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Rewiring metabolic pathways for novel organic acid production in the filamentous fungusAspergillus niger

Hossain, A.H.

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Citation for published version (APA):Hossain, A. H. (2020). Rewiring metabolic pathways for novel organic acid production in the filamentous fungusAspergillus niger.

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137

Appendices

Summary

Samenvatting

সারাাংশ

References

List of publications

Word of thanks


Summary

139

Summary Filamentous fungi are remarkable organisms, that merit their own Kingdom in the

Tree of Life. Most of these fungi are organisms that degrade and feed on (non-

living) organic matter, that is usually composed of complex biopolymers, with their

innate ability to secrete enzymes that degrade the biopolymers into saccharides.

Fungi also have the remarkable ability to grow in acidic environments and

produce organic acids, which resemble the chemical building blocks that are used

in the chemical industry for making common household products, such as

plastics. This makes fungi interesting organisms for use in industrial

biotechnology where their superior organic acid producing capacity can be fully

exploited to generate cost-effective production processes. In particular, the

species Aspergillus niger is industrially exploited for the biotechnological

production of enzymes and organic acids. This organism is widely used in

industrial biotechnological processes worldwide due to its ability to grow on a

wide variety of feedstocks and natural robustness which allow for a large

operating envelope. As such, A. niger is a suitable production organism for

bioengineering purposes.

One of the products for which A. niger is especially well suited, is itaconic acid

(IA), a dicarboxylic acid with enormous application potential. Currently, IA is used

for the production of super absorbent polymers, unsaturated polyester resins,

detergents and coatings, but if the cost price can be lowered from the current

€1.4 - €2/kg to around €1/kg, IA can serve as bio-based pre-cursor for the

synthesis of Plexiglass, which would significantly expand the IA market. The

application potential and market size of IA is hampered by the limited production

capacity of the currently used industrial production organism A. terreus, which is

sensitive towards inhibiting compounds present in cheap and unrefined

feedstocks. Therefore, improving production of IA has been proposed to increase

IA yield, titer and productivity.

The use of A. niger as a production host for IA has already been suggested earlier

by Li et al., [39], Blumhoff et al., [54] and van der Straat et al., [43]. The

introduction of the genes cadA, mttA, and mfsA, together comprising the A.

terreus IA biosynthesis cluster, in A. niger, has resulted in low-level IA production

[39,52,53]. Furthermore, significant side-production of citric acid (CA) was

observed by A. niger strains that carry the full IA biosynthesis cluster, which is

described in Chapter 2. Transcriptome analysis of IA producing A. niger strains

have led to the identification of a gene encoding a non-canonical citrate synthase

citB, which upon overexpression significantly improved IA production. Moreover,

overexpression of citB led to abolishment of CA side-product formation (Chapter


Summary

140

2). The citB gene appears to be clustered in a secondary metabolite pathway,

which has been further elucidated by Palys et al., [58] to be involved in the

biosynthesis of alkylcitric acids. However, by introducing the IA biosynthesis

cluster in A. niger, we have observed citB induction. Moreover its overexpression

ultimately led to an improvement in IA production (Chapter 2).

Chapter 3 describes the further improvement of the rewired pathway by

overexpressing two genes that encode for ATP-citrate lyase (ACL), an enzyme

responsible for citrate breakdown in its constituent components oxaloacetate and

acetyl-CoA, at the cost of ATP. ACL overexpression has led to a further

improvement of IA yield, titer and productivity, which is probably caused by a

CitB-ACL mediated futile cycle of citrate synthesis and breakdown, resulting in

increased ATP consumption. Furthermore, in Chapter 3 we also describe the

improvement of cultivation conditions for IA production by supplementing an

alkalizing nitrogen source during IA production, which resulted in an extended

production phase and higher IA titers.

IA production in A. niger led to more unexpected rewiring, as described in Chapter

4. Upon achieving high IA titers as described in Chapters 2 and 3, we have

observed reduced biomass formation and also diminishing IA titers. Moreover, in

Chapter 2 we describe the observation of reduced biomass formation upon

externally added IA to the cultivation medium. To identify and elucidate potential

IA bioconversion pathways in A. niger, we have performed transcriptome analysis

that has led to the identification of two genes, ictA and ichA, that together form

an intracellular IA bioconversion pathway. These genes are silent under non-IA

producing conditions and highly active under conditions where high IA titers are

achieved. The deletion of either ictA or ichA led to the abolishment of IA

breakdown. Furthermore, a putative second IA bioconversion pathway, mediated

by the enzyme TmtA, was also identified. The expression pattern of tmtA closely

resembles the expression patterns of ictA and ichA. However, upon deletion of

tmtA we have observed strongly reduced IA titers.

In Chapter 5, we describe the identification of the end-product of the IA

bioconversion pathway by performing HPLC analysis, which is citramalate (CM).

Furthermore, in Chapter 5, we have looked into the effects of metabolic rewiring

on the expression of genes encoding enzymes and transporters that are related

to the production of 9 industrially relevant organic acids, by transcriptome

analysis. Surprisingly, this analysis has led to the identification of a biosynthesis

cluster that consists of a citramalate synthase and MFS transporter,

overexpression of which resulted in CM production, demonstrating the possibility

of two independently regulated CM pathways in our strains.


Summary

141

In Chapter 6, the results presented in this thesis are discussed in line with results

presented in the scientific literature regarding the production of organic acids with

filamentous fungi. A frequently overlooked aspect in organic acid production is

organic acid transport. In Chapters 2, 5 and 6, we describe results that emphasize

the importance of organic acid transport and also the importance of expressing

the correct transporter to achieve high product titers, is discussed. Furthermore,

the aspect of IA-mediated weak organic acid stress is also discussed in Chapter

6, where preliminary results with S. cerevisiae are described that show

intracellular acidification after external addition of IA to the culture. The topic of

metabolic rewiring and ATP wasting as strategy to improve metabolite production

is also discussed in Chapter 6, where these aspects are discussed in line with

results presented in the scientific literature.


Samenvatting

142

Samenvatting Draadvormige schimmels zijn opmerkelijke organismen, die hun eigen rijk

verdienen in de biologie. Het merendeel van deze schimmels voedt zich door het

afbreken van (niet levend) organisch materiaal, doorgaans bestaande uit

complexe biopolymeren, middels hun natuurlijke eigenschap om enzymen uit te

scheiden die deze biopolymeren afbreken tot suikers en andere bouwstoffen.

Schimmels hebben ook het opmerkelijke vermogen om in een zure omgeving te

groeien en organische zuren te produceren, die lijken op de chemische

bouwstenen die in de chemische industrie worden gebruikt voor het maken van

huishoudelijke materialen, zoals kunststoffen. Dit maakt schimmels interessante

organismen voor gebruik in de industriële biotechnologie, waar hun superieure

productiecapaciteit van organische zuren volledig kan worden benut om

kosteneffectieve productieprocessen te genereren. De schimmelsoort

Aspergillus niger wordt met name gebruikt bij de biotechnologische productie van

enzymen en organische zuren. Dit organisme wordt wereldwijd gebruikt voor

zulke industriële biotechnologische processen, vanwege zijn robuustheid en

vermogen om te groeien op een breed scala aan verschillende grondstoffen. Dit

maakt A. niger tot een geschikt productieorganisme voor biotechnologische

doeleinden.

Een van de producten waarvoor A. niger bijzonder goed geschikt voor is om te

produceren is itaconzuur (IA), een dicarbonzuur met veel mogelijke

toepassingen. Momenteel wordt IA gebruikt voor de productie van

superabsorberende polymeren, onverzadigde polyesterharsen, wasmiddelen en

coatings. Als echter de kostprijs kan worden verlaagd van de huidige € 1,4 - € 2

/ kg tot ongeveer € 1 / kg, dan kan IA dienen als duurzame grondstof voor de

productie van plexiglas. Gezien de grote getale waarin plexiglas geproduceerd

word, zou deze ontwikkeling de IA-markt aanzienlijk vergroten. De

toepassingsmogelijkheden en de marktomvang van IA wordt momenteel

belemmerd door de beperkte productiecapaciteit van het op dit moment gebruikte

industriële productieorganisme A. terreus, die gevoelig is voor verbindingen die

aanwezig zijn in goedkope en ongeraffineerde grondstoffen. Daarom is in dit

proefschrift een strategie uitgewerkt om de IA opbrengst, titer en productiviteit te

verhogen.

Het gebruik van A. niger als productieorganisme voor IA is al eerder voorgesteld

door Li et al., [39], Blumhoff et al., [54] en van der Straat et al., [43]. De introductie

van de genen cadA, mttA en mfsA (die samen het A. terreus IA

biosynthesecluster vormen) in A. niger heeft geresulteerd in kleine hoeveelheden

IA [39,52,53]. Bovendien werd een significante nevenproductie van citroenzuur

(CA) waargenomen door A. niger stammen die het volledige IA


Samenvatting

143

biosynthesecluster bevatte, zoals wordt beschreven in hoofdstuk 2.

Transcriptoom analyse van IA producerende A. niger stammen leidde tot de

identificatie van een gen dat codeert voor een onconventioneel citraatsynthase,

citB, dat bij overexpressie de IA productie aanzienlijk verbeterde. Bovendien

verdween de nevenproductie van CA als gevolg van citB overexpressie

(hoofdstuk 2). Het citB-gen blijkt onderdeel te zijn van een genencluster dat

codeert voor een biosyntheseroute van een bepaald secundair metaboliet, dat

verder onderzocht is door Palys et al., [58] en betrokken blijkt te zijn bij de

biosynthese van alkylcitroenzuur. Door het IA-biosynthesecluster in A. niger te

introduceren, hebben we een effect op de expressie van citB waargenomen en

hebben we waargenomen dat citB overexpressie leidt tot een verbetering van de

IA-productie.

Hoofdstuk 3 beschrijft de verdere verbetering van de IA productieroute door

overexpressie van twee genen die coderen voor ATP-citraatlyase (ACL), een

enzym dat verantwoordelijk is voor de afbraak van citraat in oxaloacetaat en

acetyl-CoA, ten koste van het energie dragende molecuul ATP. Overexpressie

van heeft geleid tot een verdere verbetering van de opbrengst, titer en

productiviteit van IA, wat gedeeltelijk wordt veroorzaakt door een zogenaamde

futiele cyclus van citraatsynthese en -afbraak door CitB en ACL, die netto ATP

verbruikt. Verder beschrijven we in hoofdstuk 3 de verbetering van de

kweekomstandigheden voor IA productie door het gebruik van een stikstofbron

die de extracellulaire pH verhoogd, hetgeen resulteerde in een verlengde

productiefase en hogere IA titers.

IA productie in A. niger leidde tot meer en zelfs onverwachte aanpassing van het

metabole circuit, zoals beschreven in hoofdstuk 4. Bij het bereiken van hoge IA

titers, zoals beschreven in hoofdstuk 2 en 3, hebben we een verminderde

vorming van biomassa waargenomen en ook afnemende IA titers. Bovendien

beschrijven we in hoofdstuk 2 de waarneming van verminderde vorming van

biomassa bij extern toegevoegd IA aan het kweekmedium. Om de mogelijke

biologische omzettingsroutes van IA die hiermee samenhangen in A. niger te

identificeren hebben we transcriptoomanalyses uitgevoerd die hebben geleid tot

de identificatie van twee genen, ictA en ichA, die samen een intracellulaire

biologische omzettingsroute vormen. Deze genen zijn inactief onder

omstandigheden waarin geen IA wordt geproduceerd en zijn zeer actief onder

omstandigheden waarbij hoge IA titers worden bereikt. Het inactiveren van de

ictA of ichA genen resulteerde in het stoppen van IA afbraak. Verder werd ook

een tweede mogelijke IA-bioconversieroute geïdentificeerd, gemedieerd door het

enzym TmtA. Het expressiepatroon van tmtA lijkt sterk op de expressiepatronen

van ictA en ichA. Na inactiveren van het tmtA gen hebben we echter sterk

verlaagde IA titers waargenomen.


Samenvatting

144

In hoofdstuk 5 beschrijven we de identificatie van het eindproduct van de IA-

bioconversieroute, citramalaat (CM). Verder hebben we in hoofdstuk 5 het

transcriptoom onderzocht van IA producerende A. niger stammen op de

expressie van genen die coderen voor enzymen en transporteiwitten die

gerelateerd zijn aan de productie van 9 industrieel relevante organische zuren.

Verrassend genoeg heeft deze analyse geleid tot de identificatie van een tot dan

toe onbekend biosynthese-cluster bestaande uit een citramalaatsynthase en een

MFS-transporteiwit, overexpressie van beiden resulteerde in CM-productie.

In Hoofdstuk 6 worden de resultaten zoals gepresenteerd in dit proefschrift

besproken in lijn met de resultaten uit de wetenschappelijke literatuur met

betrekking tot de productie van organische zuren met draadvormige schimmels.

Een aspect wat vaak over het hoofd wordt gezien bij de productie van organische

zuren is het transport van deze. In de hoofdstukken 2, 5 en 6 beschrijven we

resultaten die het belang van het transport van organische zuren benadrukken

en ook het belang van het tot expressie brengen van de juiste transporteiwitten

om hoge titers te bereiken. Verder wordt ook het aspect van het ontstaan van

zwakzuur stress, veroorzaakt door IA besproken in hoofdstuk 6, waar voorlopige

resultaten met S. cerevisiae worden beschreven die intracellulaire verzuring

vertonen na externe toevoeging van IA. Het aanpassen van metabole routes en

ATP-verspilling als strategie om de metabolietproductie te verbeteren wordt

verder besproken in hoofdstuk 6, waarbij ook de resultaten uit de gerelateerde

wetenschappelijke literatuur verder worden bediscussieerd.



145


তন্তুময় ছত্রাক হল একটি অসাধারণ জীব, যা ট্রি অফ লাইফফ তাফের নিজস্ব নকিংডম এর যযাগ্য। এই

ছত্রাকগুল োর যবনিরভাগই জজনবক পোর্থফক (মৃত) ক্ষয় করর এবিং যভাজি কফর, যা সাধারণত জটিল

বাফয়াপনলমার সমন্বফয় গঠিত হয় এবিং তাফের সহজাত ক্ষমতাবফল এমি এিজাইমগুনল বাফয়াপনলমারফক

স্যাকারাইফড অবিনমত কফর । ছত্রাফকর অম্লীয় পনরফবফি বৃনি এবিং জজব অযানসড উৎপােিকরার অসাধারণ

ক্ষমতাও রফয়ফছ যা রাসায়নিক নিফে প্লানিফকর মফতা সাধারণ পণ্য জতনরফত একক নহফসফব ব্যবহৃত হয় ।

এটি নিে বাফয়াফেকফিালনজফত ব্যবহাফরর জন্য ছত্রাকফক আকর্থণীয় জীফব পনরণত কফর যযখাফি সাশ্রয়ী

উৎপােি প্রনিয়া জতনর করফত তাফের জজব অযানসড উৎপােফির উচ্চ ক্ষমতা সমূ্পণথরূফপ কাফজ লাগাফিা

যযফত পাফর। নবফির্ত, Aspergillus niger প্রজানতটি এিজাইম এবিং জজব অযানসফডর

বাফয়াফেকফিালনজকাল উৎপােফির জন্য নিেগতভাফব ব্যবহার করা হয়। এই জীবটি নবনভন্ন ধরফণর কাাঁ চামাল

এবিং প্রাকৃনতক দৃঢ়তার মফে যবফ়ে ওঠার ক্ষমতার কারফণ, নবশ্বজুফ়ে নিেজাত বাফয়াফেকফিালনজকাল

প্রনিয়াগুনলফত ব্যাপকভাফব ব্যবহৃত হয়।যযমি, A. niger হল বাফয়াইনিনিয়ানরিং এর উফেফে, অন্য

পণ্যগুনলর উৎপােি সুনবধাফর্থ একটি উপযুক্ত উৎপােিফযাগ্য জীব।

এগুফলার মফে একটি, যার জন্য A. niger নবফির্ভাফব উপযুক্ত, যসটি হল আইোফকানিক অযানসড

(আইএ), একটি ডাইকাবথনিনলক অযানসড যার ব্যবহার সম্ভাবিাসূচক ।বতথ মাফি আইএ সুপার যিার্ণকারী

পনলমার, অসমৃ্পক্ত পনলফয়িার যরনসি, নডোরফজন্টস এবিং আবরণ উৎপােি করার জন্য ব্যবহৃত হয়, তফব

ব্যফয়র মূল্য যনে বতথ মাি € 1.4 - € 2 / যকনজ যর্ফক প্রায় € 1 / যকনজ পযথন্ত িানমফয় আিা যায় তফব তা

আইএ যপ্লনিগ্লাফসর সিংফের্ফণর জন্য বাফয়া-নভনিক অগ্রদূত নহফসফব কাজ করফত পাফর, যা আইএ বাজারফক

উফেখফযাগ্যভাফব প্রসানরত করফব। আইএর প্রফয়াফগর সম্ভাবিা এবিং বাজাফরর আকার বতথ মাফি ব্যবহৃত নিে

উৎপােি জীব A. terreusএর সীনমত উৎপােি ক্ষমতার কারফণ বাধাগ্রস্ত, যা অপনরফিানধত এবিং ব্যয়বহুল

কাাঁ চামাল গুফলাফত উপনিত বাধাোিকারী যযৌগগুফলার প্রনত সিংফবেিিীল। তাই আইএ ফলি, োইোর ও

উৎপােিিীলতা বৃনির যকৌিল নহসাফব আইএর উৎপােি প্রস্তাব করা হফয়ফছ। A. niger এর সাইট্রিক

অযানসড উৎপােি এবিং বহু নিে-ব্যবহৃত কাাঁ চামাল গুফলাফত উপনিত বাধা েমিকারীফের নবরুফি প্রনতফরাফধর

উোহরণ নহসাফব এর জজব অযানসড উৎপােি করার উচ্চ ক্ষমতা, খুব উপযুক্ত বফল মফি হয়।

আইএর জন্য যপ্রাডাকিি যহাি নহসাফব A. niger ব্যবহার ইনতমফে নল et al. [39], ব্লুমহফ et al.,

[54] এবিং ভযাি যডর স্ট্র্যাে et al. দ্বারা পরামিথ যেওয়া হফয়ফছ। [43]। A. niger এ cadA, mttA,

এবিং mfsA নজিগুফলার পনরনচনতর ফফল, যারা একফত্র A. terreus জজবসিংফের্ণ গুচ্ছ জতনর কফর, নিম্ন

স্তফরর আইএ উৎপােি হফয়ফছ [39,52,53]। তদ্বযতীত, A. niger দ্বারা উপজাত নহফসফব সাইট্রিক

অযানসড (নসএ) এর উফেখফযাগ্য উৎপােি পযথফবক্ষণ করা হফয়ফছ, যযটি সমূ্পণথ আইএ জজবসিংফের্ণ গুচ্ছ



146

বহি কফর এবিং যা অোয় ২-এ বণথিা করা হফয়ফছ। A. niger এর ট্রান্সনিপফোম নবফের্ণ একটি নজি

সিাক্তকরফণর নেফক ধানবত কফরফছ, যযটি একটি িি-কযাফিানিকাল সাইফট্রে নসিফর্জ citB যক এিফকাড

কফর, যা অনতমাত্রায় প্রকাফির মােফম আইএ উৎপােফির উফেখফযাগ্যভাফব উন্ননত কফরনছল। তদুপনর, citB

এর অতযনধক প্রকাি নসএ-উপজাত গঠি বানতল কফর যেয় (নদ্বতীয় অোয়)। citB নজিটি একটি নদ্বতীয়

নবপাকীয় পফর্র মফে গুচ্ছাকাফর আফছ বফল মফি হয়, যা প্যানলস et al. [58] দ্বারা অযালকাইলসাইট্রিক

অযানসফডর জজব সিংফের্ফণ জন়েত হওয়ার কর্া বণথিা করা হফয়ফছ (অোয় 2 এবিং 6)। তফব, A. niger

এ আইএ জজবসিংফের্ণ গুচ্ছটি প্রবতথ ি কফর আমরা citB যক আইএ জজবসিংফের্ফণ পুিনিথমথাণফক পযথফবক্ষণ

কফরনছ, যা অনতমাত্রায় প্রকাফির পফর অবফিফর্ আইএ উৎপােফির উন্ননত সাধি কফর।

অোয় ৩ এ এটিনপ-র মূফল্য সাইফট্রে এর উপাোি অিাফলা এনসফেে এবিং এনসোইল-যকাএ যত এর ভাঙ্গফির

জন্য োয়ী একটি এিজাইম, এটিনপ-সাইফট্রে লাফয়জ (এনসএল)যক এিফকাড কফর এমি দুটি নজিফক

ওভারএিফপ্রস কফর পুিনিথমথাণ পফর্র আরও উন্ননতর বণথিা যেওয়া হফয়ফছ। এনসএল এর ওভারএিফপ্রিি

আইএ ফলি, োইোর এবিং উৎপােিিীলতার আরও উন্ননত ঘোয়,যা আিংনিকভাফব সাইফট্রে সিংফের্ণ এবিং

ভাঙ্গি এর একটি নিরর্থক চি নহফসফব citB-এনসএল দ্বারা পনরচানলত হয়, যযটি অবনিষ্ট এটিনপ গ্রহণ কফর

এবিং িনক্তর যিার্ণ ঘোয়। অনধকন্তু, তৃতীয় অোফয় আমরা আইএ উৎপােফির সময় ক্ষারযুক্ত িাইফট্রাফজি

উৎফসর পনরপূরক দ্বারা আইএ উৎপােফির জন্য চাফর্র অবিার উন্ননত বণথিা কফরনছ, যার ফলস্বরূপ বনধথত

উৎপােি পবথ এবিং উচ্চতর আইএ োইোর নছল।

A. niger এ আইএ উৎপােি আরও যবনি এবিং এমিনক অপ্রতযানিত পুিনিথমথাফণর নেফক পনরচানলত

কফরনছল, যযমিটি চতুর্থ অোফয় বনণথত হফয়ফছ। উচ্চ আইএ োইোর অজথ ফির পফর নদ্বতীয় এবিং তৃতীয়

অোফয় বনণথত হফয়ফছ, আমরা জজবভফরর গঠফি হ্রাস এবিং হ্রাসকারী আইএ োইোরফক পযথফবক্ষণ কফরনছ।

অনধকন্তু, নদ্বতীয় অোফয় আমরা বানহযকভাফব আবােকৃত আইএ চাফর্র মােফম হ্রাস করা জজবভর গঠফির

পযথফবক্ষণ বণথিা কনর। A. niger এ আইএ জজব রুপান্তফরর পর্ রফয়ফছ নকিা তা ব্যাখ্যা করার জন্য, আমরা

ট্রান্সনিপফোম নবফের্ণ কফরনছ যা দুটি নজি, ictA এবিং ichA নচনিত কফরফছ, যা একসাফর্ একটি

আন্তঃফকার্ীয় আইএ জজব রুপান্তফরর পর্ জতনর কফর। এই নজিগুফলা িি-আইএ উৎপােফি নিনিয় র্াফক

এবিং উচ্চ আইএ োইোর প্রানিফত অতযন্ত সনিয় র্াফক। ictA বা ichA উভয়ই মুফছ যফলার ফফল আইএর

ভাঙ্গি বানতল হফয় যায়। তদ্বযতীত, এিজাইম tmtA এর মেিতায় একটি নিনতিীল নদ্বতীয় আইএর জজব

রুপান্তফরর পর্ও নচনিত করা হফয়নছল। TmtA এর অনভব্যনক্তর িমুিাটি ictA এবিং ichA এর অনভব্যনক্তর

িমুিার সাফর্ ঘনিষ্ঠভাফব নমফল যায়। তফব, tmtA মুফছ যফলার পফর আমরা আইএ োইোফর তীব্র হ্রাস

পযথফবক্ষণ কফরনছ।



147

অোয় 5 এ, আমরা এইচনপএলনস নবফের্ণ কফর আইএর জজব রুপান্তর পফর্র যির্-পণ্যটির সিাক্তকরণ

বণথিা কনর, যা সাইট্রাম্যাফলে (নসএম)। অনধকন্তু, অোয় 5 এ, আমরা ট্রান্সনিপফোম নবফের্ণ কফর নজি

এিফকানডিং এিজাইম এবিং এমি ট্রান্সফপােথ ারগুফলা যারা ৯ টি নিে-সম্পনকথ ত প্রাসনঙ্গক জজব অযানসফডর

উৎপােফির সাফর্ সম্পনকথ ত, তাফের উপর নবপাকীয় পুিনিথমথাফণর প্রভাবগুফলা যেফখনছ। আশ্চযথজিকভাফব,

এই নবফের্ণ একটি জজব সিংফের্ণ গুচ্ছ িিাক্তকরফণর নেফক পনরচানলত কফরফছ যযটি সাইট্রাম্যাফলে সিংফের্

এবিং এমএফএস ট্রান্সফপােথ ার সমন্বফয় গঠিত,যার অতযনধক এিফপ্রিি যার ফফল নসএম উৎপােি হফয়নছল।

র্ষ্ঠ অোফয়, এই নর্নসফস উপিানপত ফলাফলগুফলা তন্তুময় ছত্রাক যর্ফক জজব অযানসড উৎপােি সম্পনকথ ত

জবজ্ঞানিক সানহফতয উপিানপত ফলাফলগুনলর সাফর্ নমল যরফখ আফলাচিা করা হফয়ফছ। জজব অযানসড

উৎপােফির একটি বোরংবোর উফপনক্ষত নেক হল জজব অযানসড পনরবহি। অোয় ২,৫ এবিং ৬ এ,আমরা জজব

অযানসড পনরবহফির গুরুত্ব সম্পনকথ ত ফলাফলগুফলাফত যজার যেই এবিং উচ্চ পণ্য োইোর অজথ ফির জন্য

সঠিক পনরবহিকারীফক প্রকাফির গুরুত্বফকও যজার নেফয় আফলাচিা করা হফয়ফছ। তদ্বযতীত,আইএ- মেনিত

দুবথল জজব অযানসড পীরফণর নেকটি র্ষ্ঠ অোফয়ও আফলাচিা করা হফয়ফছ,যযখাফি S. cerevisiae এর

প্রার্নমক ফলাফলগুফলা, যা কালচাফর আইএর বানহযক সিংফযাজফির পফর অন্তঃফকানর্ক অযানসনডনফফকিি

যেখায়, যসগুফলা বণথিা করা হফয়ফছ। নবপাকীয় পুিনিথমথাণ এবিং নবপাফকর উৎপােি উন্ননতর যকৌিল নহসাফব

এটিনপ িফষ্টর নবর্য়টিও র্ষ্ঠ অোফয় আফলাচিা করা হফয়ফছ, যযখাফি এই নেকগুফলা জবজ্ঞানিক সানহফতয

উপিানপত ফলাফফলর সাফর্ নমল যরফখ আফলাচিা করা হফয়ফছ।


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pursuit of protein targets: proteomic characterization of spore coat outer

layers. J. Proteome Res. 2013;12(10):4507-21.

2. Zha Y, Hossain AH, Tobola F, Sedee N, Havekes M, Punt PJ. Pichia

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Word of thanks

165

Word of thanks It has been quite a journey, which started in November 2013. And

although 6 years seem long, time sure has flown by very fast. Performing

the work that is presented in this thesis was challenging at times, involving

long hours and often also working in the nights and weekends. However,

during my PhD work and the years at DDNA thereafter, I have worked

with much joy and pleasure with many wonderful colleagues. I owe a great

amount of gratitude to you people for the fun times that I’ve had, learning

new things on a regular basis and making this a memorable journey.

Stanley and Gertien, I would like to thank both of you for supervising and

guiding my research with valuable tips whenever needed. I got acquainted

with you whilst pursuing my Master’s at the UvA, where I also performed

an internship in your group. It was during this internship where I got really

inspired to pursue a PhD, and I am grateful to you for this inspiration.

Furthermore, Stanley, due to your kindness and patience, I always felt I

could approach you to discuss any matters, both academic and career

related. Gertien, your energy and enthusiasm for science is contagious

and I always felt more energized after conversing with you.

Peter, you were instrumental in my development as early-career scientist.

Whenever I would find myself on a crossroads and did not know how to

navigate further, I knew I could rely on your knowledge and advise to help

me further. Also in the period after my PhD, whilst working with Dutch

DNA, I have learned many things from you, for which I am grateful. Thank

you for guiding me in the early years of my career.

Art, we met somewhere in the corridors of TNO, if I remember correctly.

Little did I know then that, like Peter, you would also be instrumental for

my development in the early stages of my career. From discussing market

trends and opportunities for biochemicals, to discussing career, religion,

business strategies, philosophy; I’ve always enjoyed these conversations

with you and learned new insights from them. Thank you for having faith

in me and mentoring me when needed.

I am also thankful to Machtelt, Wim and of course Cornelis. Machtelt, it

was always a pleasure working with you. Your calm presence and down-

to-earth approach to complex situations was often encouraging and gave

me confidence. Wim, we met little more than a year ago and unfortunately


Word of thanks

166

I haven’t had the opportunity to work with you for longer than that. Your

enormous experience in and knowledge of business development and

business management is laudable and I wish I could have learned more

from you. Cornelis, your energy and eternal enthusiasm is just

commendable. It was always nice talking to you about market

developments and how the company could maneuver itself for maximum

results.

I would also like to express my gratitude to the committee members Han,

Peter, Filipe, Hans, Teun and Martijn; who took their time to thoroughly

read and evaluate this thesis and for attending the defense.

To the students that I’ve had over the years Anna, Jos, Wouter, Roy and

Aiko; this thesis would not have been possible without your help. Anna,

thank you for your efforts and the good times that we had. Jos, next to a

very bright student you were a fun guy to hang out with. I will cherish all

our discussions that we’ve had (the topics are just too much to mention

as you undoubtedly will know) and the boxing classes that we used to

follow. Such fun times! Wouter, you were an equally bright student and

I’ve always found you way more matured beyond your years. After your

graduation it was an honor to work with you as a colleague. Roy, you were

the last student during my PhD studies, but the one with arguably the

biggest impact, which is exemplified with a co-authorship in Chapter 3,

and also providing input for two other chapters. Your structured and neat

way of working was a good fit to complement my own working style. Aiko,

also your contributions eventually resulted in a co-authorship, which is

very well-deserved. Thank you for all your efforts.

Furthermore, I wish to extend my gratitude to other colleagues of MBMFS.

Soraya, Jan, Niels, Marloes, Yanfang, Linli, Laura you people always

made me feel welcome when I would visit the UvA. Laura, I owe much

gratitude to you for helping me set-up the experiments in the lab and

inviting me for social activities that were planned. Thank all of you for your

support and friendliness.

And of course, I wish to thank my fellow colleagues at Dutch DNA. Sylvia,

Ebru, Jean-Paul, Brandon, Coen, Alexander; it was nice being office

mates with you. Somehow we always managed to combine hard work with

fun and excitement. Rowy, I enjoyed working with you on projects. Due to


Word of thanks

167

your cheerful nature you were fun to work with, although at times I had

difficulties keeping up with your pace in the lab. Thank you for being a

great colleague and a friend. Karin, Lars, Pascal, Norbert, Marian, Trifa,

Vivi, Sanaz and Hemn; thank you for your support and making my time at

DDNA memorable.

On a last note, I would like to thank my family and friends. My mother,

whom I hope to make proud with this thesis, my uncle and father, both

whom passed away too soon in 2017 and have been a great absence in

my life since then. Ronnie, you have been an example to me. You have

made great strides in your career and I have witnessed firsthand how

dedicated you are. I hope I can make the same steps in my career as you

have. I wish you all the best in your next career move to the US.

Meem, you have been a great addition to my life. Your joyful silliness is

often times the perfect ingredient to counter my bleak pessimism and

exactly what I need. Thank you for always being there for me and

supporting me all the way.


Word of thanks

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Rewiring m

etabolic pathways for novel organic acid production in the filam

entous fungus Aspergillus niger

Abeer H

. Hossain

Documents

UvA-DARE (Digital Academic Repository) Rewiring metabolic ... · observed by A. niger strains that carry the full IA biosynthesis cluster, which is ... the correct transporter to