Anaerobic and micro-aerobic 1,3-propanediol production by engineered Escherichia coli with dha genes from Klebsiella pneumoniae GLC29

Abstract

1,3-Propanediol (1,3-PDO) is a bifunctional molecule, and used in applications similar to those of ethylene glycol, propylene glycol, 1,3-butanediol and 1,4-butanediol. The use of glycerol as a feedstock is an alternative to reduce production costs for both 1,3-PDO and biodiesel, since biodiesel glycerol can be used for the production of 1,3-PDO by bacteria. Also, using metabolic engineering, it is possible to manipulate the metabolic routes and obtain high value products, reduce or eliminate the formation of undesirable byproducts. The aim of the study was to produce 1,3-propanediol in E. coli cloned with dha genes from Klebsiella pneumoniae GLC29. Six genes responsible for 1,3-PDO production in Klebsiella pneumoniae GLC29 were cloned. These genes were assembled in pSB1C3 as an expression vector: Genes dhaB1, dhaB2, dhaB3 and dhaT (pSB1C3dhaB123T), and another vector with genes dhaF and dhaG (pSB1C3dhaB123TFG) were derived using Gibson's Assembly technique. Escherichia coli TCS099 and SZ63 stains were used as hosts for 1,3-PDO production, and kept at -80°C for long-term storage. Glycerol was used as the sole or main carbon source in all experiments. Fermentations were performed in flasks in aerobic and anaerobic conditions using minimal media. Also, two stage fermentation (aerobic-anaerobic) was performed for 1,3-propanediol production. Only pSB1C3dhaB123TFG was able to produce high amounts of 1,3-PDO in shake flasks experiments, producing 2.5 g/L in micro-aerobic conditions, using E. coli TCS099 as host. Besides, E. coli SZ63 hosting pSB1C3dhaB123TFG was able to produce high amounts of 1,3-PDO, corresponding to 11.3 g/L of 1,3-PDO using a two-stage fermentation process using low concentration of vitamin B12 (1 mg/L). Plasmid pSB1C3dhaB123TFG shows potential for producing high amounts of 1,3-PDO, specially because of dhaF and dhaG, reaffirming the importance of this genes on 1,3-PDO production, especially with the addition of low amounts of vitamin B12, which is an expensive compound.

Process optimization of metabolically engineered Escherichia coliNSK015 fermentation for progressive improvement of 1,3‐propanediol production

“…3(F)). Our results were different from several previous reports in which anaerobic and microaerobic conditions were previously recommended as suitable conditions to produce 1,3‐PDO by either K. pneumoniae or Lactobacillus reuteri 8,17,18 . Those works assumed that GDht effectively functioned under anaerobic conditions 19 .…”

“…2(B)), while glycerol consumption and 1,3‐PDO production were stalled after 102 h. The results suggested that adenosyl transferase activity of E. coli may not be efficient enough to generate the adenosylated vitamin B12 to maximum level for supporting the full activity of GDHt compared to those directly utilizing coenzyme B12. Compared to other works, Neto et al 17 . used 0.25 mg L −1 (0.18 μmol L −1 ) vitamin B12 to produce 2.5 g L −1 1,3‐PDO (0.41 g g −1 yield) in batch fermentation, while Zhang et al 9 .…”

“…Those works assumed that GDht effectively functioned under anaerobic conditions 19 . Neto et al 17 . found that 1,3‐PDO at only 1.69 g L −1 from 18 g L −1 of glycerol was obtained by a recombinant E. coli under anaerobic conditions.…”

“…Our results were different from several previous reports in which anaerobic and microaerobic conditions were previously recommended as suitable conditions to produce 1,3-PDO by either K. pneumoniae or Lactobacillus reuteri. 8,17,18 Those works assumed that GDht effectively functioned under anaerobic conditions. 19 Neto et al 17 found that 1,3-PDO at only 1.69 g L −1 from 18 g L −1 of glycerol was obtained by a recombinant E. coli under anaerobic conditions.…”

“…8,17,18 Those works assumed that GDht effectively functioned under anaerobic conditions. 19 Neto et al 17 found that 1,3-PDO at only 1.69 g L −1 from 18 g L −1 of glycerol was obtained by a recombinant E. coli under anaerobic conditions. In contrast, they maintained the DO value up to 5%; 1,3-PDO was then improved to 3.15 g L −1 .…”