title: High yield 1,3-propanediol production by rational engineering of the 3-hydroxypropionaldehyde bottleneck in Citrobacter werkmanii.
abstract: BACKGROUND
  Imbalance in cofactors causing the accumulation of intermediates in biosynthesis pathways is a frequently occurring problem in metabolic engineering when optimizing a production pathway in a microorganism. In our previous study, a single knock-out Citrobacter werkmanii ∆dhaD was constructed for improved 1,3-propanediol (PDO) production. Instead of an enhanced PDO concentration on this strain, the gene knock-out led to the accumulation of the toxic intermediate 3-hydroxypropionaldehyde (3-HPA). The hypothesis was emerged that the accumulation of this toxic intermediate, 3-HPA, is due to a cofactor imbalance, i.e. to the limited supply of reducing equivalents (NADH). Here, this bottleneck is alleviated by rationally engineering cell metabolism to balance the cofactor supply.
  
  
  RESULTS
  By eliminating non-essential NADH consuming enzymes (such as lactate dehydrogenase coded by ldhA, and ethanol dehydrogenase coded by adhE) or by increasing NADH producing enzymes, the accumulation of 3-HPA is minimized. Combining the above modifications in C. werkmanii ∆dhaD resulted in the strain C. werkmanii ∆dhaD∆ldhA∆adhE::ChlFRT which provided the maximum theoretical yield of 1.00 ± 0.03 mol PDO/mol glycerol when grown on glucose/glycerol (0.33 molar ratio) on flask scale under anaerobic conditions. On bioreactor scale, the yield decreased to 0.73 ± 0.01 mol PDO/mol glycerol although no 3-HPA could be measured, which indicates the existence of a sink of glycerol by a putative glycerol dehydrogenase, channeling glycerol to the central metabolism.
  
  
  CONCLUSIONS
  In this study, a multiple knock-out was created in Citrobacter species for the first time. As a result, the concentration of the toxic intermediate 3-HPA was reduced to below the detection limit and the maximal theoretical PDO yield on glycerol was reached.
authors.affiliation: Department of Biochemical and Microbial Technology, Centre of Expertise-Industrial Biotechnology and Biocatalysis, Ghent University, Coupure links 653, 9000, Ghent, Belgium. Veerle.maervoet@UGent.be.|Department of Applied Bioscience Engineering, Laboratory of Applied Biotechnology, Ghent University, Valentin Vaerwyckweg 1, 9000, Ghent, Belgium. Veerle.maervoet@UGent.be.
authors.forename: Veerle E T
authors.initials: VE
authors.lastname: Maervoet
authors.affiliation: Department of Biochemical and Microbial Technology, Centre of Expertise-Industrial Biotechnology and Biocatalysis, Ghent University, Coupure links 653, 9000, Ghent, Belgium. Sofie.DeMaeseneire@UGent.be.
authors.forename: Sofie L
authors.initials: SL
authors.lastname: De Maeseneire
authors.affiliation: Department of Biochemical and Microbial Technology, Centre of Expertise-Industrial Biotechnology and Biocatalysis, Ghent University, Coupure links 653, 9000, Ghent, Belgium. gizemavci@gmail.com.|Bioengineering Department, Faculty of Engineering, Ege University, 35100, Bornova-Izmir, Turkey. gizemavci@gmail.com.
authors.forename: Fatma G
authors.initials: FG
authors.lastname: Avci
authors.affiliation: Department of Biochemical and Microbial Technology, Centre of Expertise-Industrial Biotechnology and Biocatalysis, Ghent University, Coupure links 653, 9000, Ghent, Belgium. Joeri.Beauprez@UGent.be.
authors.forename: Joeri
authors.initials: J
authors.lastname: Beauprez
authors.affiliation: Department of Biochemical and Microbial Technology, Centre of Expertise-Industrial Biotechnology and Biocatalysis, Ghent University, Coupure links 653, 9000, Ghent, Belgium. Wim.Soetaert@UGent.be.
authors.forename: Wim K
authors.initials: WK
authors.lastname: Soetaert
authors.affiliation: Department of Biochemical and Microbial Technology, Centre of Expertise-Industrial Biotechnology and Biocatalysis, Ghent University, Coupure links 653, 9000, Ghent, Belgium. Marjan.DeMey@UGent.be.
authors.forename: Marjan
authors.initials: M
authors.lastname: De Mey
mesh_terms: D000595:Amino Acid Sequence; D061249:Batch Cell Culture Techniques; D019149:Bioreactors / Q000382:microbiology; D002954:Citrobacter / Q000187:drug effects / Q000201:enzymology / Q000254:growth & development / Q000378:metabolism*; D005285:Fermentation / Q000187:drug effects; D055786:Gene Knockout Techniques; D005947:Glucose / Q000494:pharmacology; D005985:Glyceraldehyde / Q000031:analogs & derivatives* / Q000378:metabolism; D005990:Glycerol / Q000494:pharmacology; D005991:Glycerol Kinase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D060847:Metabolic Engineering / Q000379:methods*; D055442:Metabolome / Q000187:drug effects; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009243:NAD / Q000378:metabolism; D011407:Propane / Q000378:metabolism*; D011409:Propylene Glycols / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity / Q000187:drug effects; D013401:Sugar Alcohol Dehydrogenases / Q000737:chemistry / Q000378:metabolism
chemical_list: D011409:Propylene Glycols; D009243:NAD; C047158:3-hydroxypropionaldehyde; D005985:Glyceraldehyde; C041787:1,3-propanediol; D013401:Sugar Alcohol Dehydrogenases; C020672:glycerol dehydrogenase; D005991:Glycerol Kinase; D005947:Glucose; D005990:Glycerol; D011407:Propane
references.citation: Nucleic Acids Res. 2000 Jan 1;28(1):27-30
references.pmid: 10592173
references.citation: Int J Syst Evol Microbiol. 2002 Jul;52(Pt 4):1177-84
references.pmid: 12148625
references.citation: Metab Eng. 2002 Jul;4(3):217-29
references.pmid: 12616691
references.citation: J Bacteriol. 1996 Oct;178(20):6013-8
references.pmid: 8830700
references.citation: J Bacteriol. 1999 Apr;181(8):2351-7
references.pmid: 10197995
references.citation: Biotechnol Bioeng. 2005 Mar 5;89(5):556-64
references.pmid: 15669087
references.citation: Metab Eng. 2006 Nov;8(6):578-86
references.pmid: 16931085
references.citation: Appl Microbiol Biotechnol. 2007 Jan;73(5):1017-24
references.pmid: 16960737
references.citation: Metab Eng. 2007 May;9(3):258-67
references.pmid: 17292651
references.citation: Mol Biotechnol. 2007 Oct;37(2):112-9
references.pmid: 17914171
references.citation: Metab Eng. 2009 Mar;11(2):101-6
references.pmid: 19100856
references.citation: Appl Environ Microbiol. 2009 Mar;75(6):1628-34
references.pmid: 19139229
references.citation: Biotechnol Bioeng. 2009 Dec 1;104(5):965-72
references.pmid: 19572314
references.citation: BMC Genomics. 2010;11:579
references.pmid: 20955615
references.citation: Nucleic Acids Res. 2011 Jan;39(Database issue):D28-31
references.pmid: 20972220
references.citation: J Ind Microbiol Biotechnol. 2011 Aug;38(8):991-9
references.pmid: 20862513
references.citation: Nucleic Acids Res. 2014 Jan;42(Database issue):D199-205
references.pmid: 24214961
references.citation: Bioprocess Biosyst Eng. 2014 Apr;37(4):711-8
references.pmid: 23996279
references.citation: J Biotechnol. 2014 Jun 20;180:52-65
references.pmid: 24698847
references.citation: Microb Cell Fact. 2014;13:70
references.pmid: 24885849