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  • 1.
    Guevara-Martínez, Mónica
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
    Strain- and bioprocess-design strategies to increase production of (R)-3-hydroxybutyrate by Escherichia coli2019Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Microbial bio-based processes have emerged as an alternative to replace fossil-based processes for the production of fuels and chemicals. (R)-3-hydroxybutyrate (3HB) is a medium-value chemical that has gained special attention as a precursor of antibiotics and vitamins, as a monomer for the synthesis of tailor-made polyesters and as a nutritional source for eukaryotic cells. By integrating strain and bioprocess-design strategies the work of this thesis has aimed to improve microbial 3HB production by the well-studied platform organism Escherichia coli (strain AF1000) expressing a thiolase and a reductase from Halomonas boliviensis.

    Uncoupling growth and product formation by NH4+- or PO43-- limited fed-batch cultivations allowed for 3HB titers of 4.1 and 6.8 g L-1 (Paper I). Increasing the NADPH supply by overexpression of glucose-6-phosphate dehydrogenase (zwf) resulted in 1.7 times higher 3HB yield compared to not overexpressing zwf in NH4depleted conditions (Paper II). To increase 3HB production in high-cell density cultures, strain BL21 was selected as a low acetate-forming, 3HB-producing platform. BL21 grown in NH4limited fed-batch cultivations resulted in 2.3 times higher 3HB titer (16.3 g L-1) compared to strain AF1000 (Paper III). Overexpression of the native E. coli thioesterase “yciA”, identified as the largest contributor in 3HB-CoA hydrolysis, resulted in 2.6 times higher 3HB yield compared to AF1000 not overexpressing yciA. Overexpressing zwf and yciA in NH4depleted fed-batch experiments resulted in 2 times higher total 3HB yield (0.210 g g-1) compared to AF1000 only overexpressing zwf (Paper IV)Additionally, using 3HB as a model product, the bacterial artificial chromosome was presented as a simple platform for performing pathway design and optimization in E. coli (Paper V)While directly relevant for 3HB production, these findings also contribute to the knowledge on how to improve the production of a chemical for the development of robust and scalable processes.

  • 2.
    Guevara-Martínez, Mónica
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. Faculty of Science and Technology, Center of Biotechnology, Universidad Mayor de San Simón, Cochabamba, Bolivia.
    Perez-Zabaleta, Mariel
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. Faculty of Science and Technology, Center of Biotechnology, Universidad Mayor de San Simón, Cochabamba, Bolivia.
    Gustavsson, Martin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Quillaguamán, Jorge
    Faculty of Science and Technology, Center of Biotechnology, Universidad Mayor de San Simón, Cochabamba, Bolivia.
    Larsson, Gen
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT. KTH, Superseded Departments (pre-2005), Biotechnology.
    van Maris, Antonius J. A.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    The role of the acyl-CoA thioesterase YciA in the production of (R)-3-hydroxybutyrate by recombinant Escherichia coli2019In: Applied Microbiology and Biotechnology, p. 1-12Article in journal (Refereed)
    Abstract [en]

    Biotechnologically produced (R)-3-hydroxybutyrate is an interesting pre-cursor for antibiotics, vitamins, and other molecules benefitting from enantioselective production. An often-employed pathway for (R)-3-hydroxybutyrate production in recombinant E. coli consists of three-steps: (1) condensation of two acetyl-CoA molecules to acetoacetyl-CoA, (2) reduction of acetoacetyl-CoA to (R)-3-hydroxybutyrate-CoA, and (3) hydrolysis of (R)-3-hydroxybutyrate-CoA to (R)-3-hydroxybutyrate by thioesterase. Whereas for the first two steps, many proven heterologous candidate genes exist, the role of either endogenous or heterologous thioesterases is less defined. This study investigates the contribution of four native thioesterases (TesA, TesB, YciA, and FadM) to (R)-3-hydroxybutyrate production by engineered E. coli AF1000 containing a thiolase and reductase from Halomonas boliviensis. Deletion of yciA decreased the (R)-3-hydroxybutyrate yield by 43%, whereas deletion of tesB and fadM resulted in only minor decreases. Overexpression of yciA resulted in doubling of (R)-3-hydroxybutyrate titer, productivity, and yield in batch cultures. Together with overexpression of glucose-6-phosphate dehydrogenase, this resulted in a 2.7-fold increase in the final (R)-3-hydroxybutyrate concentration in batch cultivations and in a final (R)-3-hydroxybutyrate titer of 14.3 g L-1 in fed-batch cultures. The positive impact of yciA overexpression in this study, which is opposite to previous results where thioesterase was preceded by enzymes originating from different hosts or where (S)-3-hydroxybutyryl-CoA was the substrate, shows the importance of evaluating thioesterases within a specific pathway and in strains and cultivation conditions able to achieve significant product titers. While directly relevant for (R)-3-hydroxybutyrate production, these findings also contribute to pathway improvement or decreased by-product formation for other acyl-CoA-derived products.

  • 3.
    Guevara-Martínez, Mónica
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Univ Mayor de San Simon, Fac Sci & Technol, Ctr Biotechnol.
    Sjöberg Gällnö, Karin
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Sjöberg, Gustav
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Jarmander, Johan
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Perez-Zabaleta, Mariel
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Univ Mayor de San Simon, Fac Sci & Technol, Ctr Biotechnol.
    Quillaguamán, Jorge
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Regulating the production of (R)-3-hydroxybutyrate in Escherichia coli by N or P limitation2015In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 6, article id 844Article in journal (Refereed)
    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.

  • 4.
    Perez-Zabaleta, Mariel
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
    Guevara-Martínez, Mónica
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
    Gustavsson, Martin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
    Quillaguamán, Jorge
    Larsson, Gen
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
    van Maris, Antonius J. A.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
    Comparison of engineered Escherichia coli AF1000 and BL21 strains for (R)-3-hydroxybutyrate production in fed-batch cultivation2019In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 103, no 14, p. 5627-5636Article in journal (Refereed)
    Abstract [en]

    Accumulation of acetate is a limiting factor in recombinant production of (R)-3-hydroxybutyrate (3HB) by E. coli in high-cell-density processes. To alleviate this limitation, this study investigated two approaches: (i) Deletion of phosphotransacetylase (pta), pyruvate oxidase (poxB) and/or the isocitrate-lyase regulator (iclR), known to decrease acetate formation, on bioreactor cultivations designed to achieve high 3HB concentrations. (ii) Screening of different E. coli strain backgrounds (B, BL21, W, BW25113, MG1655, W3110 and AF1000) for their potential as low acetate-forming, 3HB-producing platforms. Deletion of pta and pta-poxB in the AF1000 strain background was to some extent successful in decreasing acetate formation, but also dramatically increased excretion of pyruvate and did not result in increased 3HB production in high-cell-density fed-batch cultivations. Screening of the different E. coli strains confirmed BL21 as a low acetate forming background. Despite low 3HB titers in low-cell density screening, 3HB-producing BL21 produced 5 times less acetic acid per mol of 3HB, which translated into a 2.3-fold increase in the final 3HB titer and a 3-fold higher volumetric 3HB productivity over 3HB-producing AF1000 strains in nitrogen-limited fed-batch cultivations. Consequently, the BL21 strain achieved the hitherto highest described volumetric productivity of 3HB (1.52 g L-1 h-1) and the highest 3HB concentration (16.3 g L-1) achieved by recombinant E. coli. Screening solely for 3HB titers in low-cell-density batch cultivations would not have identified the potential of this strain, reaffirming the importance of screening with the final production conditions in mind.

  • 5.
    Perez-Zabaleta, Mariel
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Sjöberg, Gustav
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Guevara-Martínez, Mónica
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Universidad Mayor de San Simón, Bolivia.
    Jarmander, Johan
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Gustavsson, Martin
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Quillaguamán, Jorge
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Increasing the production of (R)-3-hydroxybutyrate in recombinant Escherichia coli by improved cofactor supply2016In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 15, no 1, article id 91Article in journal (Refereed)
    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.

  • 6.
    Sjöberg, Gustav
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Guevara-Martínez, Mónica
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Gustavsson, Martin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    van Maris, Antonius J. A.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Metabolic engineering applications of the Escherichia coli bacterial artificial chromosomeManuscript (preprint) (Other academic)
    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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