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  • 1.
    Guevara-Martínez, Mónica
    et al.
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi. Univ Mayor de San Simon, Fac Sci & Technol, Ctr Biotechnol.
    Sjöberg Gällnö, Karin
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Sjöberg, Gustav
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Jarmander, Johan
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Perez-Zabaleta, Mariel
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi. Univ Mayor de San Simon, Fac Sci & Technol, Ctr Biotechnol.
    Quillaguamán, Jorge
    Larsson, Gen
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Regulating the production of (R)-3-hydroxybutyrate in Escherichia coli by N or P limitation2015Ingår i: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 6, artikel-id 844Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The chiral compound (R)-3-hydroxybutyrate (3HB) is naturally produced by many wild type organisms as the monomer for polyhydroxybutyrate (PHB). Both compounds are commercially valuable and co-polymeric polyhydroxyalkanoates have been used e.g., in medical applications for skin grafting and as components in pharmaceuticals. In this paper we investigate cultivation strategies for production of 3HB in the previously described E. coil strain AF1000 pJBGT3RX. This strain produces extracellular 3HB by expression of two genes from the PHB pathway of Halomonas boliviensis. H. boliviensis is a newly isolated halophile that forms PHB as a storage compound during carbon excess and simultaneous limitation of another nutrient like nitrogen and phosphorous. We hypothesize that a similar approach can be used to control the flux from acetylCoA to 3HB also in E coli; decreasing the flux to biomass and favoring the pathway to the product. We employed ammonium- or phosphate-limited fed-batch processes for comparison of the productivity at different nutrient limitation or starvation conditions. The feed rate was shown to affect the rate of glucose consumption, respiration, 3HB, and acetic acid production, although the proportions between them were more difficult to affect. The highest 3HB volumetric productivity, 1.5 g L-1 h(-1), was seen for phosphate-limitation.

  • 2.
    Jarmander, Johan
    et al.
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Belotserkovsky, Jaroslav
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Sjöberg, Gustav
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Guevara-Martínez, Mónica
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi. Universidad Mayor de San Simón, Bolivia .
    Zabaleta, Mariel Perez
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi. Universidad Mayor de San Simón, Bolivia .
    Quillaguaman, Jorge
    Universidad Mayor de San Simón, Bolivia .
    Larsson, Gen
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Cultivation strategies for production of (R)-3-hydroxybutyric acid from simultaneous consumption of glucose, xylose and arabinose by Escherichia coli2015Ingår i: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 14, nr 1, s. 51-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background

    Lignocellulosic waste is a desirable biomass for use in second generation biorefineries. Up to 40 % of its sugar content consist of pentoses, which organisms either take up sequentially after glucose depletion, or not at all. A previously described Escherichia coli strain, PPA652ara, capable of simultaneous consumption of glucose, xylose and arabinose was in the present work utilized for production of (R)-3-hydroxybutyric acid (3HB) from a mixture of glucose, xylose and arabinose.

    Results

    The Halomonas boliviensis genes for 3HB production were for the first time cloned into E. coli PPA652ara leading to product secretion directly into the medium. Process design was based on comparisons of batch, fed-batch and continuous cultivation, where both excess and limitation of the carbon mixture was studied. Carbon limitation resulted in low specific productivity of 3HB (< 2 mg g-1 h-1) compared to carbon excess (25 mg g-1 h-1), but the yield of 3HB/cell dry weight (Y3HB/CDW) was very low (0.06 g g-1)during excess. Nitrogen-exhausted conditions could be used to sustain a high specific productivity (31 mg g-1 h-1) and to increase the yield of 3HB/cell dry weight to 1.38 g g-1. Nitrogen-limited fed-batch process design lead to further increased specific productivity (38 mg g-1 h-1) but also to additional cell growth (Y3HB/CDW = 0.16 g g-1). Strain PPA652ara did under all processing conditions simultaneously consume glucose, xylose and arabinose, which was not the case for a reference wild type E. coli, which also gave a higher carbon flux to acetic acid.

    Conclusions

    It was demonstrated that by using the strain E. coli PPA652ara it was possible to design a production process for 3HB from a mixture of glucose, xylose and arabinose where all sugars were consumed. An industrial 3HB production process is proposed to be divided into a growth and a production phase, and nitrogen depletion/limitation is a potential strategy to maximize the yield of 3HB/CDW in the latter. The specific productivity of 3HB by E. coli reported here from glucose, xylose and arabinose is further comparable to the current state of the art for production of 3HB from glucose sources.

  • 3.
    Jarmander, Johan
    et al.
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Guevara, Mónica
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Zabaleta, Mariel Perez
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Sjöberg, Gustav
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Belotserkovsky, Jaroslav
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Quillaguaman, Jorge
    Larsson, Gen
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Production of 3-hydroxybutyrate from waste biomass by metabolically engineered Escherichia coli2014Ingår i: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 31, s. S94-S95Artikel i tidskrift (Övrigt vetenskapligt)
  • 4.
    Perez-Zabaleta, Mariel
    et al.
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Sjöberg, Gustav
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Guevara-Martínez, Mónica
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi. Universidad Mayor de San Simón, Bolivia.
    Jarmander, Johan
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Gustavsson, Martin
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Quillaguamán, Jorge
    Larsson, Gen
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Increasing the production of (R)-3-hydroxybutyrate in recombinant Escherichia coli by improved cofactor supply2016Ingår i: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 15, nr 1, artikel-id 91Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: In a recently discovered microorganism, Halomonas boliviensis, polyhydroxybutyrate production was extensive and in contrast to other PHB producers, contained a set of alleles for the enzymes of this pathway. Also the monomer, (R)-3-hydroxybutyrate (3HB), possesses features that are interesting for commercial production, in particular the synthesis of fine chemicals with chiral specificity. Production with a halophilic organism is however not without serious drawbacks, wherefore it was desirable to introduce the 3HB pathway into Escherichia coli. Results: The production of 3HB is a two-step process where the acetoacetyl-CoA reductase was shown to accept both NADH and NADPH, but where the V-max for the latter was eight times higher. It was hypothesized that NADPH could be limiting production due to less abundance than NADH, and two strategies were employed to increase the availability; (1) glutamate was chosen as nitrogen source to minimize the NADPH consumption associated with ammonium salts and (2) glucose-6-phosphate dehydrogenase was overexpressed to improve NADPH production from the pentose phosphate pathway. Supplementation of glutamate during batch cultivation gave the highest specific productivity (q(3HB) = 0.12 g g(-1) h(-1)), while nitrogen depletion/zwf overexpression gave the highest yield (Y-3HB/CDW = 0.53 g g(-1)) and a 3HB concentration of 1 g L-1, which was 50 % higher than the reference. A nitrogen-limited fedbatch process gave a concentration of 12.7 g L-1 and a productivity of 0.42 g L-1 h(-1), which is comparable to maximum values found in recombinant E. coli. Conclusions: Increased NADPH supply is a valuable tool to increase recombinant 3HB production in E. coli, and the inherent hydrolysis of CoA leads to a natural export of the product to the medium. Acetic acid production is still the dominating by-product and this needs attention in the future to increase the volumetric productivity further.

  • 5.
    Sjöberg, Gustav
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Guevara-Martínez, Mónica
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Gustavsson, Martin
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    van Maris, Antonius J. A.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Metabolic engineering applications of the Escherichia coli bacterial artificial chromosomeManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    In metabolic engineering and synthetic biology, the number of genes expressed to achieve better production and pathway regulation in each strain is steadily increasing. The method of choice for expression in Escherichia coli is usually one or several multi-copy plasmids. Meanwhile, the industry standard for long-term, robust production is chromosomal integration of the desired genes. Despite recent advances, genetic manipulation of the bacterial chromosome remains more time consuming than plasmid construction. To allow screening of different metabolic engineering strategies at a level closer to industry while maintaining the molecular-biology advantages of plasmid-based expression, we have investigated the single-copy bacterial artificial chromosome (BAC) as a development tool for metabolic engineering. Using (R)-3 hydroxybutyrate as a model product, we show that BAC can outperform multi-copy plasmids in terms of yield, productivity and specific growth rate, with respective increases of 12%, 18%, and 5%. We both show that gene expression by the BAC simplifies pathway optimization and that the phenotype of pathway expression from BAC is very close to that of chromosomal expression. From these results, we conclude that the BAC can provide a simple platform for performing pathway design and optimization. 

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