title: Increasing the production of (R)-3-hydroxybutyrate in recombinant Escherichia coli by improved cofactor supply.
abstract: BACKGROUND
  In a recently discovered microorganism, Halomonas boliviensis, polyhydroxybutyrate production was extensive and in contrast to other PHB producers, contained a set of alleles for the enzymes of this pathway. Also the monomer, (R)-3-hydroxybutyrate (3HB), possesses features that are interesting for commercial production, in particular the synthesis of fine chemicals with chiral specificity. Production with a halophilic organism is however not without serious drawbacks, wherefore it was desirable to introduce the 3HB pathway into Escherichia coli.
  
  
  RESULTS
  The production of 3HB is a two-step process where the acetoacetyl-CoA reductase was shown to accept both NADH and NADPH, but where the V max for the latter was eight times higher. It was hypothesized that NADPH could be limiting production due to less abundance than NADH, and two strategies were employed to increase the availability; (1) glutamate was chosen as nitrogen source to minimize the NADPH consumption associated with ammonium salts and (2) glucose-6-phosphate dehydrogenase was overexpressed to improve NADPH production from the pentose phosphate pathway. Supplementation of glutamate during batch cultivation gave the highest specific productivity (q3HB = 0.12 g g(-1) h(-1)), while nitrogen depletion/zwf overexpression gave the highest yield (Y3HB/CDW = 0.53 g g(-1)) and a 3HB concentration of 1 g L(-1), which was 50% higher than the reference. A nitrogen-limited fedbatch process gave a concentration of 12.7 g L(-1) and a productivity of 0.42 g L(-1) h(-1), which is comparable to maximum values found in recombinant E. coli.
  
  
  CONCLUSIONS
  Increased NADPH supply is a valuable tool to increase recombinant 3HB production in E. coli, and the inherent hydrolysis of CoA leads to a natural export of the product to the medium. Acetic acid production is still the dominating by-product and this needs attention in the future to increase the volumetric productivity further.
authors.affiliation: Division of Industrial Biotechnology, School of Biotechnology, KTH Royal Institute of Technology, AlbaNova University Center, SE 106 91, Stockholm, Sweden.|Center of Biotechnology, Faculty of Science and Technology, Universidad Mayor de San Simón, Cochabamba, Bolivia.
authors.forename: Mariel
authors.initials: M
authors.lastname: Perez-Zabaleta
authors.affiliation: Division of Industrial Biotechnology, School of Biotechnology, KTH Royal Institute of Technology, AlbaNova University Center, SE 106 91, Stockholm, Sweden.
authors.forename: Gustav
authors.initials: G
authors.lastname: Sjöberg
authors.affiliation: Division of Industrial Biotechnology, School of Biotechnology, KTH Royal Institute of Technology, AlbaNova University Center, SE 106 91, Stockholm, Sweden.|Center of Biotechnology, Faculty of Science and Technology, Universidad Mayor de San Simón, Cochabamba, Bolivia.
authors.forename: Mónica
authors.initials: M
authors.lastname: Guevara-Martínez
authors.affiliation: Division of Industrial Biotechnology, School of Biotechnology, KTH Royal Institute of Technology, AlbaNova University Center, SE 106 91, Stockholm, Sweden.
authors.forename: Johan
authors.initials: J
authors.lastname: Jarmander
authors.affiliation: Division of Industrial Biotechnology, School of Biotechnology, KTH Royal Institute of Technology, AlbaNova University Center, SE 106 91, Stockholm, Sweden.
authors.forename: Martin
authors.initials: M
authors.lastname: Gustavsson
authors.affiliation: Center of Biotechnology, Faculty of Science and Technology, Universidad Mayor de San Simón, Cochabamba, Bolivia.
authors.forename: Jorge
authors.initials: J
authors.lastname: Quillaguamán
authors.affiliation: Division of Industrial Biotechnology, School of Biotechnology, KTH Royal Institute of Technology, AlbaNova University Center, SE 106 91, Stockholm, Sweden. gen@kth.se.
authors.forename: Gen
authors.initials: G
authors.lastname: Larsson
mesh_terms: D020155:3-Hydroxybutyric Acid / Q000096:biosynthesis* / Q000737:chemistry; D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D020632:Halomonas / Q000145:classification / Q000201:enzymology / Q000235:genetics; D009243:NAD / Q000378:metabolism*; D009584:Nitrogen / Q000378:metabolism; D010802:Phylogeny; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013237:Stereoisomerism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D009243:NAD; D018698:Glutamic Acid; D000429:Alcohol Oxidoreductases; C023744:acetoacetyl-CoA reductase; D009584:Nitrogen; D020155:3-Hydroxybutyric Acid
keywords: (R)-3-hydroxybutyrate; Acetoacetyl-CoA reductase; Escherichia coli; Glutamate; Halomonas boliviensis; NADPH; Nitrogen limitation; zwf overexpression