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Pseudomonas asiatica C1, which could grow on glucose and aerobically synthesize coenzyme B12, was isolated and developed as a microbial cell factory for the production of 3-hydroxypropionic acid (3-HP) from glycerol. Three heterologous enzymes, glycerol dehydratase (GDHt), GDHt reactivase (GdrAB) and aldehyde dehydrogenase (ALDH), constituting the 3-HP synthesis pathway, were introduced, and three putative dehydrogenases, responsible for 3-HP degradation, were disrupted. In addition, the transcriptional repressor glpR and the glycerol kinase glpK were removed to increase glycerol import while eliminating the catabolic use of glycerol. Furthermore, the global regulatory protein encoded by crc and several putative oxidoreductases (PDORs) were disrupted. One resulting strain, when grown on glucose, could produce 3-HP at ~ 700 mM in 48 h in a fed-batch bioreactor experiment, with the molar yield > 0.99 on glycerol without much by-products. This study demonstrates that P. asiatica C1 is a promising host for production of 3-HP from glycerol.

The production of 1,3-propanediol (1,3-PDO) from glucose was investigated using Klebsiella pneumoniae J2B, which converts glycerol to 1,3-PDO and synthesize an essential coenzyme B12. In order to connect the glycolytic pathway with the pathway of 1,3-PDO synthesis from glycerol, i.e., to directly produce diol from glucose, glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate phosphatase from Saccharomyces cerevisiae were overexpressed. Additionally, the effect of expression levels and the use of isoforms of these two enzymes on glycerol and 1,3-PDO production were studied. Furthermore, to prevent loss of produced glycerol, the glycerol oxidation pathways were disrupted. Finally, the conversion rate of glycerol to 1,3-PDO was increased via homologous overexpression of glycerol dehydratase and 1,3-PDO oxidoreductase. The resultant strain successfully produced 1,3-PDO from glucose at a yield of 0.27mol/mol along with glycerol at 0.52mol/mol. Improvement of the engineered K. pneumoniae J2B to further increase conversion of glycerol to 1,3-PDO is discussed.

Bio-production of 1,3-propanediol (1,3-PDO) from glycerol was studied using Pseudomonas denitrificans as host, which aerobically synthesizes coenzyme B12, an essential cofactor of glycerol dehydratase (GDHt). P. denitrificans was transformed with the 1,3-PDO synthesis pathway composed of GDHt and 1,3-PDO oxidoreductase (PDOR), and its putative 3-hydroxypropionaldehyde (3-HPA) dehydrogenase(s), leading to the production of 3-hydroxypropioninc acid form the intermediary 3-HPA, was identified and deleted. In addition, to improve the availability of NADH for PDOR, oxidation of NADH in the electron transport chain was disturbed by deletion of the nuo operon and/or ndh gene. Finally, acetate formation pathway was eliminated. One resulting strain could produce 68.95 mM 1,3-PDO with the yield of 0.92 mol 1,3-PDO/mol glycerol on flask scale and 440 mM with the yield of 0.89 mol 1,3-PDO/mol glycerol in a fed-batch bioreactor experiment. This study demonstrates that P. denitrificans is a promising recombinant host for the production of 1,3-PDO from glycerol.

The necessity of costly co-enzyme B12 for the activity of glycerol dehydratase (GDHt) is considered as a major bottleneck in sustainable bioproduction of 1,3-propanediol (1,3-PD) from glycerol. Here, an E. coil Rosetta-dhaB1-dhaB2 strain was constructed by overexpressing a B12-independent GDHt (dhaB1) and its activating factor (dhaB2) from Clostridium butyricum. Subsequently, it was used in designing a co-culture with E. coli BL21-dhaT that overexpressed 1,3-PD oxidoreductase (dhaT), to produce 1,3-PD during co-fermentation of glycerol and glucose. The optimum initial ratio of BL21-dhaT to Rosetta-dhaB1-dhaB2 strains in the co-culture was 1.5. Compared to the fermentation of glycerol alone, co-fermentation approach provided 1.3-folds higher 1,3-PD. Finally, co-fermentation was done in a 10 L bioreactor that produced 41.65 g/L 1,3-PD, which corresponded to 0.69 g/L/h productivity and 0.67 mol/mol yield of 1,3-PD. Hence, the developed co-culture could produce 1,3-PD cost-effectively without requiring vitamin B12.

Poly(3-hydroxypropionate) (P3HP) is a biodegradable and biocompatible thermoplastic. In this study, a P3HP biosynthetic pathway from glycerol was constructed in recombinant Escherichia coli. The genes for glycerol dehydratase and its reactivating factor (dhaB123 and gdrAB, from Klebsiella pneumoniae), propionaldehyde dehydrogenase (pduP, from Salmonella typhimurium), and polyhydroxyalkanoate synthase (phaC1, from Cupriavidus necator) were cloned and expressed in E. coli. After culture condition optimization, the final engineered strain accumulated 10.1 g/L P3HP (46.4% of the cell dry weight) using glycerol and glucose as cosubstrates in an aerobic fed-batch fermentation. To date, this is the highest P3HP production without addition of any expensive precursor.

Fed-batch cultures of Klebsiella pneumoniae expressing Escherichia coli aldH were performed under microaerobic conditions to investigate the effects on metabolites production. Increasing the aeration rate enhanced cell growth and 3-hydroxypropionic acid (3-HP) production, but reduced 1,3-propanediol (1,3-PDO) formation. The recombinant strain K. pneumoniae/pUC18kan-aldHec produced 48.9 g/L of 3-HP and 25.3g/L of 1,3-PDO with an overall yield of 0.66 mol/mol in 28 h at an aeration rate of 1.5 vvm; however, under fully aerobic condition, no 3-HP and 1,3-PDO were produced due to the repression of dha operon. The flux through the reaction catalyzed by glycerol dehydratase and the split ratio of 1,3-PDO were negatively correlated with the aeration rate, even though the 3-HP level showed a positive trend. This study demonstrated that the relative amounts of 3-HP and 1,3-PDO can be controlled by the aeration rate.