isopropyl-thiogalactoside and hydracrylic-acid

In this study, production of 3-HP via malonyl-CoA was investigated by using metabolically engineered Escherichia coli carrying heterogeneous acetyl-CoA carboxylase (Acc) from Corynebacterium glutamicum and codon-optimized malonyl-CoA reductase (MCR) from Chloroflexus aurantiacus. Three engineered E. coli strains with different host-vector systems were constructed and investigated. The results indicated that the combination of E. coli BL21(DE3) and pET28a was the most efficient host-vector system for 3-HP production, and the highest concentration of 3-HP attained in shake flask cultivation reached 1.80g/L by the strain BE-MDA with induction at 0.25mM IPTG and 25°C, and supplementation of NaHCO3 and biotin. In fed-batch fermentation performed in a 5-L reactor, the concentration of 3-HP achieved 10.08g/L in 36h.

Topics: Acetates; Acetyl-CoA Carboxylase; Batch Cell Culture Techniques; Bioreactors; Biotin; Corynebacterium glutamicum; Escherichia coli; Fermentation; Glucose; Isopropyl Thiogalactoside; Lactic Acid; Malonyl Coenzyme A; Metabolic Engineering; Metabolic Networks and Pathways; Oxidoreductases; Sodium Bicarbonate; Time Factors

3-Hydroxypropionic acid (3-HP) is an important platform chemical proposed by the United States Department of Energy. 3-HP can be converted to a series of bulk chemicals. Biological production of 3-HP has made great progress in recent years. However, low yield of 3-HP restricts its commercialization. In this study, systematic optimization was conducted towards high-yield production of 3-HP in Klebsiella pneumoniae. We first investigated appropriate promoters for the key enzyme (aldehyde dehydrogenase, ALDH) in 3-HP biosynthesis, and found that IPTG-inducible tac promoter enabled overexpression of an endogenous ALDH (PuuC) in K. pneumoniae. We optimized the metabolic flux and found that blocking the synthesis of lactic acid and acetic acid significantly increased the production of 3-HP. Additionally, fermentation conditions were optimized and scaled-up cultivation were investigated. The highest 3-HP titer was observed at 83.8 g/L with a high conversion ratio of 54% on substrate glycerol. Furthermore, a flux distribution model of glycerol metabolism in K. pneumoniae was proposed based on in silico analysis. To our knowledge, this is the highest 3-HP production in K. pneumoniae. This work has significantly advanced biological production of 3-HP from renewable carbon sources.

Topics: Acetic Acid; Aldehyde Dehydrogenase; Bacterial Proteins; Fermentation; Gene Deletion; Gene Expression Regulation, Bacterial; Glycerol; Isopropyl Thiogalactoside; Kinetics; Klebsiella pneumoniae; Lactic Acid; Metabolic Engineering; Promoter Regions, Genetic

3-Hydroxypropionic acid (3-HP) is a value-added chemical for polymer synthesis. For biosynthesis of 3-HP from glycerol, two dhaB and dhaR clusters encoding glycerol dehydratase and its reactivating factor, respectively, were cloned from Lactobacillus brevis KCTC33069 and expressed in Escherichia coli. Coexpression of dhaB and dhaR allowed the recombinant E. coli to convert glycerol to 3-hydroxypropionaldehyde, an intermediate of 3-HP biosynthesis. To produce 3-HP from glycerol, fed-batch fermentation with a two-step feeding strategy was designed to separate the cell growth from the 3-HP production stages. Finally, E. coli JHS00947 expressing L .brevis dhaB and dhaR, and E. coli aldH produced 14.3g/L 3-HP with 0.26 g/L-h productivity, which were 14.6 and 8.53 times higher than those of the batch culture. In conclusion, overexpression of L. brevis dhaB and dhaR clusters and E. coli aldH, and implementation of the two-step feeding strategy enabled recombinant E. coli to convert glycerol to 3-HP efficiently.

Topics: Aldehyde Dehydrogenase; Amino Acid Sequence; Batch Cell Culture Techniques; Escherichia coli K12; Fermentation; Gas Chromatography-Mass Spectrometry; Genes, Bacterial; Glycerol; Hydro-Lyases; Isopropyl Thiogalactoside; Lactic Acid; Levilactobacillus brevis; Molecular Sequence Data; Multigene Family; Recombination, Genetic; Temperature

The top-valued platform chemical, 3-hydroxypropionic acid (3-HP), has a wide range of industrial applications but its biological production is not well established. Previously, the production of 3-HP from glycerol was demonstrated using a recombinant Escherichia coli strain expressing glycerol dehydratase (dhaB) and aldehyde dehydrogenase (aldH). The present investigation focuses on the effect of the culture conditions on the production of 3-HP from glycerol. The physicochemical parameters, such as pH, IPTG concentration, liquid-to-flask volume ratio, and substrate concentration, were examined in flask-scale experiments and obtained the highest titer of 3-HP at 4.4 g l(-1) in 48 h. When a fed-batch process was carried out in a bioreactor under pH-regulated conditions, the recombinant E. coli produced 3-HP at 31 g l(-1) in 72 h with a yield of 0.35 mol mol(-1) glycerol. The maximum specific rate of 3-HP production was estimated to be 3.41 mmol g(-1) cdw h(-1) between 12 and 24 h. Other than 3-HP, propionic acid (3.4 g l(-1)), 1,3-propanediol (2.4 g l(-1)), and lactic acid (1.6 g l(-1)) were produced as the major by-products. This paper reports for the first time a commercially meaningful high titer of 3-HP production.

Topics: Aldehyde Dehydrogenase; Culture Media; Escherichia coli; Gene Expression; Glycerol; Hydro-Lyases; Hydrogen-Ion Concentration; Isopropyl Thiogalactoside; Lactic Acid; Propylene Glycols; Recombinant Proteins