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- Guevara-Martínez, Mónica, 1989-, et al.
(författare)
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The role of the acyl-CoA thioesterase YciA in the production of (R)-3-hydroxybutyrate by recombinant Escherichia coli
- 2019
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Ingår i: Applied Microbiology and Biotechnology. - : Springer. - 0175-7598 .- 1432-0614. ; , s. 1-12
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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- Perez-Zabaleta, Mariel, 1987-, et al.
(författare)
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Comparison of engineered Escherichia coli AF1000 and BL21 strains for (R)-3-hydroxybutyrate production in fed-batch cultivation
- 2019
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Ingår i: Applied Microbiology and Biotechnology. - : Springer. - 0175-7598 .- 1432-0614. ; 103:14, s. 5627-5636
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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- Perez-Zabaleta, Mariel, 1987-
(författare)
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Metabolic engineering and cultivation strategies for recombinant production of (R)-3-hydroxybutyrate
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Metabolic engineering and process engineering are two powerful disciplines to design and improve microbial processes for sustainable production of an extensive number of compounds ranging from chemicals to pharmaceuticals. The aim of this thesis was to synergistically combine these two disciplines to improve the production of a model chemical called (R)-3-hydroxybutyrate (3HB), which is a medium-value product with a stereocenter and two functional groups. These features make 3HB an interesting building block, especially for the pharmaceutical industry. Recombinant production of 3HB was achieved by expression of two enzymes from Halomonas boliviensis in the model microorganism Escherichia coli, which is a microbial cell factory with proven track record and abundant knowledge on its genome, metabolism and physiology.Investigations on cultivation strategies demonstrated that nitrogen-depleted conditions had the biggest impact on 3HB yields, while nitrogen-limited cultivations predominantly increased 3HB titers and volumetric productivities. To further increase 3HB production, metabolic engineering strategies were investigated to decrease byproduct formation, enhance NADPH availability and improve the overall 3HB-pathway activity. Overexpression of glucose-6-phosphate dehydrogenase (zwf) increased cofactor availability and together with the overexpression of acyl-CoA thioesterase YciA resulted in a 2.7-fold increase of the final 3HB concentration, 52% of the theoretical product yield and a high specific productivity (0.27 g g-1 h-1). In a parallel strategy, metabolic engineering and process design resulted in an E. coli BL21 strain with the hitherto highest reported volumetric 3HB productivity (1.52 g L-1 h-1) and concentration (16.3 g L-1) using recombinant production. The concepts developed in this thesis can be applied to industrial 3HB production processes, but also advance the knowledge base to benefit design and expansion of the product range of biorefineries.
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