Inducible gene expression system by 3-hydroxypropionic acid

Zhou, Shengfang; Ainala, Satish Kumar; Seol, Eunhee; Nguyen, Trinh Thi My; Park, Sunghoon

doi:10.1186/s13068-015-0353-5

Cited by 29 publications

(28 citation statements)

References 31 publications

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“…Moreover, the enzymatic assay not only confirmed that the reductive route for 3-HP assimilation in M. extorquens AM1 was through acrylyl-CoA as intermediates, but also suggested that the genes encoding enzymes for the reductive route were unlikely able to be induced by 3-HP. The latter finding differed with the previous observation that the degradation of 3-HP by the oxidative route in P. denitrificans was increased greatly when cells were exposed to 3-HP [ 47 ]. In that study, the transcription of hpdh and hbdh involved in the oxidative route was found to be induced by a LysR-type transcriptional regulator binding with 3-HP molecule.…”

Section: Discussioncontrasting

confidence: 99%

Production of 3-hydroxypropionic acid in engineered Methylobacterium extorquens AM1 and its reassimilation through a reductive route

et al. 2017

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Background3-Hydroxypropionic acid (3-HP) is an important platform chemical, serving as a precursor for a wide range of industrial applications such as the production of acrylic acid and 1,3-propanediol. Although Escherichia coli or Saccharomyces cerevisiae are the primary industrial microbes for the production of 3-HP, alternative engineered hosts have the potential to generate 3-HP from other carbon feedstocks. Methylobacterium extorquens AM1, a facultative methylotrophic α-proteobacterium, is a model system for assessing the possibility of generating 3-HP from one-carbon feedstock methanol.ResultsHere we constructed a malonyl-CoA pathway by heterologously overexpressing the mcr gene to convert methanol into 3-HP in M. extorquens AM1. The engineered strains demonstrated 3-HP production with initial titer of 6.8 mg/l in shake flask cultivation, which was further improved to 69.8 mg/l by increasing the strength of promoter and mcr gene copy number. In vivo metabolic analysis showed a significant decrease of the acetyl-CoA pool size in the strain with the highest 3-HP titer, suggesting the supply of acetyl-CoA is a potential bottleneck for further improvement. Notably, 3-HP was rapidly degraded after the transition from exponential phase to stationary phase. Metabolomics analysis showed the accumulation of intracellular 3-hydroxypropionyl-CoA at stationary phase with the addition of 3-HP into the cultured medium, indicating 3-HP was first converted to its CoA derivatives. In vitro enzymatic assay and β-alanine pathway dependent 13C-labeling further demonstrated that a reductive route sequentially converted 3-HP-CoA to acrylyl-CoA and propionyl-CoA, with the latter being reassimilated into the ethylmalonyl-CoA pathway. The deletion of the gene META1_4251 encoding a putative acrylyl-CoA reductase led to reduced degradation rate of 3-HP in late stationary phase.ConclusionsWe demonstrated the feasibility of constructing the malonyl-CoA pathway in M. extorquens AM1 to generate 3-HP. Furthermore, we showed that a reductive route coupled with the ethylmalonyl-CoA pathway was the major channel responsible for degradation of the 3-HP during the growth transition. Engineered M. extorquens AM1 represents a good platform for 3-HP production from methanol.Electronic supplementary materialThe online version of this article (10.1186/s12934-017-0798-2) contains supplementary material, which is available to authorized users.

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Section: Discussioncontrasting

confidence: 99%

Production of 3-hydroxypropionic acid in engineered Methylobacterium extorquens AM1 and its reassimilation through a reductive route

et al. 2017

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“…RT-PCR was performed in a 48-well StepOne real-time PCR system (Thermo Fisher Scientific), and rpoD was used as an endogenous control. The experiment was performed in duplicate, and relative mRNA quantification was performed according to the Δ C T method [ 35 ].…”

Section: Methodsmentioning

confidence: 99%

Co-production of hydrogen and ethanol by pfkA-deficient Escherichia coli with activated pentose-phosphate pathway: reduction of pyruvate accumulation

Sekar

Seol

Raj

et al. 2016

Biotechnol Biofuels

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BackgroundFermentative hydrogen (H2) production suffers from low carbon-to-H2 yield, to which problem, co-production of ethanol and H2 has been proposed as a solution. For improved co-production of H2 and ethanol, we developed Escherichia coli BW25113 ΔhycA ΔhyaAB ΔhybBC ΔldhA ΔfrdAB Δpta-ackA ΔpfkA (SH8*) and overexpressed Zwf and Gnd, the key enzymes in the pentose-phosphate (PP) pathway (SH8*_ZG). However, the amount of accumulated pyruvate, which was significant (typically 0.20 mol mol−1 glucose), reduced the co-production yield.ResultsIn this study, as a means of reducing pyruvate accumulation and improving co-production of H2 and ethanol, we developed and studied E. coli SH9*_ZG with functional acetate production pathway for conversion of acetyl-CoA to acetate (pta-ackA+). Our results indicated that the presence of the acetate pathway completely eliminated pyruvate accumulation and substantially improved the co-production of H2 and ethanol, enabling yields of 1.88 and 1.40 mol, respectively, from 1 mol glucose. These yields, significantly, are close to the theoretical maximums of 1.67 mol H2 and 1.67 mol ethanol. To better understand the glycolytic flux distribution, glycolytic flux prediction and RT-PCR analyses were performed.ConclusionThe presence of the acetate pathway along with activation of the PP pathway eliminated pyruvate accumulation, thereby significantly improving co-production of H2 and ethanol. Our strategy is applicable to anaerobic production of biofuels and biochemicals, both of which processes demand high NAD(P)H.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0510-5) contains supplementary material, which is available to authorized users.

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“…3-HPA is a highly reactive compound and reacts with side chains of various amino acids, including those of Cys, Lys, His, and Arg 63 , 64 . Chemical modifications of the side chains can result in the loss of enzyme activity.…”

Section: Resultsmentioning

confidence: 99%

Engineering an aldehyde dehydrogenase toward its substrates, 3-hydroxypropanal and NAD+, for enhancing the production of 3-hydroxypropionic acid

Park

Choi

Nam

et al. 2017

Sci Rep

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3-Hydroxypropionic acid (3-HP) can be produced via the biological route involving two enzymatic reactions: dehydration of glycerol to 3-hydroxypropanal (3-HPA) and then oxidation to 3-HP. However, commercial production of 3-HP using recombinant microorganisms has been hampered with several problems, some of which are associated with the toxicity of 3-HPA and the efficiency of NAD+ regeneration. We engineered α-ketoglutaric semialdehyde dehydrogenase (KGSADH) from Azospirillum brasilense for the second reaction to address these issues. The residues in the binding sites for the substrates, 3-HPA and NAD+, were randomized, and the resulting libraries were screened for higher activity. Isolated KGSADH variants had significantly lower Km values for both the substrates. The enzymes also showed higher substrate specificities for aldehyde and NAD+, less inhibition by NADH, and greater resistance to inactivation by 3-HPA than the wild-type enzyme. A recombinant Pseudomonas denitrificans strain with one of the engineered KGSADH variants exhibited less accumulation of 3-HPA, decreased levels of inactivation of the enzymes, and higher cell growth than that with the wild-type KGSADH. The flask culture of the P. denitrificans strain with the mutant KGSADH resulted in about 40% increase of 3-HP titer (53 mM) compared with that using the wild-type enzyme (37 mM).

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Inducible gene expression system by 3-hydroxypropionic acid

Cited by 29 publications

References 31 publications

Production of 3-hydroxypropionic acid in engineered Methylobacterium extorquens AM1 and its reassimilation through a reductive route

Production of 3-hydroxypropionic acid in engineered Methylobacterium extorquens AM1 and its reassimilation through a reductive route

Co-production of hydrogen and ethanol by pfkA-deficient Escherichia coli with activated pentose-phosphate pathway: reduction of pyruvate accumulation

Engineering an aldehyde dehydrogenase toward its substrates, 3-hydroxypropanal and NAD+, for enhancing the production of 3-hydroxypropionic acid

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