07;8:2
references: 19128451
references: J Appl Microbiol. 2013 Apr;114(4):1033-45
references: 23289641
references: PLoS One. 2012;7(4):e33509
references: 22558074
references: Bioresour Technol. 2012 Jun;114:518-22
references: 22487129
references: Lett Appl Microbiol. 2012 Aug;55(2):149-54
references: 22642684
references: Appl Environ Microbiol. 2011 Aug;77(15):5467-75
references: 21685168
references: Appl Environ Microbiol. 2003 Nov;69(11):6345-53
references: 14602585
references: Appl Biochem Biotechnol. 2012 Nov;168(6):1384-93
references: 22941272
references: Appl Biochem Biotechnol. 2012 May;167(2):338-47
references: 22549584
references: J Ind Microbiol Biotechnol. 2009 Mar;36(3):319-32
references: 19137336
references: Arch Microbiol. 2013 Mar;195(3):181-8
references: 23277387
references: Appl Biochem Biotechnol. 2013 Feb;169(3):950-9
references: 23292245
references: Microb Cell Fact. 2012 Jan 20;11:11
references: 22264280
references: Bioresour Technol. 2010 Jul;101(13):5013-22
references: 20096574
references: Int J Syst Bacteriol. 1993 Apr;43(2):232-6
references: 7684239
references: Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5
references: 10829079
references: J Bacteriol. 1967 Jan;93(1):207-14
references: 4289807
references: Microbiol Mol Biol Rev. 2006 Sep;70(3):660-703
references: 16959965