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Cultivation strategies for production of (R)-3-hydroxybutyric acid from simultaneous consumption of glucose, xylose and arabinose by Escherichia coli
KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.ORCID-id: 0000-0002-7916-4731
KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi. Universidad Mayor de San Simón, Bolivia .
Visa övriga samt affilieringar
2015 (Engelska)Ingår i: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 14, nr 1, s. 51-Artikel i tidskrift (Refereegranskat) Published
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.

Ort, förlag, år, upplaga, sidor
BioMed Central, 2015. Vol. 14, nr 1, s. 51-
Nyckelord [en]
Escherichia coli, 3-Hydroxybutyric acid, 3HB, simultaneous uptake, lignocellulose, production process, nitrogen limitation
Nationell ämneskategori
Biologiska vetenskaper
Forskningsämne
Bioteknologi
Identifikatorer
URN: urn:nbn:se:kth:diva-166385DOI: 10.1186/s12934-015-0236-2ISI: 000353259300001Scopus ID: 2-s2.0-84928231166OAI: oai:DiVA.org:kth-166385DiVA, id: diva2:810678
Anmärkning

QC 20150508

Tillgänglig från: 2015-05-08 Skapad: 2015-05-08 Senast uppdaterad: 2019-05-08Bibliografiskt granskad
Ingår i avhandling
1. Strategies for improved Escherichia coli bioprocessing performance
Öppna denna publikation i ny flik eller fönster >>Strategies for improved Escherichia coli bioprocessing performance
2015 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Escherichia coli has a proven track record for successful production of anything from small molecules like organic acids to large therapeutic proteins, and has thus important applications in both R&D and commercial production. The versatility of this organism in combination with the accumulated knowledge of its genome, metabolism and physiology, has allowed for development of specialty strains capable of performing very specific tasks, opening up opportunities within new areas. The work of this thesis has been devoted to alter membrane transport proteins and the regulation of these, in order for E. coli to find further application within two such important areas.

The first area was vaccine development, where it was investigated if E. coli could be a natural vehicle for live vaccine production. The hypothesis was that the introduction and manipulation of a protein surface translocation system from pathogenic E. coli would result in stable expression levels of Salmonella subunit antigens on the surface of laboratory E. coli. While different antigen combinations were successfully expressed on the surface of E. coli, larger proteins were affected by proteolysis, which manipulation of cultivation conditions could reduce, but not eliminate completely. The surface expressed antigens were further capable of inducing proinflammatory responses in epithelial cells.

The second area was biorefining. By altering the regulation of sugar assimilation, it was hypothesized that simultaneous uptake of the sugars present in lignocellulose hydrolyzates could be achieved, thereby improving the yield and productivity of important bio-based chemicals. The dual-layered catabolite repression was identified and successfully removed in the engineered E. coli, and the compound (R)-3-hydroxybutyric acid was produced from simultaneous assimilation of glucose, xylose and arabinose.

Ort, förlag, år, upplaga, sidor
Stockholm: KTH Royal Institute of Technology, 2015. s. 104
Serie
TRITA-BIO-Report, ISSN 1654-2312 ; 2015:9
Nyckelord
E. coli, Salmonella, surface expression, autotransport, AIDA-I, lignocellulose, glucose, xylose, arabinose, simultaneous uptake, 3HB
Nationell ämneskategori
Biologiska vetenskaper
Forskningsämne
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-166387 (URN)978-91-7595-523-0 (ISBN)
Disputation
2015-06-05, FB52, AlbaNova universitetscentrum, Roslagstullsbacken 21, KTH, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Anmärkning

QC 20150508

Tillgänglig från: 2015-05-08 Skapad: 2015-05-08 Senast uppdaterad: 2015-05-08Bibliografiskt granskad
2. Metabolic engineering and cultivation strategies for recombinant production of (R)-3-hydroxybutyrate
Öppna denna publikation i ny flik eller fönster >>Metabolic engineering and cultivation strategies for recombinant production of (R)-3-hydroxybutyrate
2019 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

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.

Ort, förlag, år, upplaga, sidor
KTH Royal Institute of Technology, 2019. s. 106
Serie
TRITA-CBH-FOU ; 2019:20
Nyckelord
Escherichia coli, (R)-3-hydroxybutyrate, nitrogen limitation, nitrogen depletion, lignocellulose, fed batch, acetate, β-ketothiolase, acetoacetyl-CoA reductase, Halomonas boliviensis.
Nationell ämneskategori
Teknik och teknologier
Forskningsämne
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-251048 (URN)978-91-7873-216-6 (ISBN)
Disputation
2019-06-05, FD5, AlbaNova, Roslagstullsbacken 21, SE-11421, Stockholm, Sweden, Stockholm, 14:00 (Engelska)
Opponent
Handledare
Forskningsfinansiär
Sida - Styrelsen för internationellt utvecklingssamarbete, 70828
Anmärkning

QC 2019-05-08

Tillgänglig från: 2019-05-08 Skapad: 2019-05-08 Senast uppdaterad: 2019-05-09Bibliografiskt granskad

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Jarmander, JohanBelotserkovsky, JaroslavSjöberg, GustavGuevara-Martínez, MónicaZabaleta, Mariel PerezLarsson, Gen
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