Metabolic engineering of 3‐hydroxypropionic acid biosynthesis in Escherichia coli

3‐Hydroxypropionic acid (3‐HP) can be produced in microorganisms as a versatile platform chemical. However, owing to the toxicity of the intermediate product 3‐hydroxypropionaldehyde (3‐HPA), the minimization of 3‐HPA accumulation is critical for enhancing the productivity of 3‐HP. In this study, we identified a novel aldehyde dehydrogenase, GabD4 from Cupriavidus necator, and found that it possessed the highest enzyme activity toward 3‐HPA reported to date. To augment the activity of GabD4, several variants were obtained by site‐directed and saturation mutagenesis based on homology modeling. Escherichia coli transformed with the mutant GabD4_E209Q/E269Q showed the highest enzyme activity, which was 1.4‐fold higher than that of wild type GabD4, and produced up to 71.9 g L−1 of 3‐HP with a productivity of 1.8 g L−1 h−1. To the best of our knowledge, these are the highest 3‐HP titer and productivity values among those reported in the literature. Additionally, our study demonstrates that GabD4 can be a key enzyme for the development of industrial 3‐HP‐producing microbial strains, and provides further insight into the mechanism of aldehyde dehydrogenase activity. Biotechnol. Bioeng. 2015;112: 356–364. © 2014 Wiley Periodicals, Inc.

[1]  B. Wanner,et al.  One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[2]  H. Weiner,et al.  Identification of the cysteine residue in the active site of horse liver mitochondrial aldehyde dehydrogenase. , 1988, The Journal of biological chemistry.

[3]  H. True,et al.  A yeast prion provides a mechanism for genetic variation and phenotypic diversity , 2000, Nature.

[4]  H. Weiner,et al.  Differences in the roles of conserved glutamic acid residues in the active site of human class 3 and class 2 aldehyde dehydrogenases , 1999, Protein science : a publication of the Protein Society.

[5]  H. Weiner,et al.  Involvement of glutamate 268 in the active site of human liver mitochondrial (class 2) aldehyde dehydrogenase as probed by site-directed mutagenesis. , 1995, Biochemistry.

[6]  S. Vollenweider,et al.  3-Hydroxypropionaldehyde: applications and perspectives of biotechnological production , 2004, Applied Microbiology and Biotechnology.

[7]  C. Kim,et al.  Stimulation of reductive glycerol metabolism by overexpression of an aldehyde dehydrogenase in a recombinant Klebsiella pneumoniae strain defective in the oxidative pathway , 2011, Journal of Industrial Microbiology & Biotechnology.

[8]  K. Shimizu,et al.  Co-production of 3-hydroxypropionic acid and 1,3-propanediol by Klebseilla pneumoniae expressing aldH under microaerobic conditions. , 2013, Bioresource technology.

[9]  Vinod Kumar,et al.  Recent advances in biological production of 3-hydroxypropionic acid. , 2013, Biotechnology advances.

[10]  S. Sheikh,et al.  Involvement of Glutamate 399 and Lysine 192 in the Mechanism of Human Liver Mitochondrial Aldehyde Dehydrogenase* , 1997, The Journal of Biological Chemistry.

[11]  Johnathan E. Holladay,et al.  Top Value Added Chemicals From Biomass. Volume 1 - Results of Screening for Potential Candidates From Sugars and Synthesis Gas , 2004 .

[12]  Chelladurai Rathnasingh,et al.  Effect of process parameters on 3-hydroxypropionic acid production from glycerol using a recombinant Escherichia coli , 2009, Applied Microbiology and Biotechnology.

[13]  Vinod Kumar,et al.  Aldehyde dehydrogenase activity is important to the production of 3-hydroxypropionic acid from glycerol by recombinant Klebsiella pneumoniae , 2012 .

[14]  H. Weiner,et al.  Horse liver aldehyde dehydrogenase. II. Kinetics and mechanistic implications of the dehydrogenase and esterase activity. , 1972, The Journal of biological chemistry.

[15]  M. Holtzapple,et al.  Effect of biodiesel glycerol type and fermentor configuration on mixed-acid fermentations. , 2010, Bioresource technology.

[16]  K. Shimizu,et al.  Simultaneous production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol by a recombinant strain of Klebsiella pneumoniae. , 2012, Bioresource technology.

[17]  W. Wackernagel,et al.  Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. , 1995, Gene.

[18]  Samir Kumar Khanal,et al.  Biodiesel-derived crude glycerol bioconversion to animal feed: a sustainable option for a biodiesel refinery. , 2011, Bioresource technology.

[19]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[20]  H. Weiner,et al.  Horse liver aldehyde dehydrogenase. I. Purification and characterization. , 1972, The Journal of biological chemistry.

[21]  R. H. Hall,et al.  96. Acid-catalysed hydration of acraldehyde. Kinetics of the reaction and isolation of β-hydroxypropaldehyde , 1950 .

[22]  Chelladurai Rathnasingh,et al.  Cloning, expression, and characterization of an aldehyde dehydrogenase from Escherichia coli K-12 that utilizes 3-Hydroxypropionaldehyde as a substrate , 2008, Applied Microbiology and Biotechnology.

[23]  K. L. Smiley,et al.  Production of 3-Hydroxypropionaldehyde from Glycerol , 1983, Applied and environmental microbiology.

[24]  L. Jarboe.  YqhD: a broad-substrate range aldehyde reductase with various applications in production of biorenewable fuels and chemicals , 2010, Applied Microbiology and Biotechnology.

[25]  I. Berregi,et al.  Glycerol metabolism and bitterness producing lactic acid bacteria in cidermaking. , 2008, International journal of food microbiology.

[26]  Nicole A. Leal,et al.  PduP is a coenzyme-a-acylating propionaldehyde dehydrogenase associated with the polyhedral bodies involved in B12-dependent 1,2-propanediol degradation by Salmonella enterica serovar Typhimurium LT2 , 2003, Archives of Microbiology.

[27]  S. Sheikh,et al.  The Potential Roles of the Conserved Amino Acids in Human Liver Mitochondrial Aldehyde Dehydrogenase* , 1997, The Journal of Biological Chemistry.

[28]  Vinod Kumar,et al.  Production of 3‐hydroxypropionic acid from glycerol by recombinant Klebsiella pneumoniae ΔdhaTΔyqhD which can produce vitamin B12 naturally , 2013, Biotechnology and bioengineering.

[29]  Seonghun Kim,et al.  Production of 3-hydroxypropionic acid through propionaldehyde dehydrogenase PduP mediated biosynthetic pathway in Klebsiella pneumoniae. , 2012, Bioresource technology.

[30]  G. Bennett,et al.  Enhancement of lactate and succinate formation in adhE or pta‐ackA mutants of NADH dehydrogenase‐deficient Escherichia coli , 2005, Journal of applied microbiology.

[31]  Jin-Ho Seo,et al.  Biosynthesis of 3-hydroxypropionic acid from glycerol in recombinant Escherichia coli expressing Lactobacillus brevis dhaB and dhaR gene clusters and E. coli K-12 aldH. , 2013, Bioresource technology.

[32]  S. M. Raj,et al.  Production of 3-hydroxypropionic acid from glycerol by a novel recombinant Escherichia coli BL21 strain , 2008 .