Biosynthetic Production of Human Milk Oligosaccharides
Oligosaccharides Huʻakau ʻa e tangata
Ko e oligosaccharides huʻakau fakaetangata (HMOs), ʻa ia ʻoku toe ʻiloa pe ko e huʻakau ʻa e tangata ko e glycans, ko ha konga ia ʻo e kulupu oligosaccharides. Ko e ngaahi sipinga ʻiloa ʻo e HMOs kau ai ʻa e 2 ʻ-fucosyllactose (2′-ʻikai l), lacto-N-neotetraose (LNnT), 3 ʻ-galactosyllactose (3′-GL), mo difucosyllactose (TOTOL).
Neongo ʻoku ʻi ai ha huʻakau ʻi he iscomposed ʻa e tangata ʻo lahi ange ia ʻi he ngaahi HMO ʻe 150 ki he fale, ka ko e 2-fucosyllactose pe (2 ʻoku ʻikai ʻasi ai e l) mo e lacto-N-neotetraose (LNnT) ʻoku lolotonga faʻu ʻaki ia ha tuʻunga fakakomesiale pea fakaʻaongaʻi ia ko ha hono additives ʻi he founga ʻa e valevale.
ʻoku ʻiloa e huʻakau oligosaccharides (HMOs) ʻi hono mahuʻinga ʻo e meʻakai fakatupu moʻui lelei. Ko e faʻahinga ivi makehe ʻa e huʻakau fakaetangata ko e oligosaccharides, ʻa ia ʻoku hoko ko e prebiotics, meʻa fakapipiki ki he antimicrobials, pea mo e immunomodulators ʻi he gut ʻa e valevale pea tokoni ʻaupito ki he fakalakalaka ʻo e ʻuto. ʻoku maʻu ʻa e HMOs ʻi he huʻakau ʻa e tangata; mammalian kehe milks (e.g. pulu, kosi, fanga sipi, kameli mo e ala meʻa pehe) ʻoku ʻikai ke nau maʻu ʻa e faʻahinga oligosaccharides ko ʻeni.
Ko e oligosaccharides ʻa e huʻakau ʻa e tangata ko e foʻi faiva malohi taha ia ʻi he huʻakau ʻa e tangata, ʻa ia ʻe lava ke maʻu ʻo kapau ʻe veteki pe emulsified pe ʻi he vai. ʻoku lahi taha e solids ʻoku maʻu ʻe he ngaahi Lactose mo e faʻafaʻesiti ʻi he huʻakau fakaetangata. ʻoku HMOs e kau 0.35 – ʻaunise 0.88 (9.9 – 24.9 g)/L. fakafuofua ki he 200 ʻoku ʻiloa ʻa e huʻakau mo e faʻahinga kehekehe ʻo e tangata oligosaccharides. Ko e oligosaccharide mahuʻinga ʻi he 80% ʻo e kakai fefine kotoa pe ko e 2′-fucosyllactose, ʻa ia ʻoku ʻi he huʻakau mei he faʻahinga ʻo e tangata ʻoku ne fakatahatahaʻi ʻa e 2.5 g/L.
Koeʻuhi ʻoku ʻikai ko ha ha HMOs, ʻoku ʻikai ke nau calorically tokoni ai ki he meʻakai fakatupu moʻui lelei. Indigestible kelolii me'akai mahoa'a, ʻoku nau ngaue prebiotics pea ʻoku nau filifili fermented kinautolu ʻoku nau fie maʻua gut microflora, tautautefito ki he bifidobacteria.
Ngaahi lelei fakaemoʻui lelei ʻo e Huʻakau fakaetangata Oligosaccharides (HMOs)
- paotoloaki e fakalakalaka ʻa e fanau valevale
- ʻoku mahuʻinga ki he fakalakalaka e ʻuto
- Kuo fakafili ki he laukovi mo
- ngaahi ola ʻo e fakapipiki-Kulu ʻi he tohitufa gastro-ngakau
- poupouʻi e founga maluʻi mei he kakai lalahi
Biosynthesis ʻo e Huʻakau ʻa e tangata Oligosaccharides
ʻoku fakaʻaongaʻi e ngaahi founga ngaue mo e enzymatic/na'e kimo ai enzymatic ngaahi tekinolosia lolotonga ki he synthesis ʻo e HMOs. Ki he ngaahi meʻa HMO maʻu ʻi he meʻafua ki he ngaahi koloa lalahi, ʻoku lava ʻe he fermentation ʻo e microbial ki he ngaohiʻanga meʻa toʻotoʻo, piokalafi-kemikale synthesis, pea mo e founga kehekehe ʻo e ʻa e ʻo ENZYMATIC ai. Koeʻuhi ko e ngaahi ʻuhinga fakaʻekonomika, ʻoku lolotonga ngaue ʻaki ʻa e piokalafi synthesis ʻi he microbial e ngaahi ngaohiʻanga toʻotoʻo ʻi he founga pe ko ia ʻoku fakaʻaongaʻi ki he tuʻunga ʻo e ngaahi meʻa fakapisinisi ko e HMOs.
Fermentation ʻo HMOs fakaʻaongaʻi e ngaahi ʻene Microbial Toʻotoʻo
Ko e. kololi, ko Saccharomyces cerevisiae mo Lactococcus lactis ʻoku fakaʻaongaʻi angamaheni ia ko e ngaahi ngaohiʻanga toʻotoʻo ʻa ia ʻoku fakaʻaongaʻi ki he piokalafi ʻo e molecules totonu hange ko e HMOs. Ko e Fermentation ko ha founga biochemical ia ʻo fakaʻaongaʻi ʻa e ngaahi organisms Micro ke fakaului ʻaki ha substrate totonu ki he molecules totonu. ʻoku ngaue ʻaki ʻe Microbial e ngaahi meʻa toʻotoʻo ha sugars faingofua ko substrate, ʻa ia naʻa nau ului ai ki HMOs. Koeʻuhi ko ha kiʻi sugars faingofua (hange ko lactose) ko ha substrate lahi, maʻamaʻa, ʻoku tauhi ai ʻa e totongi ʻo e founga ngaue piokalafi e synthesis.
ʻoku takiekina ʻa e tupulaki mo e vave ʻo e bioconversion ʻe he lahi hono feʻaveʻaki ʻo e ngaahi ivi (substrate) ki he microorganisms. Ko e lahi ʻo e paʻanga ʻoku hiki ko ha tefitoʻi meʻa ia ʻoku ne uesia ʻa e koloa synthesis lolotonga fermentation. ʻoku ʻiloa ʻa e Ultrasonication ke poupouʻi ʻa e fehikitaki lahi.
During fermentation, the conditions in the bioreactor must be constantly monitored and regulated so that the cells can grow as quickly as possible in order to then produce the targeted biomolecules (e.g. oligosaccharides such as HMOs; insulin; recombinant proteins). Theoretically, the product formation starts as soon as the cell culture begins to grow. However especially in genetically modified cells such as engineered microorganisms it is usually induced later by adding a chemical substance to the substrate, which upregulates the expression of the targeted biomolecule. Ultrasonic bioreactors (sono-bioreactor) can be precisely controlled and allow for the specific stimulation of microbes. This results in an accelerated biosynthesis and higher yields.
Ultrasonic lysis and extraction: Fermentation of complex HMOs might be limited by low fermentation titers and products remaining intracellular. Ultrasonic lysis and extraction is used to release intracellular material before purification and down-stream processes.
Ultrasonically Tuʻuaki Fermentation
The growth rate of microbes such as Escherichia coli, engineered E.coli, Saccharomyces cerevisiae and Lactococcus lactis can be accelerated by increasing the mass transfer rate and cell wall permeability by applying controlled low-frequency ultrasonication. As a mild, non-thermal processing technique, ultrasonication applies purely mechanical forces into the fermentation broth.
Acoustic Cavitation: The working principle of sonication is based on acoustic cavitation. The ultrasonic probe (sonotrode) couples low-frequency ultrasound d waves into the medium. The ultrasound waves travel through the liquid creating alternating high-pressure (compression) / low-pressure (rarefaction) cycles. By compressing and stretching the liquid in alternating cycles, minute vacuum bubbles arise. These small vacuum bubbles grow over several cycles until they reach a size where they cannot absorb any further energy. At this point of maximum growth, the vacuum bubble implodes violently and generates locally extreme conditions, known as the phenomenon of cavitation. In the cavitational “hot-spot”, high pressure and temperature differentials and intense shear forces with liquid jets of up to 280m/sec can be observed. By these cavitational effects, thorough mass transfer and sonoporation (the perforation of cell walls and cell membranes) is achieved. The nutrients of the substrate are floated to and into the living whole cells, so that the cell factories are optimally nourished and growth as well as conversion rates are accelerated. Ultrasonic bioreactors are a simple, yet highly effective strategy to process biomass in a one-pot biosynthesis process.
Ko e sonication ʻoku mapuleʻi lelei, ʻoku ʻiloa ia ʻi ha ngaahi founga fakatupulaki fermentation.
ʻoku hanga ʻe Sonication ʻo fakalakalaka ʻa e "ngaue ʻa e tokolahi bioprocesses kau ai ʻa e ngaahi selo ʻo e substrate ʻi hono fai ha ngaue lahi ange, fakaleleiʻi ʻa e lahi pe tupulaki ʻaki hono fakatupulaki ʻo e porosity (), pea mo maʻu ʻa e ngaahi konga ʻo e kongokonga." (Naveena et Al. 2015)
Read more about ultrasonically-assisted fermentation!
Ngaahi faingamalie ʻo Ultrasonically fakalalahi Fermentation
- 'Oku 'omi lahi ange
- Fakavaveʻi Fermentation
- Fakaʻaiʻai fakahangatonu
- Fakatupulaki Substrate hono Toʻo hake
- Fakalahi ʻa e Porosity Toʻotoʻo
- faingofua ke ngaue
- Faingofua Retro feʻunga
- ngaahi me'afua hake
- Kulupu (batch) pe InIine ki he ngaue
- ROI 'o e 'aukai
Naveena et Al. (2015) ne maʻu ʻe he ultrasonic intensification ha ngaahi faingamalie lahi lolotonga ʻa bioprocessing, kau ai ʻa e fakamole ʻi he ngaue ki hono fakaleleiʻi ʻo e ngaahi fili ʻoku fai, ʻa e faingofua ʻo e ngaue mo e ngaahi fie maʻu ʻa e malohi ʻoku feʻunga.
Kau ultrasonic-High Fermentation kau faiva
ʻoku fekauʻaki ʻa e Fermentation mo e moʻui microorganisms hange ko e pekitilia pe ʻisite, ʻa ia ʻoku hoko ko ha ngaahi founga toʻotoʻo. ʻi heʻene sonication ʻoku fakaʻaongaʻi ia ke ne tuʻuaki ʻa e lahi ʻo e paʻanga mo fakatupulaki e tupulaki ʻa e ului ʻa e microorganism, ʻoku mahuʻinga leva ke puleʻi e ultrasonic malohi kae lava ke fakaʻehiʻehi mei he fakaʻauha ʻo e ngaahi faʻunga toʻotoʻo.
ʻoku mataotao ʻa Hielscher Ultrasonics ʻi hono teuteuʻi, ngaohi mo tufaki ʻo e ultrasonicators ʻoku maʻolunga, ʻa ia ʻe lava pe ke mapuleʻi mo vakaiʻi ke fakapapauʻi ʻoku fermentation lelei.
ʻoku ʻikai ngata pe ʻi hono mahuʻinga ke mapuleʻi e founga ki he maʻolunga mo e lelei ange, ka ʻoku lava pe ke toutou fakahoko mo faʻu e ngaahi ola. Tautefito ki he taimi ʻoku fakaʻaiʻai ai e ngaahi founga fakaist, ʻoku mahuʻinga ke fulihi e ngaahi fakangatangata ʻi he founga toʻotoʻo, ke maʻu ha tuʻunga maʻolunga ke tokoniʻi mo taʻofi ʻaki ha konga maʻulalo. Ko ia ai, ʻoku fakanaunau ʻa e ngaahi sipinga fakaʻilekitulonika kotoa ʻo Hielscher ultrasonicators ʻaki ha polokalama ʻoku fakapotopoto, ʻa ia ʻoku ne fakaʻata koe ke ke liliu, vakaiʻi mo fakaleleiʻi ʻa e ngaahi fakangatangata sonication. Ultrasonic ʻoku mahuʻinga ʻa e fakangatangata ʻo e founga ngaue, hange ko e amplitude, mafana, malohi, sonication taimi, siakale ʻo e fatongia, mo e ngaahi ivi ki hono poupouʻi HMO ngaue ʻi he fermentation.
ʻoku ʻotometiki pe hono fakangatangata ʻo e polokalama fakakomipiuta Hielscher ultrasonicators ki he founga hono fakafekauʻaki ʻo e kaati SD. Ko e fakamatala ʻotometiki ki hono hiki ʻo e sonication, ko e fakavaʻe ia ʻo e founga ngaue standardization mo e reproducibility/repeatability, ʻa ia ʻoku fie maʻu ki he ngaahi founga ngaohi ʻo e Ngaohiʻanga koloa (GMP).
Ultrasonic Rectors for Fermentation
Hielscher offers ultrasonic probes of various size, length and geometries, which can be used for batch as well as continuous flow-through treatments. Ultrasonic reactors, also known as sono-bioreactors, are available for any volume covering the ultrasonic bioprocessing from small lab samples to pilot and fully-commercial production level.
ʻoku ʻiloʻi lelei pe ko e tuʻuʻanga ʻo e sonotrode ʻo e ultrasonic ʻi he vaka te ne takiekina hono tufaki ʻo e cavitation mo e streaming-Micro ʻi he founga Lotoloto. ʻoku totonu ke fili ʻa e Sonotrode mo ultrasonic Seinopolo ʻo fakatatau ki he fakahokohoko ʻo e broth toʻotoʻo. ʻi he lolotonga ʻo e sonication ʻe lava ke fakahoko ia ʻi he kulupu (batch) ʻe taha ʻi he maʻu meʻatokoni, ki he ngaahi polokalama maʻolunga ʻoku fokotuʻu atu ke fakaʻaongaʻi maʻu pe. Ko ʻetau hu atu ʻi he ultrasonic ki he feituʻu ko ia, ʻoku maʻu ʻe he ngaahi meʻa toʻotoʻo kotoa pe ʻa e ʻilo tatau ki sonication fakapapauʻi ʻa e faitoʻo lelei taha. Hielscher Ultrasonics ʻoku ʻi ai ha ngaahi ultrasonic probes ʻe niʻihi ʻoku nau haʻu fakataha ke fakatahatahaʻi ʻa e fokotuʻutuʻu lelei taha ultrasonic bioprocessing.
Ultrasonics 'o e Hielscher – Mei he loki fakatotolo ki he pailate ki he faiva
Hielscher Ultrasonics ʻoku ne kapui e tuʻunga maʻolunga ʻo e ngaahi meʻangaue ultrasonic ʻoku fai ki ai e tokoni ultrasonic homogenisers ki he sipinga teuteu ki he sea ʻi ʻolunga mo e pailate pea mo ha ngaahi ʻiuniti ngaue malohi ʻi he ultrasonic ʻa ia ʻoku faingofua hono fakahoko fonufonu loli 'emau he houa. ʻe lava ke fakakau ngofua ʻa e Hielscher ultrasonicators ʻi hono fokotuʻu mo fakaleleiʻi ʻa e ngaahi tafaʻaki kehekehe ʻo e paʻanga, ʻa ia ʻoku kau ki ai ʻa e niʻihi kotoa pe ʻoku nau toutou fakahoko, kuo ʻosi fafangaʻi-(batches) ʻo fakafou ʻi he hulu fokotu'utu'u.
ʻoku fakaʻata ʻe he ngaahi founga kehekehe mo e ngaahi konga fakapatonu ʻa e ola lelei hono fulihi hoʻo ultrasonic ki he ngaahi fie maʻu ki hoʻo founga ngaue.
ʻoku Hielscher ʻa e ngaue ko ia ki he 24/7 ʻi he ngaue kakato mo e fatongia mamafa ʻi he ngaahi tukunga ʻoku faingataʻa, pea ʻoku fie maʻu leva ke tokangaʻi ʻa e ultrasonic processors.
'Oku 'omi 'e he e tepile 'i lalo ha faka'ilonga ia 'o e tu'unga 'o e ngaue ki he fakafuofua'i 'o e hotau ultrasonicators:
|Kulupu (batch) 'o e tohi||'Oku tafe mai 'a e 'ea||'Oku fokotu'u atu 'a e ngaahi me'angaue|
|mL 'o e 1 ki he 500||10 ki he 200mL/miniti 'e||UP100H|
|mL 'i he 10 ki he 2000||20 ki he 400mL/miniti 'e||UP200Ht, UP400St|
|0.1 ki he 20L||0.2 ke 4L/miniti 'e||UIP2000hdT|
|10 ki he 100L||2 ki he 10L/miniti 'e||UIP4000hdT|
|n.a.||10 ki he 100L/miniti 'e||UIP16000|
|n.a.||lalahi||fakataha'i 'o e UIP16000|
Fetu'utaki mai kiate kimautolu! / Kole kiate kitautolu!
Ngaahi tohi/fakamo'oni fakafolofola
- Muschiol, Jan; Meyer, Anne S. (2019): A chemo-enzymatic approach for the synthesis of human milk oligosaccharide backbone structures. Zeitschrift für Naturforschung C, Volume 74: Issue 3-4, 2019. 85-89.
- Birgitte Zeuner, David Teze, Jan Muschiol, Anne S. Meyer (2019): Synthesis of Human Milk Oligosaccharides: Protein Engineering Strategies for Improved Enzymatic Transglycosylation. Molecules 24, 2019.
- Yun Hee Choi, Bum Seok Park, Joo‐Hyun Seo, Byung‐Gee Ki (2019): Biosynthesis of the human milk oligosaccharide 3‐fucosyllactose in metabolically engineered Escherichia coli via the salvage pathway through increasing GTP synthesis and β‐galactosidase modification. Biotechnology and Bioengineering Volume 116, Issue 12. December 2019.
- Balakrishnan Naveena, Patricia Armshaw, J. Tony Pembroke (2015): Ultrasonic intensification as a tool for enhanced microbial biofuel yields. Biotechnology of Biofuels 8:140, 2015.
- Shweta Pawar, Virendra K. Rathod (2020): Role of ultrasound in assisted fermentation technologies for process enhancements. Preparative Biochemistry & Biotechnology 50(6), 2020. 1-8.
Ngaahi mo'oni'i me'a 'oku mahu'inga ke 'ilo'i
Biosynthesis using Cell Factories
A microbial cell factory is a method of bioengineering, which utilizes microbial cells as a production facility. By genetically engineering microbes, the DNA of microorganisms such as bacteria, yeasts, fungi, mammalian cells, or algae is modified turning microbes into cell factories. Cell factories are used to convert substrates into valuable biological molecules, which are used e.g. in food, pharma, chemistry and fuel production. Different strategies of cell factory-based biosynthesis aim at the production of native metabolites, expression of heterologous biosynthetic pathways, or protein expression.
Cell factories can be used to either synthesize native metabolites, to express heterologous biosynthetic pathways, or to express proteins.
Biosynthesis of native metabolites
Native metabolites are defined as biological molecules, which the cells used as cell factory produce naturally. Cell factories produce these biological molecules either intracellularly or a secreted substance. The latter is preferred since it facilitates the separation and purification of the targeted compounds. Examples for native metabolites are amino and nucleic acids, antibiotics, vitamins, enzymes, bioactive compounds, and proteins produced from anabolic pathways of cell.
Heterologus Biosynthetic Pathways
When trying to produce an interesting compound, one of the most important decisions is the choice of production in the native host, and optimize this host, or transfer of the pathway to another well-known host. If the original host can be adapted to an industrial fermentation process, and there are no health-related risks in doing so (e.g., production of toxic by-products), this can be a preferred strategy (as was the case e.g., for penicillin). However, in many modern cases, the potential of using an industrially preferred cell factory and related platform processes out-weighs the difficulty of transferring the pathway.
The expression of proteins can be achieved via homologous and heterologous ways. In homologous expression, a gene that is naturally present in an organism is over-expressed. Through this over-expression, a higher yield of a certain biological molecule can be produced. For heterologous expression, a specific gene is transferred into a host cell in that the gene is not present naturally. Using cell engineering and recombinant DNA technology, the gene is inserted into the host’s DNA so that the host cell produces (large) amounts of a protein that it would not produce naturally. Protein expression is done in a variety of hosts from bacteria, e.g. E. coli and Bacillis subtilis, yeasts, e.g., Klyuveromyces lactis, Pichia pastoris, S. cerevisiae, filamentous fungi, e.g. as A. niger, and cells derived from multicellular organisms such as mammals and insects. Innummerous proteins are of great commercial interest, including from bulk enzymes, complex bio-pharmaceuticals, diagnostics and research reagents. (cf. A.M. Davy et al. 2017)