Transport of Dicarboxylic Acids in Saccharomyces cerevisiae

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Transport of Dicarboxylic Acids in

Saccharomyces cerevisiae

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Transport of Dicarboxylic Acids in

Saccharomyces cerevisiae

PROEFSCHRIFT

ter verkrijging van de graad van doctor aan de Technische Universiteit Delft,

op gezag van de Rector Magnificus Prof. ir. K.C.A.M. Luyben, voorzitter van het College voor Promoties,

in het openbaar te verdedigen op maandag 18 november 2013 om 15:00 uur

door

Elaheh JAMALZADEH

Master of Science in Chemical Engineering (Biotechnology) Iranian University of Science and Technology, Iran

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proefschrift is goedgekeurd door de promotor: Prof. dr. ir. J.J. Heijnen

Copromotor: Dr. W.M. van Gulik

Samenstelling promotiecommissie:

Rector Magnificus Voorzitter

Prof. dr. Ir. J.J. Heijnen Technische Universiteit Delft, promotor Dr. Ir. W.M. van Gulik Technische Universiteit Delft Prof. dr. Ir. H.J. Noorman Technische Universiteit Delft

Prof. dr. E. Heinzle University of Saarland

Prof. dr. Ing. H.C.M. Reuss University of Stuttgart

Dr. Ir. L.H. de Graaf Wageningen University

Dr. M.L.A. Jansen DSM Biotechnology Center

Prof. Dr. J.T. Pronk Technische Universiteit Delft, Reservelid

The research presented in this thesis was performed at the Cell Systems engineering group, Department of Biotechnology, Faculty of Applied Sciences, Delft University of Technology (The Netherlands).

This project is financially supported by the Netherlands Ministry of Economic Affairs and the B-Basic partner organizations (www.b-basic.nl) through B-Basic, a public-private NWO-ACTS program (NWO-ACTS = Advanced Chemical Technologies for Sustainability). This project was carried out within the research programme of Kluyver Center for Genomics of Industrial Fermentation which is part of the Netherlands Genomics Initiative / Netherlands organization for Scientific Research.

ISBN: 978-90-8891-734-9

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to my parents

to Ali

to Tara

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vii

Acknowledgements

This thesis would not have been possible without supports and contributions by many people who deserve special mention.

In the first place, I am sincerely thankful to Sef Heijnen, my promoter, for giving me the opportunity to work in CSE group, for his supervision, and his constant guidance during my PhD, as well as encouragement and advice for choosing my career after PhD. Dear Sef, I was very lucky to be your PhD student. Thank you for advising me to follow my career in industry. I learnt a lot about yeast physiology during my PhD, and now I am enjoying every day working with yeast to make it tasty.

I am grateful to Walter van Gulik, my daily supervisor and co-promoter, for his support on scientific aspects, practical work, and writing. Thank you Walter for looking critically at my reports and data and helping me to become an independent researcher. I don’t forget the days I ran from lab to your office asking for help. You always listened to me calmly and helped me to solve the problems. Thank you for translating the summary of thesis.

I am very thankful to Peter Verheijen for his support on modeling and data analysis. I learnt a lot about statistics and modeling while I was working with you on my first publication. I also enjoyed our non-scientific discussions during coffee breaks and sharing ideas for propositions since the beginning of PhD. Thank you for translating my propositions.

I sincerely thank Prof. Heinzle, Prof. Reuss, Dr. de Graaf, Dr. Jansen, Prof. Noorman, and Prof. Pronk for accepting to be part of my PhD defense committee and for their valuable comments on my thesis.

I also thank Adrie Straathof, Cagri Efe, and Carol Roa Engel for their collaboration in dicarboxylic acid project and for exchanging ideas and valuable discussions during the B-Basic meetings.

Before I start my PhD at former BPT group (CSE), I joined the PDEng program as guest and could join many courses at Biotechnology Department, while applying for PhD position. I should thank Janine Kiers for giving me the opportunity to apply for PDEng program in the summer of 2006, though after the deadline and arranging the interview session very quick during the holiday period. I also thank Lies van der Meer at PDEng office for her support. I started the PDEng program with a group of Dutch and international students (Anne Marieke, Magdalena R., Magdalena S., Yeoson, Leila, and Nirmal). It was great knowing you all and I enjoyed working with you. You helped me feel at home in the first months of my arrival in the Netherlands.

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I spent five great years at the Department of Biotechnology in room 2.513 in which I shared the office with Rutger, Marija, Sreekanth, and Rob and later with Aljoscha, and Camilo. Thanks for the great atmosphere and fruitful discussions. Special thank to Rob (Brooijmans) for 1.5 years collaborations on fumaric acid project. You started as postdoc in our group in the beginning of 2009. We could test the strains you constructed by combining modeling and genetic engineering tools and performed adaptive evolution experiment. The results of our work are published in Chapter 5 of this thesis.

I would like to thank Hilal and Amin for their collaboration on performing the thermodynamic feasibility study for dicarboxylic acid production. The results of our work are published as book chapter and in Chapter 2 of this thesis.

I had a chance to supervise some students with their final thesis during my PhD. Marilia and Shirmey, the results of your work are published in Chapter 4 of this thesis. It was a great pleasure working with you. Marilia, you did a great job during your master’s thesis and managed to formulate those difficult thermodynamic equations for transport perfectly. Many thanks to Rudy for his hard work in the lab and performing more than 6 chemostat experiments in such a short time during Bachelor internship. You were a fast learner and very precise in the lab and I could easily rely on your data. The results of your work are published in Chapter 5 of this thesis. Last but not the least, I thank Edwin for performing validation experiments for leakage of metabolites in anaerobic conditions under supervision of Luisa and I. I wish you good luck with your PhD thesis.

I could not perform and complete my experiments without technical support and proper analysis. I thank the technical team, Rob Kerste, Dirk Geerts, and Mario Pronk, for their support and help in the fermentation lab. Rob, I still use your advice, the inoculation dance, for enzymatic hydrolysis at work. I am very thankful to the analysis team Jan van Dam, Reza Maleki Seifar, Cor Ras, Angela ten Pierick, Zhen Zheng, and Patricia van Dam for their support during my PhD. I also thank Johan for performing TOC measurements.

Jenifer, thanks a lot for your administrative support. It is amazing how efficient you work and how fast you respond to our emails and calls and help the group running.

I also thank Sjaak for his support for lab requirements and also for creating nice atmosphere in the building by organizing many beautiful events together with his team.

Thanks to all BPT members who I had the chance to know them during my PhD and some of them became my good friends and colleagues at DSM. Andre, Emrah, Hilal, Rutger, Lodewijk, Zheng, Katelijne, Luisa, Amit, Camilo, Hugo, Mihir, Fredrik, Marco, Sergio, Domenico, and Marjan; Thanks for your help in rapid samplings and good discussions in the group meetings.

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Aknowledgments ix

I thank my DSP friends Maria, Rosario, Carol, Cagri, Sreekanth, Beckley, Kedar, David, Camilo, and Sushil (our rapid sampling member) for sharing and celebrating good moments at the coffee table.

I would like to thank my friends Luisa, Hilal, Katelijne, Fanni, Irina, and Lucie for the beautiful hours we spent together in Delft every month. I gained a lot of positive energy every time I met you girls.

Thanks to all my Iranian friends who I had the opportunity to know them in the Netherlands and to DISAA members for all the great times we spent together; with special thanks to Fatemeh & Amir Abbas, Ehsan & Maryam, Mehdi & Somayeh, Nasim & Farzad, Sanaz & Hamed, and Laleh & Poorya.

I am very thankful to Peter and Anja for helping us to improve our Dutch language, interesting discussions about the Dutch culture and Delft history, and their supports.

Finally I wish to express my love and gratitude to my family members back in Iran, for their love and courage; my parents, Aydin Jamalzadeh and Fatemeh Azarang, who raised me, couraged me to study, prayed for me, and supported me; my husband, Ali, who has always stood by me, believed me, and helped me; and Tara, our little star, who brought us laughter, joy, and new prospects in life. To them I dedicate this thesis.

Delft, November 2013 Elaheh Jamalzadeh

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xi

Summary

The production of chemicals from renewable feedstocks using microorganisms receives considerable attention due to the ever-increasing possibilities of genetic engineering and the contribution to decreasing CO2 emissions. C-4 dicarboxylic acids (succinic, fumaric, and

malic) are mentioned as top 12 chemical building blocks manufactured from bio-feed-stocks. Production of organic acids such as fumaric acid and succinic acid is preferably performed in absence of oxygen, leading to theoretical production yields, and at low pH (lower than pKa value of acids) in order to reduce the recovery costs (Chapter 2). However, in current naturally producing bacterial strains, fermentations are performed at neutral pH, requiring the addition of calcium carbonate, which leads to formation of byproducts during the recovery and downstream processing. The yeast Saccharomyces cerevisiae is an organism that is able to grow at low pH (Chapter 3). Additional advantages of this organism (as model organism) are that its genome has been fully sequenced and it has been proposed, in several recent studies, as cell factory for the production of bulk chemicals from renewable feedstocks (Chapter 3).

An important aspect of organic acid production in microbial cell factories is the export of the product over the cell membrane with a sufficient capacity. High extracellular organic acid concentrations are required for economical production, leading to export of the produced acid over the cellular membrane against a concentration gradient, which can only be accomplished via energy requiring active transport mediated by specific proteins in the cell membrane. The crucial question is the amount of energy that those transport processes need in order to be accomplished. Energetics of fumaric acid and succinic acid export under low cultivation pH has been studied theoretically using a thermodynamic approach and flux analysis in this thesis (Chapter 2) and based on the obtained results metabolic engineering strategies for microbial production of these carboxylic acids are suggested. Based on theoretical calculations, the most likely export mechanism for dicarboxylic acids at low cultivation pH is a Fum2-_/proton

antiporter, importing one proton for each fumarate anion exported.

Considering antiport mechanisms for export of succinic acid and fumaric acid and translating the Gibbs energy of succinic acid and fumaric acid formation from glucose to the corresponding ATP requirement, we concluded that succinic acid can be produced at neutral as well as at low cultivation pH under anaerobic conditions. However, according to our analysis fumaric acid can only be produced under anaerobic conditions at neutral pH, while at low pH the fermentation must be carried out under aerobic conditions due to the ATP requirement for fumaric acid export.

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It should be realized, however, that in a fermentation process carried out at low pH, the organic acid is predominantly present in the undissociated form. Active export of a dicarboxylic acid from the cells into the extracellular environment will thus result in a steep concentration gradient of the undissociated form over the plasma membrane. Assuming that the plasma membrane is permeable for the undissociated species, this will create a futile cycle of active export and diffusion back into the cells at the expense of metabolic energy (ATP). Uptake of fumaric acid was measured at different cultivation pH in a unique experimental set-up (step experiment) in Chapter 3 under anaerobic conditions and in Chapter 4 under aerobic conditions, both in glucose limited chemostats. The cell physiology (aerobic and anaerobic) did not significantly change at pH range of 3.5 to 5.0, but at lower pH (< 3.5) lower cell growth occurred, possibly due to H+_{cycling. Under anaerobic conditions the}

fumaric acid concentration in the feed, as well as the initial concentration in the fermentation broth, was increased stepwise. It was found that the uptake rate of fumaric acid increased at decreasing cultivation pH, indicating that the undissociated form is most likely the main species that is taken up by the yeast cells. From the observation that the fumaric acid uptake profile versus the undissociated acid concentration was asymmetrical, it was concluded that uptake of fumaric acid is likely to be facilitated by two or more membrane transporter proteins with a maximum capacity and different affinities (Chapter 3). Fumaric acid metabolism was observed under these conditions from the lower level of fumaric acid in the total broth. The metabolism of fumaric acid can occur via the mal-ethanolic pathway, which might be activated at presence of high level of fumaric acid.

Fumaric acid step experiments under aerobic conditions in presence of 1 mmol.L-1 _fumaric

acid in the feed medium showed that fumaric acid uptake increased at decreasing pH, which would indicate that the undissociated form of the acid is likely to be the main transported species. However, the uptake rates were much lower than in a similar experiment under anaerobic conditions. Besides, at pH 5, where the fraction of undissociated acid is negligible, there was still fumaric acid uptake measured. This means that the diffusion coefficient is by far higher than reported values for other carboxylic acids such as malic acid. It is unlikely that passive diffusion (especially at pH 5.0) is the only transport mechanism and it is therefore likely that the import is mediated by a membrane transporter protein (Chapter 4).

Fumaric acid uptake resulted in a significant increase in concentrations of other TCA-cycle acids in total broth, while the intracellular concentrations increased and remained unchanged when the cultivation pH was decreased further. This might indicate that fumaric acid was converted, after entering the cytosol, to other organic acids, where after these compounds were secreted from the cells. Based on theoretical calculations, a transporter is involved in transport of these organic acids and it seems to be an H+_{-symporter and possibly a reversible}

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Summary xiii

In Chapter 5, metabolic flux analysis using a yeast whole genome model was used to select potential genetic engineering targets to achieve ATP-based stoichiometrically coupled fumaric acid production to growth under aerobic conditions. Two deletion mutants were constructed. In the first mutant the FUM1 gene encoding Fumarase and in the second mutant the FUM1 and ZWF1 genes (encoding G6P dehydrogenase) were deleted. These strains were tested in glucose-limited chemostats under aerobic conditions. The observed biomass and fumaric acid yields were much lower than the model predictions, because ethanol was the unexpected dominant produced metabolite and not fumaric acid. In order to increase the fumaric acid productivity (which leads to highest ATP production), the double deletion mutant was chosen for a further evolution experiment that was carried out in a continuous mode for 300 days. However the fumaric acid titer did not increase but acetate was produced instead at very high level. Analysis suggests that evolution selected for a pathway with much more ATP production per mole glucose, as expected. Accumulation of fumaric acid inside the cells indicates that an effective fumaric acid exporter might not be present and moreover the selective pressure for evolution was decreased due to futile cycling of acetic acid, which leads to ATP dissipation. Recommendations for process and strain improvement are to introduce a fumarate exporter.

We have focused on transport of succinic acid in Chapters 6 and 7. The physiology of wild type yeast (S.cerevisiae) was studied under low pH (3.0) fermentation conditions (Chapter 6). We investigated the possible presence of succinic acid futile cycling at the presence of a high level of succinic acid in the fermenter (227 mmol.L-1_{) as will occur in the production scale.}

This was studied by a so-called succinic acid wash-in experiment where the succinic acid level in the fermenter was increased gradually after exchanging the feed medium with an identical medium containing 254 mmol.L-1_{succinic acid. A transport model was applied,}

wherein the undissociated acid was considered to be taken up by irreversible passive diffusion and is subsequently dissociated inside the cells to Suc2-_{and 2H}+ _{and subsequent export of}

dissociated acid (Suc2-_{) by a proton antiporter. This would result in the export of three protons}

per Suc2-_{exported, via the proton ATPase at the expense of one ATP per H}+_{. In this study}

both succinic acid metabolism and active export (seen clearly by the strong increase in ATP production) occurred and resulted in futile cycling at a high rate. The succinic acid wash-in resulted in 19% decrease in biomass formation due to the large energy demand from the futile cycle. The rate of succinic acid metabolism was 6.04±1.07 mmol.Cmol-1_.h-1_{after a new}

steady state was reached in the wash-in experiment. The presence of the high extracellular level of undissociated acid caused a cycle of influx and export resulting in an observed large extra ATP dissipation rate. The rate of succinic acid export, which cycled continuously into the cells, was estimated from the extra ATP dissipation rate to increase from 0 to 54 mmol.Cmol-1_.h-1_{. Measurement of the out/in total succinic acid ratios lead to a value of 8}

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irrespective of the succinic acid cycling rate. This indicated that not an antiporter but a reversible uniporter for the succinate transport might be present.

Using the obtained results on export mechanism and metabolism of succinic acid in Chapter 6, we studied the physiology and metabolism of a succinic acid producing mutant strain (SUC501) in Chapter 7. The effect of the carbon dioxide level on succinic acid production and metabolism was studied by increasing the level of CO2 in the inflow gas to the fermenter

from 0 (100% air) to 50%. The production rate of succinic acid increased about 5 times at 0.65 bar CO2 pressure, probably due to stimulation of the pyruvate carboxylation reactions.

The intercellular succinate level was higher in the mutant compared to the wild type strain and increased at increasing level of CO2. The constant ratio of 4.3 of extracellular succinate to intracellular succinate under production conditions showed that the transporter works close to equilibrium with a uniport as most likely transport mechanism as was also concluded in Chapter 6 for the wild type strain.

Futile cycling of succinic acid at low pH and high succinic acid concentrations in this mutant strain was studied in a similar set-up as described in Chapter 6. Succinic acid concentrations in the broth increased to 169 mmol.L-1_{, which was lower than in the wild type strain (227}

mmol.L-1_{). The respiration rate (q}

O2 and qCO2) increased during wash in of succinic acid and

the biomass concentration increased, which clearly indicated the metabolism and futile cycling of succinic acid. The net succinic acid uptake was measured to be about 3 times higher than the for wild type strain. The ATP dissipation rate increased at increasing level of extracellular succinate as observed for the wild type strain. The succinic acid cycling rate and respectively the succinic acid uptake rate were estimated using the ATP dissipation rate assuming the transport model (Model 1) described in Chapter 6 and a new model (Model 2 wherein the reversible uniporter imports Suc2-_{which is exported by an irreversible ABC}

transporter). Model 1 turned out to be thermodynamically infeasible, whereas Model 2 was thermodynamically feasible. Model 2 should be investigated further by performing a similar wash-in experiment at higher pH where the higher fraction Suc2-_{species is present in the}

broth.

The main question remains to be answered is the mechanism of dicarboxylic acid cycling. The suggested cycling model (Model 2) should be confirmed by proper biochemical investigation. Besides, performing transport studies at a higher cultivation pH, where the fraction of undissociated acid and passive diffusion is negligible, can assist us identify the uptake mechanism and species which is taken up by yeast cells. Finally the futile cycling must be prevented, which occurs based on Model 2, by identifying and knocking out of the succinic acid ABC transporter.

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xv

Samenvatting

De productie van chemicaliën uit hernieuwbare grondstoffen met behulp van micro-organismen krijgt veel aandacht vanwege de steeds toenemende mogelijkheden van genetische manipulatie en de bijdrage aan het verminderen van CO2-uitstoot. C-4

dicarbonzuren (barnsteenzuur, fumaarzuur en appelzuur) worden genoemd als de top 12 chemische bouwstenen vervaardigd uit bio-grondstoffen.

Fermentatieve productie van organische zuren zoals fumaarzuur en barnsteenzuur wordt bij voorkeur uitgevoerd in afwezigheid van zuurstof, wat leidt tot productie met theoretische opbrengst, en bij lage pH om de opwerkingskosten (Hoofdstuk 2) te verminderen. Echter, in de huidige natuurlijk producerende bacteriestammen worden fermentaties uitgevoerd bij neutrale pH, door toevoeging van calciumcarbonaat, wat leidt tot de vorming van bijproducten zoals gips tijdens de opwerking. De gist Saccharomyces cerevisiae is een organisme dat kan groeien bij lage pH (Hoofdstuk 3). Bijkomende voordelen van dit organisme (als modelorganisme) zijn dat het genoom volledig is gesequenced waardoor gist als een geschikte cel fabriek wordt beschouwd voor de productie van bulkchemicaliën uit hernieuwbare grondstoffen (Hoofdstuk 3).

Een belangrijk aspect van organische zuurproductie in de microbiële cel fabriek is de export van het product over de celmembraan met een voldoende capaciteit. Verhoogde extracellulaire organisch zuur concentraties is nodig voor economische productie, en dus zal export van het geproduceerde zuur via de celmembraan tegen een concentratiegradiënt geschieden, en is dus alleen mogelijk door energie verbruikend actief transport gemedieerd door specifieke eiwitten in de celmembraan. De cruciale vraag is hoeveel energie voor deze transportprocessen nodig is. De energetica van fumaarzuur en barnsteenzuur export bij lage pH werd theoretisch bestudeerd met behulp van een thermodynamische benadering en flux analyse (Hoofdstuk 2). Op basis van de verkregen resultaten werden metabolic engineering strategieën voor de microbiële productie van deze carbonzuren voorgesteld. Op basis van theoretische berekeningen werd gevonden dat het meest waarschijnlijke export mechanisme voor dicarbonzuren bij lage pH een Fum2-_{/proton antiporter is, waarbij voor elk fumaraat}

anion een proton wordt geëxporteerd.

Aannemende dat de export van barnsteenzuur en fumaarzuur geschiedt via een antiport mechanisme en na omrekenen van de Gibbs energie van barnsteen en fumaarzuur vorming vanuit glucose naar de benodigde hoeveelheid ATP, concludeerden we dat barnsteenzuur

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geproduceerd kan worden bij neutrale en lage pH onder anaerobe kweek omstandigheden. Volgens onze analyse is fumaarzuur productie onder anaërobe omstandigheden alleen mogelijk bij neutrale pH.

Men moet zich echter realiseren dat indien een fermentatieproces bij lage pH wordt uitgevoerd, het organische zuur voornamelijk aanwezig is in de ongedissocieerde vorm. Actieve export van een dicarbonzuur vanuit de cellen naar de extracellulaire omgeving zal dus resulteren in een steile concentratiegradiënt van de gedissocieerde vorm over de plasmamembraan. Ervan uitgaande dat het plasmamembraan permeabel is voor de ongedissocieerde vorm, zal dit leiden tot een futiele cyclus van actieve export en import via passieve diffusie ten koste van metabolische energie.

Opname van fumaarzuur werd gemeten bij verschillende pH in een unieke experimentele set-up (stap experiment) (Hoofdstuk 3) onder anaërobe omstandigheden en onder aërobe omstandigheden (Hoofdstuk 4), in glucose gelimiteerde chemostaten. De cell fysiologie (aëroob en anaëroob) bleek niet significant te veranderen binnen een pH range van 3,5-5,0, maar lagere pH (<3,5) bleek invloed te hebben op de celgroei, mogelijk door een futiele cyclus. Onder anaërobe omstandigheden werd de fumaarzuur concentratie in de voeding, en de initiële concentratie in het fermentatiemedium stapsgewijs verhoogd van 0-60 mmol.L-1_.

Gevonden werd dat de opnamesnelheid van fumaarzuur toenam bij afnemende pH, wat aangeeft dat de gedissocieerde vorm waarschijnlijk de belangrijkste species is die wordt opgenomen door de gistcellen. Uit de observatie dat het profiel van de fumaarzuur opnamesnelheid versus de ongedissocieerde zuurconcentratie asymmetrisch was, werd geconcludeerd dat de opname van fumaarzuur waarschijnlijk wordt gemedieerd door twee of meer membraan transport eiwitten met een maximum capaciteit en verschillende affiniteiten (Hoofdstuk 3). Fumaarzuur metabolisme werd waargenomen onder deze omstandigheden vanwege de lagere fumaarzuur concentratie in het effluent van de chemostaat.

Uit fumaarzuur stap experimenten onder aërobe omstandigheden in aanwezigheid van 1 mmol.L-1_{fumaarzuur in het voedingsmedium bleek dat de fumaarzuur opname toenam bij}

afnemende pH, hetgeen erop wijst dat de ongedissocieerde vorm van het zuur waarschijnlijk de belangrijkste getransporteerde species is . De opnamesnelheden waren veel lager dan in een soortgelijk experiment onder anaërobe omstandigheden. Bovendien werd zelfs bij pH 5, waarbij de fractie van gedissocieerde zuur verwaarloosbaar was nog opname van fumaarzuur gemeten. Dit betekent dat de diffusiecoëfficiënt hoger is dan de gerapporteerde waarden voor andere carbonzuren zoals appelzuur. Het is onwaarschijnlijk dat passieve diffusie (vooral bij pH 5,0) het enige transportmechanisme is en het is derhalve waarschijnlijk dat bij deze pH de opname tevens wordt gemedieerd door een membraan transport eiwit (Hoofdstuk 4). Fumaarzuur opname resulteerde in een significante toename van de concentratie van andere TCA-cyclus zuren in het totale fermentatie beslag, terwijl ook de intracellulaire concentraties

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Samenvatting xvii

stegen, maar bleven gelijk wanneer de pH verder werd verlaagd. Dit zou betekenen dat fumaarzuur wordt omgezet in andere organische zuren, na binnenkomst in het cytosol, waarna deze verbindingen werden uitgescheiden door de cellen. Echter, het totaal van de uitscheidingssnelheden van deze organische zuren verminderde bij stijgende opnamesnelheid van fumaarzuur, wat zou kunnen betekenen dat alle TCA cyclus zuren zoals fumaarzuur worden vervoerd met hetzelfde type membraan transporter.

In Hoofdstuk 5, werd metabole flux analyse, met behulp van een compleet genoom-model voor gist, gebruikt om potentiële genetische manipulatie doelwitten te selecteren om ATP-gebaseerde stoichiometrisch gekoppeld fumaarzuur productie onder aërobe omstandigheden te realiseren. Twee deletiemutanten werden geconstrueerd. In de eerste mutant werd het FUM1 gen dat codeert voor furmarase en in de tweede mutante de genen FUM1 en ZWF1 (coderend G6P dehydrogenase) uitgeschakeld. Deze stammen werden getest in glucose gelimiteerde chemostaten onder aërobe omstandigheden. De waargenomen biomassa en fumaarzuur opbrengsten waren veel lager dan de voorspellingen, omdat ethanol de meest geproduceerde metaboliet was en niet fumaarzuur. Om de productiviteit van fumaarzuur (wat in theorie leidt tot hoogst ATP productie) te verhogen, werd een dubbele deletie mutant gekozen voor verdere ontwikkeling in een evolutie experiment dat in een continue wijze werd uitgevoerd gedurende 300 dagen. Het bleek echter dat de fumaarzuur titer niet hoger werd maar in plaats daarvan meer acetaat werd geproduceerd en minder ethanol. Analyse suggereerde dat selectie was opgetreden voor een route met een hogere ATP productie per mol glucose (acetate ipv ethanol), zoals verwacht. De gemeten ophoping van fumaarzuur in de cel duidt op het ontbreken van een effectieve fumaarzuur exporter terwijl bovendien de evolutionaire selectiedruk afnam door een futiele cyclus van azijnzuur diffusie en export, leidend tot een toegenomen ATP dissipatie.

We hebben ons gericht op het transport van barnsteenzuur in de Hoofdstukken 6 en 7. We hebben eerst de fysiologie van wild-type gist (S.cerevisiae) bij lage pH (3,0) (Hoofdstuk 6) en het effect van hoge concentraties barnsteenzuur in de chemostaat (227 mmol.L-1_{) zoals zal}

optreden op productieschaal, op ATP dissipatie in futiele cycli onderzocht. Dit werd gedaan middels een zogenaamde barnsteenzuur inwas experiment, waarbij het barnsteenzuur niveau in de chemostaat geleidelijk steeg na vervanging van het voedingsmedium met een overigens identiek medium dat 254 mmol.L-1_{barnsteenzuur bevatte. Een theoretisch model voor}

barnsteenzuur werd toegepast, waarbij werd aangenomen dat het ongedissocieerde zuur wordt opgenomen middels passieve diffusie, daarna wordt omgezet in de cellen in SUC2- _{en 2H}+_en

vervolgens geexporteerd als volledig gedissocieerd zuur (SUC2-_{) door een proton antiporter.}

In dit onderzoek werd zowel barnsteenzuur metabolisme als actieve export (duidelijk te zien door de sterke toename van de ATP productie) waargenomen, wat resulteerde in aanzienlijke ATP dissipatie in futiele cyclus. Tijdens het barnsteenzuur inspoel experiment werd een 19% afname in biomassa formatie waargenomen, waarschijnlijk door de grote vraag naar energie

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in de futiele cyclus. De snelheid van barnsteenzuur metabolisme was 6,04 ± 1,07 mmol.Cmol -1_.h-1_{nadat een nieuwe steady state werd bereikt in het inspoel experiment. De aanwezigheid}

van een hoge extracellulaire concentratie van ongedissocieerd zuur veroorzaakt een cyclus van instroom en export war resulteerde in een waargenomen grote extra ATP dissipatie. De energetische kosten van barnsteenzuur export, konden worden geschat op basis van de extra ATP dissipatie, die steeg van 0 to 54 mmol.Cmol-1_.h-1_{. Meting van de uit/in ratio van totaal}

barnsteenzuur resulteerde in een waarde van 8, ongeacht de export snelheid. Dit gaf aan dat in plaats van een antiporter een reversibele uniporter voor succinaat transport meer voor de hand zou liggen.

Met behulp van de verkregen resultaten betreffende het export mechanisme en het metabolisme van barnsteenzuur (Hoofdstuk 6) bestudeerden we de fysiologie en het metabolisme van een barnsteenzuur producerende stam (SUC501) (Hoofdstuk 7). Het effect van het kooldioxidegehalte op de productie van barnsteenzuur en metabolisme werd bestudeerd door het verhogen van het CO2 gehalte in het beluchtingsgas van de fermentor van

0 tot 50%. De productiesnelheid van barnsteenzuur nam toe met ongeveer een factor 5 onder deze omstandigheden, waarschijnlijk door stimulatie van de pyruvaat carboxylerings reacties. De constante verhouding van 4,3 van extracellulaire succinaat tot intracellulaire succinaat onder productieomstandigheden bleek dat de transporter dicht bij evenwicht werkt met een Uniport als meest waarschijnlijke transportmechanisme zoals ook in Hoofdstuk 6 werd geconcludeerd voor de wild-type stam.

Futile cycling van barnsteenzuur bij lage pH en hoge concentraties barnsteenzuur in deze mutant stam werd onderzocht in een soortgelijke opstelling zoals beschreven in Hoofdstuk 6. Barnsteenzuur concentraties in de fermentatie stegen tot 169 mmol.L-1_{, wat lager was dan in}

de wild type stam (227 mmol.L-1_{). De ademhaling (q}

O2 en qCO2) was verhoogd tijdens

inspoelen van barnsteenzuur en de concentratie biomassa was toegenomen, waaruit duidelijk bleek dat barnsteenzuur werd gemetaboliseerd maar dat tevens futile cyclus optrad. De netto barnsteenzuur opname was ongeveer 3 maal hoger dan voor het wild type stam. De ATP dissipatie nam toe bij toenemende concentratie extracellulair succinaat zoals ook werd waargenomen voor de wild-type stam. De snelheid van deze futiele cyclus werd geschat aan de hand van de ATP dissipatiesnelheid gebruik makend van het transport-model (Model 1) In Hoofdstuk 6 en een nieuw model (Model 2, waarin import van SUC2- _{optreedt via een}

reversibel uniport mechanisme en export middels een irreversibele ABC transporter). Model 1 bleek thermodynamisch niet mogelijk, terwijl Model 2 thermodynamisch wel mogelijk was. De juistheid van Model 2 moet verder worden onderzocht door het uitvoeren van soortgelijke inspoel experimenten bij hogere pH waarbij de fractie van het volledig gedissocieerde zuur hoger is.

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Samenvatting xix

De belangrijkste vraag die nog moet worden beantwoord is wat het precieze mechanisme van de futiele dicarbonzuur cyclus is. Het voorgestelde model (Model 2) dient te worden bevestigd door biochemische onderzoek. Daarnaast zouden transport studies bij een hogere pH moeten worden uitgevoerd, waarbij de fractie ongedissocieerde zuur, en dus het aandeel van passieve diffusie, verwaarloosbaar zijn. Dit kan ons helpen het opnamemechanisme en de getransporteerde species te identificeren. Uiteindelijk dienen dergelijke futiele cycli te worden geëlimineerd, wat, indien Model 2 juist zou zijn, zou betekenen dat de barnsteenzuur ABC-transporter zou moeten worden verwijderd.

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xxi

A.3. In vivo energy aspects of ATP, proton motive force and fumarate reductase ATP ... 170 A.4. Proton motive force (pmf) ... 171 A.5. Fumarate reductase in succinate production ... 171 A.6. Effect of acid back diffusion on product yield of dicarboxylic acid ... 173 Appendix B. Acetic Acid Permeability Coefficient Calculation ... 175 Appendix C. Succinic Acid Transport Calculations ... 177 C.1. Succinic acid transport calculations ... 178 C.2. Octanol-water partition coefficient of carboxylic acids ... 178

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Contents xxiii

C.3. Equilibrium constants for transport ... 179 Appendix D. Intracellular Metabolite Data and Specific Rates for SUC501 Strain ... 181 D.1. Intracellular metabolites ... 182 D.2. Biomass specific rates during succinic acid wash-in experiment ... 183 Bibliography ... 185 About the Author ... 195

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1

CHAPTER 1: General Introduction

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1.1.Microbial production of dicarboxylic acids at low pH

The production of chemicals from renewable feedstocks using microorganisms receives considerable attention due to the ever-increasing possibilities of genetic engineering and the contribution to decreasing CO2 emissions. Especially multifunctional molecules, containing

alcoholic and carboxylic acid groups, are of interest because of their application in polymer production, which represents a very large market (Sauer et al. 2007;Song and Lee, 2006). Four carbon 1,4-diacids (succinic, fumaric, and malic) were mentioned as top 12 chemical building blocks manufactured from bio-feed-stocks in a report from the USDOE (US Department of Energy, 2004). These dicarboxylic acids are chemically malleable and can be readily converted into a range of commodity and specialty chemicals (Zeikus et al. 1999).

Saccharomyces cerevisiae has been selected as production platform for several organic acids

such as lactic and malic acid (Abbott et al. 2009;van Maris et al. 2004b;Zelle et al. 2008;Zelle et al. 2010a). It is a robust microorganism and can grow under stressful conditions such as low pH conditions or in presence of high concentrations of organic acids.

S.cerevisiae also plays a major role in applied research due to its outstanding capacity to

produce ethanol and carbon dioxide from sugars with high productivity, titer, and yield. Moreover, S.cerevisiae has been used as a host organism for pharmaceutical protein production in the past. S.cerevisiae is relatively tolerant to low pH values and high sugar and ethanol concentrations, i.e., properties that lower the risk of contamination in industrial fermentation. Moreover, this yeast is fairly resistant to inhibitors present in biomass hydrolysates and is able to grow both aerobically and anaerobically. These have been the major reasons for increased exploration of S.cerevisiae in industrial (“white”) biotechnology, focusing on the fermentative production of industrially relevant biochemicals, e.g., glycerol, propanediol, organic acids, sugar alcohols, L-glycerol-3-phosphate (LG3P), steroids, and isoprenoids. (Nevoigt, 2008;Sauer et al. 2007).

Production of organic acids such as fumaric acid and succinic acid is preferably performed in absence of oxygen, leading to theoretical production yields of 2 and 1.71 mole fumaric acid and succinic acid (per mole glucose) respectively (Chapter 2 of this thesis). However, in current bacterial anaerobic fermentations, performed at neutral pH, this yield is not achieved due to formation of byproducts such as acetic acid and ethanol, which are probably needed for ATP production. Furthermore, efforts have been made to improve the productivity of these bacterial strains, which can tolerate high concentration of organic acids and are able to consume cheaper carbon sources (Zeikus et al. 1999). However, the recovery and downstream processing costs account for 60-70% of the total production costs (Chen and Patel, 2012) . Therefore, a more cost effective way is to produce the undissociated dicarboxylic acid in the fermentor. This avoids extra alkali cost in fermentation for pH control

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Transport 3

and use of inorganic acid in down stream processing to recover the dicarboxylic acid from the salt form produced in the fermentor. This requires that the fermentation be carried out at low pH (lower than the pKa values of organic acid). The growth stoichiometry and physiology of

Saccharomyces cerevisiae hardly changes under low pH conditions (Jamalzadeh et al.

2012;Verduyn et al. 1990b), which is advantageous for production of dicarboxylic acids in undissociated form.

1.2.Transport

Microbial production of organic acids involves various membrane transport processes, such as substrate uptake and product export. These processes are of major importance for the production of organic acids via microorganisms (Sauer et al. 2007). The crucial question is the amount of energy that those transport processes need in order to be accomplished. High extracellular organic acid concentrations are required for economical production, leading to export of the produced acid over the cellular membrane against a concentration gradient, which can only be accomplished via energy requiring active transport mediated by specific proteins in the cell membrane. However, few studies have been reported in this area. Van Maris et al. (van Maris et al. 2004c) calculated the energy requirement for export of monocarboxylic acids such as lactic acid at different extracellular pH, and concluded that under typical process conditions, export of lactic acid is unavoidably an energy-consuming process. Energetics of dicarboxylic acids such as fumaric acid and succinic acid export under low cultivation pH has been studied theoretically by Jamalzadeh and Taymaz-Nikerel (Chapter 2 of this thesis) and based on the obtained results metabolic engineering strategies for microbial production of these carboxylic acids are suggested.

Nevertheless, very few studies have been reported on the transport of dicarboxylic acids in yeast and most of them do not come from the near past. Focusing on acid uptake, in 1987 it was reported that S.cerevisiae can import dicarboxylic acids only via non-facilitated simple diffusion of the undissociated acid (Salmon, 1987) .

It should be realized, however, that in a fermentation process carried out at low pH, the organic acid is predominantly present in the undissociated form. Active export of dicarboxylic acid from the cells into the extracellular environment will thus result in a steep concentration gradient of the undissociated form over the plasma membrane. Assuming that the plasma membrane is permeable for the undissociated species, this will create a futile cycle of active export and diffusion back into the cells at the expense of metabolic energy (ATP). Therefore this futile cycling is an important, but hardly studied, aspect in microbial dicarboxylic acid production at low pH.

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Yeast species differ remarkably in their ability to transport and utilize intermediates of the TCA cycle (Saayman et al. 2000). Hence, specific research on S.cerevisiae is needed in order to draw safe conclusions for that species. In all reported transport studies cells were grown in batch cultures under glucose excess and therefore repression conditions in mineral mediums with mechanical shaking. The uptake rates were measured in two ways: either after the addition of labeled acids in yeast suspension or by measuring proton uptake with a pH meter (Casal et al. 2008;Cassio and Leao, 1993;Corte-Real et al. 1989;Corte-Real and Leao, 1990;Osothsilp and Subden, 1986;Saayman et al. 2000;Salmon, 1987;Sousa et al. 1992). However, the results obtained from such experiments have plenty room for improvement. It becomes thus obvious that the underlying transport mechanisms of organic acids in

S.cerevisiae are not very well studied under glucose-limited conditions. Well-controlled

continuous fermentations are suitable methods for transport studies and measuring the uptake rate of dicarboxylic acids. Besides, metabolomics tools assist us to measure the intracellular levels of dicarboxylic acids under dynamic and steady state conditions in order to obtain insight about out/in ratios of acid. Thus we can identify the possible transport mechanism under different cultivation pH by comparing the experimental in/out dicarboxylic acid concentration ratio with equilibrium ratios derived from thermodynamic equilibrium calculations for candidate transport mechanisms (This thesis).

The plasma membrane is of crucial importance for the cell viability and performance. On the one hand, it must retain the dissolved materials of the cells, so that they do not simply leak out into the environment. On the other hands, it must control import and export of nutrients and metabolic products and thus maintain the concentrations of solutes in the cytosol at the desired levels (Barnett, 2008). A protein-free lipid bilayer would perfectly prevent the loss of charged and polar solutes from a cell but it would not allow transport of many required molecules into cytosol. In such a case hydrophobic and uncharged polar molecules would all diffuse across the membrane down their concentration gradient. The rate of transport then, would depend on the size of the molecules and their solubility in oil (smaller, hydrophobic and less polar molecules would diffuse faster). Furthermore, protein-free lipid bilayers would be highly impermeable to charged molecules (ions) since their charge and high degree of degradation would prevent them from entering the hydrocarbon phase of the bilayer (Kaback, 1976;Walter et al. 1982;Walter and Gutknecht, 1984).

However, cell membranes need to allow the passage of various polar molecules, such as ions, amino acids, nucleotides, and many cells metabolites as well as to transport molecules against their concentration gradients. As a result, special transport mechanisms are responsible for transferring such solutes across the membranes. Specific membrane transport proteins occur in many forms and each one of them transports a particular class of molecules or there are even more selective proteins that are activated with certain molecular species of one class. The importance of the above functions is indicated by the large numbers of genes in all

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Transport 5

organisms that code for transport proteins, which make up between 15 and 30% of the membrane proteins in the cells (Barnett, 2008).

Several mechanisms are employed to achieve transmembrane transport and in order to study them a classification is helpful (Figure 1.1). There are two main categories of transport mechanisms: passive and active.

Figure 1.1 Schematic view of different transport mechanisms (online source)

Passive transport occurs only down hill a concentration gradient for hydrophobic non-charged compounds and thus no energy is required for it. It includes simple diffusion and facilitated transport. In simple diffusion, as its name implies, molecules simply pass directly through the cells membrane. In facilitated transport though, specific proteins are used to transport charged and hydrophilic compounds, either facilitative transporters (also known as carriers) or channel proteins (Andre, 1995;Kaback, 1976).

The second wider category, active transport, includes also two classes; primary active transport and secondary active transport. Those can occur against a concentration gradient but since that is thermodynamically unfavorable, they require input of energy. Primary active transport directly utilizes chemical energy to move molecules through a membrane. Most of the proteins that perform this type of transport are transmembrane ABC-transporters. In secondary active transport, molecules are moved through a membrane as the direct result of the diffusion of another substance (possibly H+_{). Specifically, there is no direct coupling of}

Energy Simple diffusion Channel-mediated Transporter -mediated

Passive Transport Active Transport

C oncen tra tio n Grad ien t Transported Molecule Channel Transporter Out In Energy Simple diffusion Channel-mediated Transporter -mediated

Passive Transport Active Transport

C oncen tra tio n Grad ien t Transported Molecule Channel Transporter Out In Lipid Bilayer

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ATP; instead, the electrochemical potential difference created by pumping ions out of cells is used. (Niemietz et al. 1981;Sigler and Hofer, 1997)

Three different kinds of carriers exist: uniporters, symporters and antiporters (Figure 1.2). Uniport carriers mediate transport of a single solute. Symport (or cotransport) carriers bind two dissimilar solutes and transport them together across the membrane. Antiport carriers exchange one solute for another across the membrane(Andre, 1995). As already mentioned , the B-species in Figure 1.2 is usually H+_.

Figure 1.2 Schematic view of different carrier transporter proteins.

1.3.Metabolic engineering and evolutionary approach for

production of dicarboxylic acids

Metabolic engineering helps to solve defined problems, such as limiting by-product formation, broadening the range of carbon sources used, and increasing product fluxes. However, the focus in the future should move towards influencing complex cellular processes and

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Metabolic Engineering and Evolutionary Approach 7

properties, such as transport obstacles, because these are inherently connected with process performance, however we do not know enough yet for rational modification in these areas (Sauer, 2001).

Since the first introduction of metabolic engineering, there have been tremendous enhancements of its toolbox, and several related disciplines have emerged, such as inverse metabolic engineering and evolutionary engineering (Nielsen, 1998;Shimizu, 2009;Stephanopoulos, 1999). These developments have strongly influenced yeast strain improvement programs in the past few years and have greatly enhanced the potential for using yeast in biotechnological production processes (Zeikus et al. 1999).

Stoichiometric genome scale models assist us to choose the best metabolic engineering strategy in order to produce the desired organic acid. These whole genome models are available for S.cerevisiae (Lewis et al. 2012;Patil et al. 2004) and the recent version incorporates more than 1400 reactions, based on 904 genes and considers aspects of compartmentalization of cytosol, mitochondria, nucleus, extracellular space, vacuole, peroxisome, golgi and endoplasmatisch reticulum (Mo et al. 2009). Such models aim to incorporate all known metabolic reactions of S.cerevisiae in a mathematically accessible format to simulate phenotypes (wild-type and mutants) in-silico. This approach is applied in this thesis to select the best metabolic engineering approach for creating a high fumaric acid producing strain. However, Flux Balance Analysis generally gives a quantitative prediction of wild-type phenotypic behavior for optimized growth. It can be argued that evolution has in fact optimized the wild-type flux distribution towards optimal growth. However, it is not to be expected that a constructed mutant strain for product formation is also directly fully adapted to this new genetic change and has optimally redistributed its metabolic fluxes. It will require a certain amount of (evolutionary) time to allow the fine-tuning of enzyme activity to optimally redistribute the metabolic fluxes again (Segre et al. 2002a).

A strain with specific properties obtained by rational design can be subjected to evolutionary engineering for further improvement (Sauer, 2001). This approach has been applied recently by some researchers in Saccharomyces cerevisiae (Van Maris et al. 2004a;Wisselink et al. 2009). In order to obtain the most effective strain, a selective pressure is required to adapt the cells in the desired direction, which is production of our desired product. This can be achieved by coupling the product formation with energy production and growth. This approach is studied in detail in Chapter 5 of this thesis.

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Figure 1.3 Application of genome scale models in metabolic engineering (Patil et al. 2004).

1.4.Thesis Structure

We have started the thesis by a short introduction to microbial production of dicarboxylic acids, transport mechanisms in microorganisms, metabolic engineering, and evolutionary approaches in biochemical engineering for production of organic acids in Saccharomyces

cerevisiae.

This thesis addresses fumaric acid (Chapters 2, 3, 4, and 5) and succinic acid (Chapter 2, 6, and 7). In Chapter 2, a thermodynamic analysis of the conversion of glucose to fumaric and succinic acid is presented with respect to Gibbs energy aspects of fumaric acid production and export of acid. Thermodynamic analysis assisted us to study the feasibility of anaerobic production of fumaric acid and succinic acid at low pH, by considering the energy cost for export of fumaric acid. The energy-based bottlenecks for production of these organic acids are discussed in this chapter and possible metabolic engineering strategies are suggested. In Chapter 3, the uptake of fumaric acid in S.cerevisiae is studied in carbon limited chemostat cultures under anaerobic conditions. The effect of the presence of fumaric acid at

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Thesis Structure 9

different pH values (3–5) has been investigated in order to obtain more knowledge about possible uptake mechanisms and to obtain information on the type of transported species. Also the effect of the extracellular pH and fumaric acid concentration on the anaerobic physiology of the cells was taken into account.

Based on the results obtained from the thermodynamic feasibility study, the pH-dependent uptake of fumaric acid was studied in aerobic conditions in Chapter 4 since, according to Chapter 2, production of fumaric acid at low pH seemed to be not possible under fully anaerobic conditions. We quantified the uptake rate of fumaric acid under aerobic conditions under different cultivation pH (3-5) and presence of 1 mmol.L-1_{fumaric acid in the feed}

medium and studied the effect of fumaric acid uptake on intracellular TCA cycle organic acids and their secretion rates. In addition the effect of low pH on aerobic growth stoichiometry/physiology is discussed in this chapter.

In Chapter 5, we discuss the evolutionary approach for production of fumaric acid under aerobic conditions. We have constructed two yeast mutants based on predictions provided by genome scale modeling. Physiology of the constructed yeast mutants are studied in glucose-limited chemostat and compared with the wild type strain under similar fermentation conditions. One genetically modified strain was selected for further screening by evolutionary experiment in continuous culture mode. The results of the evolutionary experiments and the possible bottlenecks for fumaric acid production are discussed in this chapter.

In Chapter 6, the growth stoichiometry and physiology of wild type strain Saccharomyces

cerevisiae (CENPK 113-7D) was investigated in aerobic glucose-limited chemostats in the

presence of high feed concentrations of succinic acid at low culture pH (3.0) by performing a succinic acid wash-in experiment to simulate the actual production conditions in industrial scale. The focus was to measure the specific uptake rate of succinic acid and study the possible existence of an energy consuming futile cycle of passive diffusion of undissociated succinic acid into the cells and its subsequent active export. Metabolic network analysis assisted us to estimate the ATP dissipation rates under succinic acid wash-in conditions and respectively the succinic acid flux. Using the observed transport rate and the measured in/out concentration ratios of succinic acid the succinic acid uptake mechanism is studied here and possible mechanisms are discussed. Moreover, the effect of succinic acid metabolism on central carbon metabolism is studied during the wash-in experiment.

Cell physiology and intracellular flux distribution of a succinic acid producing

Saccharomyces cerevisiae mutant strain (SUC501) is studied, in Chapter 7, under aerobic

low pH conditions (pH=3.0) by performing glucose-limited chemostat experiments at different levels of carbon dioxide. Succinic acid is produced via the CO2-consuming reductive

TCA cycle and a dicarboxylic acid transporter was expressed in the membrane in this new strain that was kindly provided by Royal DSM. Effect of carbon dioxide is studied on specific

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production rate of succinic acid. In addition, information on the mechanism and the energetics of succinic acid transport and metabolism of succinic acid was obtained by performing a succinic acid wash-in experiment, similar set up as applied in Chapter 6, where the organism was exposed to increasing concentration of succinic acid in the broth. Possible back flux of succinic acid into the cells in the presenceof high extracellular concentration of undissociated succinic acid was investigated and the effect of succinic acid uptake on metabolism was studied. Succinic acid concentration ratios (out/in) have been applied as a tool to understand the export mechanism of succinic acid by assuming possible transport models under succinic acid production and secretion in wash-in experiment.

Finally, the conclusions and recommendations for future work in dicarboxylic acid transport study and metabolic engineering of Saccharomyces cerevisiae for production of dicarboxylic acids are given in Chapter 8.

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Published as: Jamalzadeh E§_{, Taymaz-Nikerel H}§_{, Heijnen JJ, van Gulik WM, Verheijen PJT. 2013. A} thermodynamic analysis of dicarboxylic acid production in microorganisms; Chapter 21 in: Biothermodynamics: the role of thermodynamics in biochemical engineering. Ed: Urs van Stockar, EFPL Press, Lausanne Switzerland, P 547-579 (§: equal contribution).

CHAPTER 2: Analysis of Dicarboxylic Acids Production in

Microorganisms

Equation Chapter 2 Section 1

roduction of dicarboxylic acids from microorganisms, and hence searching for genetic engineering targets to further increase the product yields, receives substantial interest. Therefore it is of importance that the limits of the production pathways are known. This can be analyzed stoichiometrically and thermodynamically as shown here for the production of succinic and fumaric acids from glucose at 25 °C for pH 1-8. Considering the different extent of each species (solid, undissociated, dissociated) of the organic acids at each pH, very negative Gibbs energy of reaction for the theoretical anaerobic reaction (-22 to -70 kJ.mol-1_{fumaric acid and -125 to -159}

kJ.mol-1_{succinic acid), with more Gibbs energy production at higher pH were found,}

promising that the maximal theoretical product yields (1.71 and 2.00 mol.mol-1_for

succinic and fumaric, respectively) were possible, also at low pH (< 3).

Thermodynamically feasible transport mechanisms were found to be 1 H+_{antiport at pH}

3 and 1 H+_{-symport at pH 7 for both acids, to achieve sufficient driving force for export}

of the organic acid, assuming an intracellular pH of 7. Alternative aerobic and anaerobic networks for fumaric acid and anaerobic network for succinic acid are proposed with energy (ATP) characteristics considered. The effect of acid back diffusion, which occurs at low pH by the undissociated organic acid, on the product yield was found to be different for the studied organic acids, being positively correlated with succinic acid (due to less growth) and negatively correlated with fumaric acid (due to increased aerobic catabolism). Taking the energy aspects of the optimal network into account, genetic engineering targets are suggesteted.

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2.1.Introduction

The production of chemicals from renewable feedstocks using microorganisms receives considerable attention due to the ever-increasing possibilities of genetic engineering and the contribution to decreasing CO2 emissions. Especially multifunctional molecules, containing alcoholic and carboxylic acid groups, are of interest because of their application in polymer production, which represents a very large market (Sauer et al. 2007). Four carbon 1,4-diacids (succinic, fumaric, and malic) are top 12 chemical building blocks manufactured from bio-feed-stocks in a report from the USDOE (US Department of Energy, 2004)

As a consequence the biotechnological processes will occur on very large scale, where from a cost point of view one desires the highest yield of product on substrate, low cost of auxiliary chemicals (e.g. pH control) and low cost of downstream processing (DSP). A general approach to evaluate these aspects, leading to relevant targets for genetic engineering, is therefore of interest and is the focus of this contribution. The approach will be illustrated using two dicarboxylic acids (succinic and fumaric acid) and will be thermodynamically-based, ensuring its general applicability to any other product of interest.

Our approach uses a thermodynamic framework to analyze the production of dicarboxylic acids (succinic and fumaric) from glucose at low pH with respect to stoichiometry and energy aspects of the black box theoretical product reaction and of candidate metabolic networks.

2.2.Outline of the approach

For dicarboxylic acids, one has to consider several aspects:

• There is production of acid, which requires the consumption of alkali for pH control in the fermentation process. In DSP the conventional method to obtain the undissociated acid (needed in the final polymerization processes) requires the addition of an inorganic acid solution. Overall, this leads to the consumption of stoichiometric amounts of alkali (e.g. CaCO3) and acid (e.g. H2SO4) leading to

production of stoichiometric amounts of salt (e.g. CaSO4). Purchase of the alkali and

acid and disposal of the salt pose a significant cost factor of about € 0.30 per kg acid (see Appendix A.1). Economically it is therefore desirable to perform the fermentation process at low pH, producing the undissociated acid, which avoids the need for these auxiliary agents.

• The dicarboxylic acids are produced in metabolic reaction networks, which require that the product be exported over the membrane. While this is no problem for alcohols (passive diffusion is possible), intracellular organic acid anions (charge -2) require special transporters which must achieve the required large out/in

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Black Box Analysis 13

concentration ratio of the acid which is typical order of 103 (because typical intracellular concentrations of the dicarboxylic acids are order 10-3_mol.L-1_{and an}

economically viable process requires about 1 mol.L-1_{concentration in the broth).}

Therefore, the export of the acid has an energy aspect. This energy aspect is even augmented because all the produced H+_{must also be exported against the H}+_gradient

(proton motive force).

• Organic acids, especially at the preferred low extracellular pH, can only be exported using energy consuming active transport mechanisms as explained above. However, at low pH the richly available extracellular undissociated organic acid can freely diffuse back into the cell, leading to an energy consuming (futile) cycle of export and import of undissociated acid. This energy drain leads to a changed product yield (Verduyn et al. 1990a;Verduyn et al. 1990b).

• At low pH organic acids often reach a solubility limit.

This analysis will present for the two organic acids the maximal theoretical yield, the theoretical product reaction, the consumption of alkali, the osmotic strength and amount of solid product as function of pH, and finally the calculation of ∆rG as function of pH.

2.3.Black box thermodynamic of the theoretical dicarboxylic acid

product reaction

2.3.1.Maximal theoretical product yield

A product made by microorganisms is the result of a metabolic network. Theoretically, the highest product yield is obtained when the organism does not spend substrate for growth, maintenance or external electron acceptor (as O2). This leads to a theoretical situation where

one envisages that the cell only makes the intended product under anaerobic conditions where all electrons from the substrate end in the product. The theoretical maximal yield then follows from a simple balance of degree of reduction (Roels 1983):

theor s sp p γ mol P Y = mol S γ ⎛ ⎞ ⎜ ⎟ ⎝ ⎠ . (2.1)

Assuming glucose (C6H12O6, γs = 24) as a renewable substrate and considering succinic acid

(C4H6O4, γp = 14) or fumaric acid (C4H4O4, γp = 12) as products gives 24/14=1.71 and

24/12=2.00 as maximal theoretical molar yields for succinic and fumaric acid per mole glucose. This theoretical maximal yield would be achieved anaerobically and is not based on any metabolic reaction network assumption. In principle, this black box result poses a

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theoretical maximum for any conceivable network, which starts with glucose and produces the acid as only product. The feasibility of this yield needs however first to be checked from a thermodynamic point of view, for which the stoichiometry of the theoretical product reaction is needed.

2.3.2.Stoichiometry of the theoretical product reaction

A dicarboxylic acid in aqueous solution is composed of 4 different species which are undissociated acid dissolved in water (aq), due to a limited solubility (see Figure 2.1) there is solid undissociated acid (s) present and the dissolved undissociated acid dissociates at increasing pH into its mono (-1) and di (-2) anion.

Considering the four product species, and using succinic acid as an example, we can write the product reaction as (note that consumed compounds have negative coefficients and that CO2

is assumed to be in equilibrium with the gas phase):

aC6H12O6 _{aq + bCO}2 _{g + cC}4H6O4 _{s + dC}4H6O4 _{aq +}

cC4H5O4-‐1_{aq + fC}₄_H₄_O₆-‐2_{aq + gH}₂_{O + hH}+_. _(2.2)

The 8 coefficients (a to h) can be calculated as follows, where we distinguish two regimes.

Dissolved acid regime

In this regime there is absence of solid product, hence c = 0 in equation (2). 7 stoichiometric coefficients remain to be calculated. The required 7 equations are:

4 conservation (C,H,O, charge) equations:

6a + b + 4(d + e + f ) = 0_, _(2.3)

12a + 6d + 5e + 4 f + 2g + h = 0_, _(2.4)

6a + 2b + 4(d + e + f ) + g = 0_, _(2.5)

-e - 2 f + h = 0_. _(2.6)

For the product sum relation, we assume that the sum of all acid species equals 1mole (knowing c = 0):

d + e + f = 1_. _(2.7)

The dicarboxylic acids have two dissociation equilibria with dissociation equilibrium constants of pK1 and pK2: