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Engineering short-chain carboxylic-acid metabolism in the model microorganism Escherichia coli
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.ORCID iD: 0000-0002-7916-4731
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 [en]
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: urn:nbn:se:kth:diva-273006ISBN: 978-91-7873-502-0 (print)OAI: oai:DiVA.org:kth-273006DiVA, id: diva2:1428318
Public defence
2020-05-29, https://kth-se.zoom.us/webinar/register/WN_r0Ns_mQkT5ujvMv0h4fb9Q, Stockholm, 14:00 (English)
Opponent
Supervisors
Note

QC 2020-05-07

Available from: 2020-05-07 Created: 2020-05-05 Last updated: 2020-05-25Bibliographically approved
List of papers
1. Cultivation strategies for production of (R)-3-hydroxybutyric acid from simultaneous consumption of glucose, xylose and arabinose by Escherichia coli
Open this publication in new window or tab >>Cultivation strategies for production of (R)-3-hydroxybutyric acid from simultaneous consumption of glucose, xylose and arabinose by Escherichia coli
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2015 (English)In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 14, no 1, p. 51-Article in journal (Refereed) 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.

Place, publisher, year, edition, pages
BioMed Central, 2015
Keywords
Escherichia coli, 3-Hydroxybutyric acid, 3HB, simultaneous uptake, lignocellulose, production process, nitrogen limitation
National Category
Biological Sciences
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-166385 (URN)10.1186/s12934-015-0236-2 (DOI)000353259300001 ()25889969 (PubMedID)2-s2.0-84928231166 (Scopus ID)
Note

QC 20150508

Available from: 2015-05-08 Created: 2015-05-08 Last updated: 2020-05-05Bibliographically approved
2. Characterization of volatile fatty acid utilization in Escherichia coli aiming for robust valorisation of food residues
Open this publication in new window or tab >>Characterization of volatile fatty acid utilization in Escherichia coli aiming for robust valorisation of food residues
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Valorisation of food residues would greatly benefit from development of robust processes that create added value compared to current feed- and biogas applications. Recent advances in membrane-bioreactor-based open mixed microbial cultures, enable robust conversion of fluctuating streams of food residues to a mixture of volatile fatty acids (VFAs). In this study, such a mixed stream of VFAs was investigated as a substrate for Escherichia coli, a well studied organism suitable for application in further conversion of the acids into compounds of higher value, and/or that are easier to separate from the aqueous medium. E. coli was cultured in batch on a VFA-rich anaerobic digest of food residues, tolerating up to 40 mM of total VFAs without any reduction in growth rate. In carbon-limited chemostats of E. coli W3110 ΔFadR on a simulated VFA mixture, the straight chain VFAs (C2-C6) in the mixture were readily consumed simultaneously. At the dilution rate 0.1 h-1, mainly acetic-, propionic- and caproic acid were consumed, while consumption of all the provided acids were observed at 0.05 h-1. Interestingly, also the branched isovaleric acid was consumed through a hitherto unknown mechanism. In total, up to 80% of the carbon supplied from VFAs was consumed by the cells, and approximately 2.7% was excreted as nucleotide precursors in the medium. These results suggest that VFAs derived from food residues are a promising substrate for E. coli.

Keywords
Volatile fatty acids, VFA, Food waste, Escherichia coli, Anaerobic digest
National Category
Industrial Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-273004 (URN)
Note

QC 20200506

Available from: 2020-05-05 Created: 2020-05-05 Last updated: 2020-05-06Bibliographically approved
3. Regulating the production of (R)-3-hydroxybutyrate in Escherichia coli by N or P limitation
Open this publication in new window or tab >>Regulating the production of (R)-3-hydroxybutyrate in Escherichia coli by N or P limitation
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2015 (English)In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 6, article id 844Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Frontiers Research Foundation, 2015
Keywords
E. coil, 3-hydroxybutyrate (3HB), polyhydroxybutyrate (PHB), fed-batch, phosphate, ammonium, limitation, depletion
National Category
Microbiology
Identifiers
urn:nbn:se:kth:diva-173438 (URN)10.3389/fmicb.2015.00844 (DOI)000360116800001 ()2-s2.0-84941053409 (Scopus ID)
Funder
Swedish Research Council FormasSida - Swedish International Development Cooperation Agency
Note

QC 20150918

Available from: 2015-09-18 Created: 2015-09-11 Last updated: 2020-05-05Bibliographically approved
4. Increasing the production of (R)-3-hydroxybutyrate in recombinant Escherichia coli by improved cofactor supply
Open this publication in new window or tab >>Increasing the production of (R)-3-hydroxybutyrate in recombinant Escherichia coli by improved cofactor supply
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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
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
Keywords
Escherichia coli, Halomonas boliviensis, (R)-3-hydroxybutyrate, Acetoacetyl-CoA reductase, NADPH, zwf overexpression, Glutamate, Nitrogen limitation
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-189084 (URN)10.1186/s12934-016-0490-y (DOI)000377167900001 ()27245326 (PubMedID)2-s2.0-84971577878 (Scopus ID)
Note

QC 20160808

Available from: 2016-08-08 Created: 2016-06-27 Last updated: 2020-05-05Bibliographically approved
5. Metabolic engineering applications of the Escherichia coli bacterial artificial chromosome
Open this publication in new window or tab >>Metabolic engineering applications of the Escherichia coli bacterial artificial chromosome
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
Keywords
3-Hydroxybutyrate, Chromosomal expression, Pathway optimization, F plasmid, Bacterial artificial chromosome, Metabolic engineering
National Category
Industrial Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-261932 (URN)10.1016/j.jbiotec.2019.09.002 (DOI)000487564300007 ()31505217 (PubMedID)2-s2.0-85072060887 (Scopus ID)
Note

QC 20191015

Available from: 2019-10-15 Created: 2019-10-15 Last updated: 2020-05-05Bibliographically approved

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