Pathway design for mixotrophic production of biochemicals from CO2 and methanol in yeasts

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Lactic acid production in the synthetic autotroph Komagataella phaffii

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Increasing the carbon efficiency of citric acid production

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Conversion of CO2 into organic acids by engineered autotrophic yeast

The increase of CO2 emissions due to human activity is one of the preeminent reasons for the present climate crisis. In addition, considering the increasing demand for renewable resources, the upcycling of CO2 as a feedstock gains an extensive importance to establish CO2-neutral or CO2-negative industrial processes independent of agricultural resources. Here we assess whether synthetic autotrophic Komagataella phaffii (Pichia pastoris) can be used as a platform for value-added chemicals using CO2 as a feedstock by integrating the heterologous genes for lactic and itaconic acid synthesis. 13C labeling experiments proved that the resulting strains are able to produce organic acids via the assimilation of CO2 as a sole carbon source. Further engineering attempts to prevent the lactic acid consumption increased the titers to 600 mg L−1, while balancing the expression of key genes and modifying screening conditions led to 2 g L−1 itaconic acid. Bioreactor cultivations suggest that a fine-tuning on CO2 uptake and oxygen demand of the cells is essential to reach a higher productivity. We believe that through further metabolic and process engineering, the resulting engineered strain can become a promising host for the production of value-added bulk chemicals by microbial assimilation of CO2, to support sustainability of industrial bioprocesses

Slow Growth and Increased Spontaneous Mutation Frequency in Respiratory Deficient afo1- Yeast Suppressed by a Dominant Mutation in ATP3

A yeast deletion mutation in the nuclear-encoded gene, AFO1, which codes for a mitochondrial ribosomal protein, led to slow growth on glucose, the inability to grow on glycerol or ethanol, and loss of mitochondrial DNA and respiration. We noticed that afo1- yeast readily obtains secondary mutations that suppress aspects of this phenotype, including its growth defect. We characterized and identified a dominant missense suppressor mutation in the ATP3 gene. Comparing isogenic slowly growing rho-zero and rapidly growing suppressed afo1- strains under carefully controlled fermentation conditions showed that energy charge was not significantly different between strains and was not causal for the observed growth properties. Surprisingly, in a wild-type background, the dominant suppressor allele of ATP3 still allowed respiratory growth but increased the petite frequency. Similarly, a slow-growing respiratory deficient afo1- strain displayed an about twofold increase in spontaneous frequency of point mutations (comparable to the rho-zero strain) while the suppressed strain showed mutation frequency comparable to the repiratory-competent WT strain. We conclude, that phenotypes that result from afo1- are mostly explained by rapidly emerging mutations that compensate for the slow growth that typically follows respiratory deficiency

Metabolik mühendislik yaklaşımıyla geliştirilen Pichia Pastoris ile hücre dışı termostabil glukoz izomeraz üretimi.

The aim of this study is to develop a metabolically engineered P. pastoris strain for production of an active extracellular thermostable glucose isomerase (GI) enzyme by using genetic engineering techniques. For this purpose, research program was performed in two sub-programs. In the first sub-program, xylA gene from Thermus thermophilus was amplified and inserted into pPICZα-A expression vector. Thereafter, this pPICZα-A::xylA vector was cloned into AOX1 locus in P. pastoris genome and expressed under alcohol oxidase promoter which is induced by methanol. After constructing the recombinant P. pastoris strains, the best producing strain was selected according to the specific enzyme activity assay and SDS-PAGE analyses in batch shaker-bioreactor experiments. The selected recombinant P. pastoris clone carrying xylA gene in its genome was named as eP20. Using recombinant P. pastoris eP20 clone, effects of salt and sorbitol concentration on the cell growth and recombinant GI activity were investigated. The data obtained from the experiments showed that the maximum cell and GI activity values were obtained in production medium that contained 30 g L-1 sorbitol, 4.35 g L-1 ammonium sulphate, 0.1 M potassium phosphate buffer (pH 6.0), 14.9 g L-1 MgSO4•7H2O, 1.17 g L-1 CaSO4 •2H2O, 1 ml L-1 chloramphenicol and 4.35 ml L-1 PTM1; where, the maximum biomass and recombinant GI activity were calculated , respectively, as 6.3 g L-1 and 3.21 U L-1. Moreover, the research program related with the effect of initial sorbitol concentration shows that optimum initial sorbitol concentration, CS0 is 50 g L-1 that resulted a cell concentration and recombinant GI activity which are 7.32 g L-1 and 3.6 U L-1, respectively. In the second part of the M.Sc. of the study, a pilot scale bioreactor experiment in a working volume of 1 L was performed in controlled bioreactor system. The variations in the cell growth, recombinant GI activity, AOX activity, total protease activity and organic acid concentrations throughout the fermentation were analyzed whereas the specific growth rates, yield coefficients and specific consumption rates were also calculated. The results showed that a pH and oxygen controlled operation enabled an important increase in recombinant GI activity. In this context, recombinant GI activity was increased as 56.1-fold and resulted in 202.8 U L-1 at t=12 whereas the maximum biomass concentration was obtained as 85.2 g L-1 at t=36. In this study, an active thermostable recombinant GI enzyme was produced extracellularly by a yeast cell, i.e. recombinant P. pastoris, for the first time.M.S. - Master of Scienc

Transkripsiyon mühendisliği yöntemleriyle tasarlanan GAP promotoru altında pichia pastoris ile rekombinant protein üretiminin geliştirilmesi.

The objective of this PhD thesis is to enhance the expression strength of glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP) for improved recombinant protein (r-protein) production through modifying the transcription factors (TF) that regulate the functioning of PGAP by transcriptional engineering. PGAP was analyzed in terms of putative TF binding sites. A synthetic library was constructed with distinct regulatory properties through deletion and duplication of these putative transcription factor binding sites and selected TFs genes were overexpressed or deleted to understand their roles on r-protein production. Using enhanced green fluorescent protein (eGFP) as the first model protein, an expression strength in a range between 0.35- and 3.10-fold of the wild-type PGAP was obtained in Pichia pastoris. Another model protein, recombinant human growth hormone (rhGH) was produced under control of selected promoter variants, i.e. P9 (Gal4-like binding site was duplicated) and P10 (Rfx1 binding site was deleted), and 2.4- to 1.6-fold higher product titers were reached compared to wild-type PGAP in 24 deep well plates, respectively. Finally, combining two approaches resulted in 2.2-fold increase in wet cell weight specific rhGH yield with P9 that was coupled with overexpression of a Gal4-like TF compared to PGAP in fed-batch bioreactor cultivation. In addition to its role on enhancing r-protein production under PGAP, the effect of overexpression of Gal4-like TF on central carbon metabolism of P. pastoris was investigated. The specific glucose consumption rate and ethanol production rates were increased by 1.7- and 8-fold in P. pastoris Gal4-like transcription factor overexpression mutants compared to wild-type P. pastoris, respectively. 13C labelled metabolic flux and RNASeq analyses results exhibited that overexpression of Gal4-like TF caused downregulation in pentose phosphate pathway and increased the fluxes through lower glycolysis pathway significantly. This higher and imbalanced glycolytic flux caused an overflow metabolism which triggered the fermentative pathway. Taken together, it was revealed that overexpression of Gal4-like transcription factor resulted in a switch from Crabtree negative to Crabtree positive behavior.Ph.D. - Doctoral Progra

Kuzu ayaklarından bazı ekstrasellüler matriks bileşenlerinin (Kolajen ve GAG) ultrason destekli ekstraksiyonu ve karakterizasyonu

Bu tez çalışmasında, ekstrasellüler matriks bileşenlerinden kolajen ve glukozaminoglikanların (GAG) ultrason destekli esktraksiyonu ve saflaştırılması hedeflenmiştir. Çalışma kapsamında, kuzu ayakları, hammadde kaynağı olarak kullanılmış ve en yüksek düzeyde verim eldesi için uygun ekstraksiyon koşulları belirlenmiştir. Farklı sıc aklık ve sürelerde uygulanan ultrason işleminin kolajen ve GAG yapısı üzerine etkileri incelenmiştir. Çalışmanın ilk aşamasında, kolajen eldesi için uygun ekstraksiyon parametreleri belirlenmiştir. Ekstraksiyon işleminde enzimatik hidroliz yöntemi seçilmiş ve pepsin enzimi kullanılmıştır. Ön işlemleri tamamlanan kuzu ayakları, ekstraksiyon işlemi için % 0.1 (w/v) pepsin içeren 1:20 (w/v) oranda %5'lik laktik asit çözeltisi içinde 6 saat bekletilmiştir. Ardından 3 farklı sıcaklık (20± 2°C, 25± 2°C ve 30± 2°C) ve sürede (20, 40, 60 dk.) ultrason destekli ekstraksiyon gerçekleştirilmiştir. Ekstraksiyon işleminin ardından örnekler saflaştırma işlemi için diyaliz edilerek -56°C'de 32 sa boyunca dondurarak kurutulmuştur. En yüksek kolajen konsantrasyonuna 25°C-60 dk parametresinde (5061,75±171,41 μg/μl) ulaşılmıştır. SDS-PAGE elektroforez ve FTIR analizi sonuçları, farklı sıcaklık ve sürelerde uygulanan ultrason işleminin, kolajen yapısına zarar vermediğini ve tüm kolajenlerin yapısal bütünlüğünü koruduğunu göstermiştir. SEM analizi sonucunda, tüm kolajen örneklerinin lifli ve gözenekli yapıya sahip olduğu gözlenmiştir. viii Kolajen örneklerinin b* ve L* değerleri arasındaki fark istatistiksel olarak anlamlı bulunmuştur. Örneklerin a* değerleri arasındaki fark ise sadece dört grup için anlamlı bulunmuştur (p0.05). Çalışmanın ikinci aşamasında, GAG eldesi için uygun ekstraksiyon parametreleri belirlenmiştir. Ekstraksiyon işleminde papain enzimi ve sodyum asetat çözeltisi kullanılmıştır. Katı:çözgen oranı 1:20 (w/v), enzim miktarı (5 mg papain / 1 gr kuzu ayağı), sıcaklık (60°C) ve ekstraksiyon süresi 20 saat olarak belirlenmiştir. 20 saat ekstraksiyon işleminin ardından ultrason uygulamasının etkilerinin incelenmesi amacıyla, 3 farklı sıcaklık (60± 2°C, 65± 2°C ve 70± 2°C) ve sürede (20, 40, 60 dk.) ultrason destekli ekstraksiyon gerçekleştirilmiştir. Ekstraksiyon işleminin ardından örnekler saflaştırma işlemi için diyaliz edilerek -56°C'de 24 sa boyunca dondurarak kurutulmuştur. En yüksek kondroitin sülfat konsantrasyonuna 60°C-20 dk parametresinde (471,38±49,96 mg/L) ulaşılmıştır. FTIR analizi sonucunda, farklı ekstraksiyon koşulları altında elde edilen GAG örnekleri ile standarda ait spektrumlarının benzerlik gösterdiği görülmüştür. SEM analizi sonuçları, GAG örneklerindeki kümeleşmiş yapının ultrason süresi ve sıcaklığına bağlı olarak değiştiğini göstermiştir. Genel olarak farklı sıcaklık ve sürelerde uygulana ultrason işleminin GAG örneklerinin renk değerleri üzerine anlamlı bir etkisi olmadığı görülmüştür.In this thesis, ultrasound-assisted extraction and purification of collagen and glucosaminoglycans (GAG), one of the extracellular matrix components, was aimed. Within the scope of the study, lamb legs were used as raw material source and suitable extraction conditions were determined for obtaining the highest yield The effects of ultrasound treatment on collagen and GAG structure were investigated at different temperatures and durations. In the first stage of the study, suitable extraction parameters for the production of collagen were determined. Enzymatic hydrolysis method was selected for extraction and pepsin enzyme was used. The lambs which were pre-treated were left for 6 hours in a 1:20 (w / v) 5% lactic acid solution containing 0.1% (w / v) pepsin for extraction. Then, ultrasound-assisted extraction was performed at 3 different temperatures (20± 2°C, 25± 2°C and 30± 2°C) and time (20, 40, 60 min). After extraction, the samples were dialyzed for purification and freeze-dried at -56 ° C for 32 hours. The highest collagen parameter was reached at 25°C-60 min (5061,75±171,41 μg/μl). The results of SDS-PAGE electrophoresis and FTIR analysis showed that ultrasound applied at different temperatures and times did not damage the collagen structure and retain the structural integrity of all collagen. As a result of SEM analysis, it was observed that all collagen samples have fibrous and porous structure. The difference between b * and L * values of collagen samples was statistically significant x (p0.05). The difference between the a * values of the samples was significant only for the four groups. In the second stage of the study, suitable extraction parameters for GAG were determined. Papain enzyme and sodium acetate solution were used in the extraction process. The solid to solvent ratio was 1:20 (w / v), the amount of enzyme (5 mg papain / 1 g lamb's foot), temperature (60°C) and extraction time was determined as 20 hours. After 20 hours of extraction, ultrasound assisted extraction was performed in 3 different temperatures (60± 2°C, 65± 2°C and 70± 2°C) and time (20, 40, 60 min) to examine the effects of ultrasound application. After extraction, the samples were dialyzed for purification and freeze-dried at -56°C for 24 hours. The highest chondroitin sulfate concentration was reached at 60°C-20 min (471,38±49,96 mg/L). As a result of FTIR analysis, it was seen that GAG samples obtained under different extraction conditions were similar to the spectra of the standard. The results of SEM analysis showed that the clustered structure of GAG samples changed depending on the ultrasound time and temperature