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Metabolic engineering applications of the Escherichia coli bacterial artificial chromosome
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.ORCID iD: 0000-0002-7916-4731
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. Univ Mayor San Simon, Ctr Biotechnol, Fac Sci & Technol, Cochabamba, Bolivia..ORCID iD: 0000-0001-6501-9886
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.ORCID iD: 0000-0001-5319-7511
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.ORCID iD: 0000-0002-3314-6060
2019 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 305, p. 43-50Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
ELSEVIER , 2019. Vol. 305, p. 43-50
Keywords [en]
3-Hydroxybutyrate, Chromosomal expression, Pathway optimization, F plasmid, Bacterial artificial chromosome, Metabolic engineering
National Category
Industrial Biotechnology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-261932DOI: 10.1016/j.jbiotec.2019.09.002ISI: 000487564300007PubMedID: 31505217Scopus ID: 2-s2.0-85072060887OAI: oai:DiVA.org:kth-261932DiVA, id: diva2:1361261
Note

QC 20191015

Available from: 2019-10-15 Created: 2019-10-15 Last updated: 2020-05-05Bibliographically approved
In thesis
1. 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_r0Ns_mQkT5ujvMv0h4fb9Q, Stockholm, 14:00 (English)
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Note

QC 2020-05-07

Available from: 2020-05-07 Created: 2020-05-05 Last updated: 2020-05-25Bibliographically approved

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Sjöberg, GustavGuevara-Martínez, Mónicavan Maris, Antonius J. A.Gustavsson, Martin

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