Improved p-hydroxybenzoate production by engineered Pseudomonas putida S12 by using a mixed-substrate feeding strategy

Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
Applied Microbiology and Biotechnology (Impact Factor: 3.81). 02/2011; 90(3):885-93. DOI: 10.1007/s00253-011-3089-6
Source: PubMed

ABSTRACT The key precursors for p-hydroxybenzoate production by engineered Pseudomonas putida S12 are phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P), for which the pentose phosphate (PP) pathway is an important source. Since PP pathway fluxes are typically low in pseudomonads, E4P and PEP availability is a likely bottleneck for aromatics production which may be alleviated by stimulating PP pathway fluxes via co-feeding of pentoses in addition to glucose or glycerol. As P. putida S12 lacks the natural ability to utilize xylose, the xylose isomerase pathway from E. coli was introduced into the p-hydroxybenzoate producing strain P. putida S12palB2. The initially inefficient xylose utilization was improved by evolutionary selection after which the p-hydroxybenzoate production was evaluated. Even without xylose-co-feeding, p-hydroxybenzoate production was improved in the evolved xylose-utilizing strain, which may indicate an intrinsically elevated PP pathway activity. Xylose co-feeding further improved the p-hydroxybenzoate yield when co-fed with either glucose or glycerol, up to 16.3 Cmol% (0.1 g p-hydroxybenzoate/g substrate). The yield improvements were most pronounced with glycerol, which probably related to the availability of the PEP precursor glyceraldehyde-3-phosphate (GAP). Thus, it was demonstrated that the production of aromatics such as p-hydroxybenzoate can be improved by co-feeding different carbon sources via different and partially artificial pathways. Moreover, this approach opens new perspectives for the efficient production of (fine) chemicals from renewable feedstocks such as lignocellulose that typically has a high content of both glucose and xylose and (crude) glycerol.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Background Achieving a sustainable society requires, among other things, the use of renewable feedstocks to replace chemicals obtained from petroleum-derived compounds. Crude glycerol synthesized inexpensively as a byproduct of biodiesel production is currently considered a waste product, which can potentially be converted into value-added compounds by bacterial fermentation. This study aimed at evaluating several characterized P. putida strains to produce medium-chain-length poly(3-hydroxyalkanoates) (mcl-PHA) using raw glycerol as the only carbon/energy source.ResultsAmong all tested strains, P. putida KT2440 most efficiently synthesized mcl-PHA under nitrogen-limiting conditions, amassing more than 34% of its cell dry weight as PHA. Disruption of the PHA depolymerase gene (phaZ) in P. putida KT2440 enhanced the biopolymer titer up to 47% PHA (%wt/wt). The low biomass and PHA titer found in the mutant strain and the wild-type strain KT2440 seems to be triggered by the high production of the side-product citrate during the fermentation process which shows a high yield of 0.6 g/g.Conclusions Overall, this work demonstrates the importance of choosing an appropriate microbe for the synthesis of mcl-PHA from waste materials, and a close inspection of the cell metabolism in order to identify undesired compounds that diminish the availability of precursors in the synthesis of biopolymers such as polyhydroxyalkanoates. Future metabolic engineering works should focus on reducing the production of citrate in order to modulate resource allocation in the cell¿s metabolism of P. putida, and finally increase the biopolymer production.
    BMC Biotechnology 12/2014; 14(1):110. DOI:10.1186/s12896-014-0110-z · 2.59 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Carotenoids, a subfamily of terpenoids, are yellow- to red-colored pigments synthesized by plants, fungi, algae, and bacteria. They are ubiquitous in nature and take over crucial roles in many biological processes as for example photosynthesis, vision, and the quenching of free radicals and singlet oxygen. Due to their color and their potential beneficial effects on human health, carotenoids receive increasing attention. Carotenoids can be classified due to the length of their carbon backbone. Most carotenoids have a C40 backbone, but also C30 and C50 carotenoids are known. All carotenoids are derived from isopentenyl pyrophosphate (IPP) as a common precursor. Pathways leading to IPP as well as metabolic engineering of IPP synthesis and C40 carotenoid production have been reviewed expertly elsewhere. Since C50 carotenoids are synthesized from the C40 carotenoid lycopene, we will summarize common strategies for optimizing lycopene production and we will focus our review on the characteristics, biosynthesis, glycosylation, and overproduction of C50 carotenoids.
    Applied Microbiology and Biotechnology 04/2014; 98(10). DOI:10.1007/s00253-014-5693-8 · 3.81 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Background Production of monoterpenoids as valuable chemicals using recombinant microbes is a growing field of interest. Unfortunately, antimicrobial activity of most monoterpenoids hampers a wide application of microorganisms for their production. Strains of Pseudomonas putida, a fast growing and metabolically versatile bacterium, often show an outstanding high tolerance towards organic solvents and other toxic compounds. Therefore, Pseudomonas putida constitutes an attractive alternative host in comparison to conventionally used microorganisms. Here, metabolic engineering of solvent tolerant Pseudomonas putida as a novel microbial cell factory for de novo production of monoterpenoids is reported for the first time, exemplified by geranic acid production from glycerol as carbon source. The monoterpenoic acid is an attractive compound for application in the flavor, fragrance, cosmetics and agro industries.ResultsA comparison between Escherichia coli, Saccharomyces cerevisiae and Pseudomonas putida concerning the ability to grow in the presence of geranic acid revealed that the pseudomonad bears a superior resilience compared to the conventionally used microbes. Moreover, Pseudomonas putida DSM 12264 wildtype strain efficiently oxidized externally added geraniol to geranic acid with no further degradation. Omitting external dosage of geraniol but functionally expressing geraniol synthase (GES) from Ocimum basilicum, a first proof-of-concept for de novo biosynthesis of 1.35 mg/L geranic acid in P. putida DSM 12264 was achieved. Doubling the amount of glycerol resulted in twice the amount of product. Co-expression of the six genes of the mevalonate pathway from Myxococcus xanthus to establish flux from acetyl-CoA to the universal terpenoid precursor isopentenylpyrophosphate yielded 36 mg/L geranic acid in shake flask experiments. In the bioreactor, the recombinant strain produced 193 mg/L of geranic acid under fed-batch conditions within 48 h.Conclusion Metabolic engineering turned Pseudomonas putida DSM 12264, a versatile monoterpenoid oxidation biocatalyst, into an efficient microbial cell factory for de novo geranic acid production. Improvements by metabolic and process engineering are expected to further increase the product concentration. To the best of the authors¿ knowledge, this is the first example of a de novo production of a monoterpenoid with Pseudomonas putida and of a microbial monoterpenoic acid synthesis in general.
    Microbial Cell Factories 12/2014; 13(1):170. DOI:10.1186/s12934-014-0170-8 · 4.25 Impact Factor

Full-text (4 Sources)

Available from
May 26, 2014