The industrial yeast Pichia pastoris is converted from a heterotroph into an autotroph capable of growth on CO2

Gassler, Thomas; Sauer, Michael; Gasser, Brigitte; Egermeier, Michael; Troyer, Christina; Causon, Tim J.; Hann, Stephan; Mattanovich, Diethard; Steiger, Matthias G.

doi:10.1038/s41587-019-0363-0

Cited by 238 publications

(199 citation statements)

References 53 publications

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“…Many reasons may lead to this problem, but inefficiency intermediates supplement may be an important reason. Therefore, Clarify intermediates recycle regulation mechanism in methanol utilization pathway maybe not only beneficial for other non-native methylotrophic microbes construction but also would promote other one-carbon source utilization [4,26]. In Saccharomyces cerevisiae, Tkl1 has been shown as the rate-limiting factor in NOP pathway [27].…”

Section: Discussionmentioning

confidence: 99%

“…As an important heterologous protein expression system, more than 1,000 proteins have been successfully expressed via P. pastoris, including angiostatin, a-glucosidase [1]and Anti-CEACAM5 nanobody [2,3]. Moreover, as the rare methylotrophic microbes, detailed methanol metabolic pathway especially important intermediates recycle pathway not only significantly promotes other 3 methylotrophic microbes construction but also promotes autotrophic microorganism design [4]. Many results showed that the robust alcohol oxidase 1 promoter (P AOX1 ) contributes most to this successful expression system [5].…”

Section: Introductionmentioning

confidence: 99%

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Methanol expression regulator 1 (Mxr1p) promotes xylulose 5-phosphate recycle via increaseing transketolase activity in Pichia pastoris

Zhan

Pan

Liu

et al. 2020

Preprint

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Background Methanol expression regulator 1 (Mxr1p) is a key transcription factor that plays a vital role in the methanol utilization pathway in Pichia pastoris ( P. pastoris ). Most genes referred to the methanol utilization pathway were regulated by Mxr1p. However, some genes did not show a significant difference between methanol and glycerol even though they play an important role in the methanol utilization pathway. So far, the regulation mechanism about these genes and the relationship with Mxr1p are still unknown. Results Methanol metabolic pathway analysis revealed that most of the methanol-induced genes were upregulated in transcriptional level when cultured in methanol. Whereas some genes like tkl1 (transketolase 1) did not show significant up-regulation in methanol even though it plays a very important role in Xu5P recycle, the reason is still not clear. To clarify this point, firstly, pull-down and MS experiments were performed. The result shows that Tkl1p protein combined with Mxr1p in vitro . Subsequently, this result was further confirmed by yeast two-hybrid in vivo , and the binding region mainly located from 150AA to 400AA. Moreover, Ser215 phosphorylation did not affect this interaction. In addition, Mxr1p-400AA integration into Δmxr1 could rescue cell growth in methanol. All the above results proved that Mxr1p played a post-translational role in the methanol utilization pathway and Mxr1p-400AA may achieved most of Mxr1p functions. Secondly, the function of Mxr1p-Tkl1p complex was expounded by detecting formaldehyde consumption and xylulose production in cell-free systems. Results showed that Mxr1p-Tkl1p mixture could promote formaldehyde consumption and xylulose production in vitro . Conclusion Mxr1p promotes methanol utilization via combining with Tkl1p to accelerate Xu5P recycle and this interaction was not affected by Ser215 phosphorylation.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Methanol expression regulator 1 (Mxr1p) promotes xylulose 5-phosphate recycle via increaseing transketolase activity in Pichia pastoris

Zhan

Pan

Liu

et al. 2020

Preprint

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“…Engineered compartmentalization has garnered considerable attention in recent years 7,[14][15][16][17][18] .…”

Section: Introductionmentioning

confidence: 99%

“…Organelles have also been harnessed for the reactions that they natively perform. For example, the endoplasmic reticulum and Golgi in Chinese Hamster Ovary cells are used for the glycosylation of heterologous proteins 26 , and recently the methanol assimilation pathway in the peroxisomes of Pichia pastoris was engineered into a carbon dioxide fixation pathway where six native peroxisome enzymes were utilized 17 . Advances in the development of synthetic compartments have also been reported, including the synthesis of prokaryotic encapsulin nanocompartments in eukaryotes 15,27 , bacterially-derived proteinaceous gas vesicles in mammalian cells 28 , and light-inducible phase-separated organelles in yeast 16 .…”

Section: Introductionmentioning

confidence: 99%

Repurposing the yeast peroxisome to compartmentalize a toxic enzyme enables improved (S)-reticuline production

Grewal¹,

Samson²,

Baker

et al. 2020

Preprint

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Eukaryotic cells compartmentalize metabolic pathways in organelles to achieve optimal reaction conditions and avoid crosstalk with other factors in the cytosol. Increasingly, engineers are researching ways in which synthetic compartmentalization could be used to address challenges in metabolic engineering. Here, we identified that norcoclaurine synthase (NCS), the enzyme which catalyzes the first committed reaction in benzylisoquinoline alkaloid (BIA) biosynthesis, is toxic when expressed cytosolically in Saccharomyces cerevisiae and, consequently, restricts (S)-reticuline production. We developed a compartmentalization strategy that alleviates NCS toxicity while promoting increased (S)-reticuline titer, achieved through efficient targeting of toxic NCS to the peroxisome while, crucially, taking advantage of the free flow of metabolite substrates and product across the peroxisome membrane. We identified that peroxisome protein capacity in S. cerevisiae becomes a limiting factor for further improvement of BIA production and demonstrate that expression of engineered transcription factors can mimic the oleate response for larger peroxisomes, further increasing BIA titer without the requirement for peroxisome induction with fatty acids. This work specifically addresses the challenges associated with toxic NCS expression and, more broadly, highlights the potential for engineering organelles with desired characteristics for metabolic engineering.

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“…Recent studies have made considerable progress in establishing carbon fixation pathways in heterotrophic organisms, with the longterm goal of achieving synthetic autotrophy, which could pave the way towards sustainable bioproduction schemes rooted in CO2 and renewable energy 4,5 . Most notably, overexpression of phosphoribulokinase and Rubisco, followed by long-term evolution, enabled the industrial hosts Escherichia coli 6,7 and Pichia pastoris 8 to synthesize all biomass from CO2 via the RuBP cycle. Also, overexpression of enzymes of the 3hydroxypropionate bicycle established the activity of different modules of this carbon fixation pathway in E. coli 9 .…”

Section: Introductionmentioning

confidence: 99%

Awakening a latent carbon fixation cycle inEscherichia coli

Satanowski

Dronsella

Noor

et al. 2020

Preprint

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Carbon fixation is one of the most important biochemical processes. Most natural carbon fixation pathways are thought to have emerged from enzymes that originally performed other metabolic tasks. Can we recreate the emergence of a carbon fixation pathway in a heterotrophic host by recruiting only endogenous enzymes? In this study, we address this question by systematically analyzing possible carbon fixation pathways composed only of Escherichia coli native enzymes. We identify the GED (Gnd-Entner-Doudoroff) cycle as the simplest pathway that can operate with high thermodynamic driving force. This autocatalytic route is based on reductive carboxylation of ribulose 5-phosphate (Ru5P) by 6-phosphogluconate dehydrogenase (Gnd), followed by reactions of the Entner-Doudoroff pathway, gluconeogenesis, and the pentose phosphate pathway. We demonstrate the in vivo feasibility of this new-to-nature pathway by constructing E. coli gene deletion strains whose growth on pentose sugars depends on the GED shunt, a linear variant of the GED cycle which does not require the regeneration of Ru5P. Several metabolic adaptations, most importantly the increased production of NADPH, assist in establishing sufficiently high flux to sustain this growth. Our study exemplifies a trajectory for the emergence of carbon fixation in a heterotrophic organism and demonstrates a synthetic pathway of biotechnological interest.

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The industrial yeast Pichia pastoris is converted from a heterotroph into an autotroph capable of growth on CO2

Cited by 238 publications

References 53 publications

Methanol expression regulator 1 (Mxr1p) promotes xylulose 5-phosphate recycle via increaseing transketolase activity in Pichia pastoris

Methanol expression regulator 1 (Mxr1p) promotes xylulose 5-phosphate recycle via increaseing transketolase activity in Pichia pastoris

Repurposing the yeast peroxisome to compartmentalize a toxic enzyme enables improved (S)-reticuline production

Awakening a latent carbon fixation cycle inEscherichia coli

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