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Increasing the production of (R)-3-hydroxybutyrate in recombinant Escherichia coli by improved cofactor supply
KTH, School of Biotechnology (BIO), Industrial Biotechnology.ORCID iD: 0000-0003-3873-4977
KTH, School of Biotechnology (BIO), Industrial Biotechnology.ORCID iD: 0000-0002-7916-4731
KTH, School of Biotechnology (BIO), Industrial Biotechnology. Universidad Mayor de San Simón, Bolivia.ORCID iD: 0000-0001-6501-9886
KTH, School of Biotechnology (BIO), Industrial Biotechnology. (Industrial Biotechnology)
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2016 (English)In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 15, no 1, article id 91Article in journal (Refereed) Published
Resource type
Text
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.

Place, publisher, year, edition, pages
Springer, 2016. Vol. 15, no 1, article id 91
Keywords [en]
Escherichia coli, Halomonas boliviensis, (R)-3-hydroxybutyrate, Acetoacetyl-CoA reductase, NADPH, zwf overexpression, Glutamate, Nitrogen limitation
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-189084DOI: 10.1186/s12934-016-0490-yISI: 000377167900001PubMedID: 27245326Scopus ID: 2-s2.0-84971577878OAI: oai:DiVA.org:kth-189084DiVA, id: diva2:951344
Note

QC 20160808

Available from: 2016-08-08 Created: 2016-06-27 Last updated: 2020-05-05Bibliographically approved
In thesis
1. Metabolic engineering and cultivation strategies for recombinant production of (R)-3-hydroxybutyrate
Open this publication in new window or tab >>Metabolic engineering and cultivation strategies for recombinant production of (R)-3-hydroxybutyrate
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2019. p. 106
Series
TRITA-CBH-FOU ; 2019:20
Keywords
Escherichia coli, (R)-3-hydroxybutyrate, nitrogen limitation, nitrogen depletion, lignocellulose, fed batch, acetate, β-ketothiolase, acetoacetyl-CoA reductase, Halomonas boliviensis.
National Category
Engineering and Technology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-251048 (URN)978-91-7873-216-6 (ISBN)
Public defence
2019-06-05, FD5, AlbaNova, Roslagstullsbacken 21, SE-11421, Stockholm, Sweden, Stockholm, 14:00 (English)
Opponent
Supervisors
Funder
Sida - Swedish International Development Cooperation Agency, 70828
Note

QC 2019-05-08

Available from: 2019-05-08 Created: 2019-05-08 Last updated: 2019-05-09Bibliographically approved
2. Strain- and bioprocess-design strategies to increase production of (R)-3-hydroxybutyrate by Escherichia coli
Open this publication in new window or tab >>Strain- and bioprocess-design strategies to increase production of (R)-3-hydroxybutyrate by Escherichia coli
2019 (English)Doctoral 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.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2019. p. 100
Series
TRITA-CBH-FOU ; 2019:25
Keywords
E. coli, (R)-3-hydroxybutyrate, metabolic engineering, bioprocess design, NADPH, acetic acid, thioesterase, BAC
National Category
Industrial Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-251096 (URN)978-91-7873-205-0 (ISBN)
Public defence
2019-06-03, F3, Lindstedtsvägen 26, Sing-Sing, våningsplan 2, KTH Campus, Stockholm, 14:00 (English)
Opponent
Supervisors
Funder
Sida - Swedish International Development Cooperation AgencySwedish Research Council Formas
Note

QC 2019-05-09

Available from: 2019-05-09 Created: 2019-05-09 Last updated: 2019-05-09Bibliographically approved
3. Engineering short-chain carboxylic-acid metabolism in the model microorganism Escherichia coli
Open this publication in new window or tab >>Engineering short-chain carboxylic-acid metabolism in the model microorganism Escherichia coli
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The ever-increasing concern about carbon dioxide emissions has created an urgent need to develop alternative methods to cheaply and renewably produce materials, chemicals and fuels. The biorefinery is uniquely suited to deliver these products from sustainable biomass. However, cheaply and efficiently converting the dispersed, heterogenous and recalcitrant biomass to useful products requires further technical development. To address some of these challenges, the aim of this thesis was to investigate methods to improve the economic viability of the microbial biorefinery by evaluating short chain carboxylic acids as substrates (volatile fatty acids) and products ((R)-3-hydroxybutyrate, 3HB).

Initially, two renewable and cheap sources of carbon were investigated as substrates for E. coli. It was determined that E. coli is a suitable microorganism for valorization of volatile fatty acids derived from food waste. Also, it was shown that lignocellulosic sugars with a composition based on a hydrolysate of wheat straw can be converted to 3HB in E. coli with similar yields and productivities as from pure glucose. To improve the yield of the model product 3HB, and thereby the potential gross profit, substrate depletion was used as a strategy throughout the thesis to control bioprocesses. Specifically, nutrient depletion was shown to decouple growth from 3HB production in nitrogen and phosphorous depleted batches, increasing the yield of 3HB. To further improve 3HB production, metabolic engineering was used to improve the availability of NADPH. Additionally, the bacterial artificial chromosome (BAC) was investigated as a robust single-copy vector for metabolic engineering in E. coli. The expression of a large operon from the BAC was shown to be comparable to chromosomal expression. Then, the specific growth rate, productivity and yield of 3HB producing strains was increased by expression of the 3HB production pathway from the BAC instead of a multi-copy plasmid. Finally, the BAC was shown to be a useful tool for the optimization of enzyme expression levels in metabolic pathways. While directly beneficial for 3HB production, the methods and strategies employed in this thesis are broadly applicable to increase the economic viability of microbial biorefineries.

Abstract [sv]

Koldioxidutsläppens påverkan på klimatet utgör en av vår tids största utmaningar och det finns ett omedelbart behov av att utveckla kostnadseffektiva produktionsmetoder för framställning av förnyelsebara material, kemikalier och bränslen från biomassa. Här spelar bioraffinaderier en unikt viktig roll. Biomassan är dock geografiskt utspridd med heterogena egenskaper och svår att bryta ned. Det krävs tekniska framsteg för att utveckla en konkurrenskraftig produktion. Syftet med denna avhandling var att undersöka metoder för att öka det mikrobiella bioraffinaderiets ekonomiska bärkraft, med fokus på korta karboxylsyror som substrat (volatila fettsyror) och produkter ((R)-3- hydroxybutyrate, 3HB).

Initialt undersöktes två prismässigt konkurrenskraftiga och förnyelsebara kolkällor som substrat för E. coli. Det slogs fast att E. coli är lämpad för valorisering av volatila fettsyror framställda från matavfall. Dessutom bevisades det att lignocellulosabaserade sockerarter, med en sammansättning motsvarande ett vetestråhydrolysat, kan konverteras till 3HB i E. coli med utbyte och produktivitet motsvarande de från ren glukos. För att öka utbytet av modellprodukten 3HB, och den potentiella bruttovinsten, användes substratbegränsning för att styra bioprocesser genomgående i avhandlingen. Det kunde uttryckligen påvisas att det var det möjligt att koppla loss 3HB-produktion från tillväxt i kväve- och fosforbegränsade batch-odlingar, och därmed öka 3HB-utbytet. För att ytterligare öka 3HB-produktionen användes metabolic engineering för att öka tillgången på NADPH. Dessutom undersöktes den bakteriella artificiella kromosomen (BAC) för användning i metabolic engineering av E. coli, då det är en robust vektor som replikerar med kopietal 1–2. Genom att överföra ett stort operon från kromosomen till BAC gick det att bevisa att genuttrycket motsvarar kromosomalt integrerade gener. Därefter visades det att den specifika tillväxthastigheten, produktiviteten och utbytet av en 3HB-producerande stam kunde ökas genom uttryck av 3HB-gener från BAC istället för en plasmid med medelhögt kopietal. Slutligen kunde det fastställas att BAC är ett användbart verktyg för att optimera nivåer av enzymuttryck i metabola reaktionsvägar. Metoderna och strategierna som användes i avhandlingen bidrog till förbättrad 3HB produktion, men de kan också tillämpas i ett bredare sammanhang och därmed öka möjligheten för mikrobiella bioraffinaderier att förbättra den ekonomiska bärkraften.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2020. p. 57
Series
TRITA-CBH-FOU ; 2020:19
Keywords
metabolic engineering, (R)-3-hydroxybutyrate, volatile fatty acids, bioprocess design, Escherichia coli, bacterial artificial chromosome
National Category
Natural Sciences Industrial Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-273006 (URN)978-91-7873-502-0 (ISBN)
Public defence
2020-05-29, https://kth-se.zoom.us/webinar/register/WN_BfA8UJa9TpyPf7oprzShDA, Stockholm, 14:00 (English)
Opponent
Supervisors
Note

QC 2020-05-07

Available from: 2020-05-07 Created: 2020-05-05 Last updated: 2020-05-12Bibliographically approved

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Perez-Zabaleta, MarielGuevara-Martínez, MónicaGustavsson, Martin

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