ATP drives direct photosynthetic production of 1-butanol in cyanobacteria.

Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA 90095, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 04/2012; 109(16):6018-23. DOI: 10.1073/pnas.1200074109
Source: PubMed

ABSTRACT While conservation of ATP is often a desirable trait for microbial production of chemicals, we demonstrate that additional consumption of ATP may be beneficial to drive product formation in a nonnatural pathway. Although production of 1-butanol by the fermentative coenzyme A (CoA)-dependent pathway using the reversal of β-oxidation exists in nature and has been demonstrated in various organisms, the first step of the pathway, condensation of two molecules of acetyl-CoA to acetoacetyl-CoA, is thermodynamically unfavorable. Here, we show that artificially engineered ATP consumption through a pathway modification can drive this reaction forward and enables for the first time the direct photosynthetic production of 1-butanol from cyanobacteria Synechococcus elongatus PCC 7942. We further demonstrated that substitution of bifunctional aldehyde/alcohol dehydrogenase (AdhE2) with separate butyraldehyde dehydrogenase (Bldh) and NADPH-dependent alcohol dehydrogenase (YqhD) increased 1-butanol production by 4-fold. These results demonstrated the importance of ATP and cofactor driving forces as a design principle to alter metabolic flux.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Butanol is a promising next generation fuel and a bulk chemical precursor. Although clostridia are the primary industrial microbes for the fermentative production of 1-butanol, alternative engineered hosts have the potential to generate 1-butanol from alternative carbon feedstocks via synthetic metabolic pathways. Methylobacterium extorquens AM1, a facultative methylotrophic α-proteobacterium, is a model system for assessing the possibility of generating products such as 1-butanol from one-carbon and two-carbon feedstocks. Moreover, the core methylotrophic pathways in M. extorquens AM1 involve unusual coenzyme A (CoA)-derivative metabolites, such as crotonyl-CoA, which is a precursor for the production of 1-butanol.
    Biotechnology for Biofuels 01/2014; 7(1):156. · 6.22 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Background Recent efforts demonstrated the potential application of cyanobacteria as a ¿microbial cell factory¿ to produce butanol directly from CO2. However, cyanobacteria have very low tolerance to the toxic butanol, which limits the economic viability of this renewable system.ResultsThrough a long-term experimental evolution process, we achieved a 150% increase of the butanol tolerance in a model cyanobacterium Synechocystis sp. PCC 6803 after a continuous 94 passages for 395 days in BG11 media amended with gradually increased butanol concentration from 0.2% to 0.5% (v/v). To decipher the molecular mechanism responsible for the tolerance increase, we employed an integrated GC-MS and LC-MS approach to determine metabolomic profiles of the butanol-tolerant Synechocystis strains isolated from several stages of the evolution, and then applied PCA and WGCNA network analyses to identify the key metabolites and metabolic modules related to the increased tolerance. The results showed that unstable metabolites of 3-phosphoglyceric acid (3PG), D-fructose 6-phosphate (F6P), D-glucose 6-phosphate (G6P), NADPH, phosphoenolpyruvic acid (PEP), D-ribose 5-phosphate (R5P), and stable metabolites of glycerol, L-serine and stearic acid were differentially regulated during the evolution process, which could be related to tolerance increase to butanol in Synechocystis.Conclusions The study provided the first time-series description of the metabolomic changes related to the gradual increase of butanol tolerance, and revealed a metabolomic basis important for rational tolerance engineering in Synechocystis.
    Microbial Cell Factories 11/2014; 13(1):151. · 4.25 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Flavonoid metabolism and its fascinating molecules that are natural products in plants, have attracted the attention of industry and researchers involved in plant science, nutrition, bio/chemistry, chemical bioengineering, pharmacy, medicine, etc., since flavonoids were found to be directly or indirectly connected to health. Subsequently, in the last few years flavonoids became top stories in pharmaceutical industry, which is continually seeking for novel ways to produce safe and efficient drugs. Microbial cell cultures can act as workhorse bio-factories by offering their metabolic machinery for the benefit of optimizing the conditions and increasing the productivity of a selective flavonoid. Furthermore, metabolic engineering methodology came to reinforce what nature does best by tuning inadequacies and dead-ends of a metabolic pathway. Combinatorial biosynthesis techniques led to discovery of novel ways to produce plant natural and even unnatural flavonoids, while on top of that metabolic engineering gave the opportunity to industry to invest in synthetic biology to overcome restricted diversification and productivity issues existing so far in synthetic chemistry protocols. In this review, we present an update on rationalized approaches for the production of natural or unnatural flavonoids through biotechnology, analyzing the significance of combinatorial biosynthesis of agricultural/ pharmaceutical compounds produced in heterologous organisms. We also quote strategies and achievements thrived so far in the area of synthetic biology, with emphasis on metabolic engineering targeting the cellular optimization of microorganisms and plants producing flavonoids, stressing the advances in flux dynamic control and optimization. The involvement of the rapidly increasing numbers of assembled genomes that contribute to the gene- or pathway- mining to identify gene(s) responsible for producing species-specific secondary metabolites is finally considered.
    Frontiers in Plant Science 01/2015; 6. · 3.64 Impact Factor


Available from