Towards systems metabolic engineering in Pichia pastoris

Schwarzhans, Jan Philipp; Luttermann, Tobias; Geier, Martina; Kalinowski, Jörn; Friehs, Karl

doi:10.1016/j.biotechadv.2017.07.009

Cited by 124 publications

(85 citation statements)

References 292 publications

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“…1a), targeting our search to the yeast P. pastoris . Long favored as a host for heterologous protein production 28 , P. pastoris has recently emerged as a promising chassis for metabolic engineering applications owing to its growth to high cell densities and its excellent protein expression capabilities 29 . In addition, its methanol utilization (MUT) pathway represents one of the largest sets of tightly co-regulated genes in nature, offering transcriptional repression via glucose and inducibility via methanol 30 , making it an ideal target for BDP mining.…”

Section: Resultsmentioning

confidence: 99%

Engineered bidirectional promoters enable rapid multi-gene co-expression optimization

et al. 2018

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Numerous synthetic biology endeavors require well-tuned co-expression of functional components for success. Classically, monodirectional promoters (MDPs) have been used for such applications, but MDPs are limited in terms of multi-gene co-expression capabilities. Consequently, there is a pressing need for new tools with improved flexibility in terms of genetic circuit design, metabolic pathway assembly, and optimization. Here, motivated by nature’s use of bidirectional promoters (BDPs) as a solution for efficient gene co-expression, we generate a library of 168 synthetic BDPs in the yeast Komagataella phaffii (syn. Pichia pastoris), leveraging naturally occurring BDPs as a parts repository. This library of synthetic BDPs allows for rapid screening of diverse expression profiles and ratios to optimize gene co-expression, including for metabolic pathways (taxadiene, β-carotene). The modular design strategies applied for creating the BDP library could be relevant in other eukaryotic hosts, enabling a myriad of metabolic engineering and synthetic biology applications.

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Section: Resultsmentioning

confidence: 99%

Engineered bidirectional promoters enable rapid multi-gene co-expression optimization

et al. 2018

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“…In contrast to its well‐known function as a protein expression system, the potential of P. pastoris as a cell factory for the production of chemicals has been largely underestimated. Nevertheless, increasing number of studies reported using P. pastoris as a microbial cell factory for the production of chemicals either via biocatalysis or more recently via fermentation . This paper aims to outline the major progress made in recent years toward developing P. pastoris as an efficient yeast cell factory with emphasis on its role as a whole‐cell catalyst for chemical production.…”

Section: Introductionmentioning

confidence: 99%

Pichia pastoris as a Versatile Cell Factory for the Production of Industrial Enzymes and Chemicals: Current Status and Future Perspectives

Zhu

Sun

2019

Biotechnology Journal

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With nearly three decades of development, Pichia pastoris (P. pastoris) has become a powerful eukaryotic protein expression system for the expression of thousands of proteins both on a laboratory and industrial scale. In addition, it has also been extensively used as a cell factory for the production of a variety of chemicals. This review summarizes the bottlenecks and solutions in achieving high-level secretory protein expression with P. pastoris and then outlines its applications on chemical production with an emphasis on its role as whole-cell biocatalyst. Furthermore, current challenges and future directions of this important system are also discussed.

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“…In many previous studies, the classic AOX1 promoter (P AOX1 ), including P AOX1 ‐derived variants, was mostly used for recombinant protein expression in P. pastoris owing to its strong and tightly regulated features . From the industrial perspective, however, the main drawbacks of this type of strain are the high amount of heat production and oxygen requirement as well as the presence of methanol as a toxic and flammable substance in the upstream and sometimes downstream stages . Glyceraldehyde 3‐phosphate dehydrogenase (GAP) promoter is one of the alternative natural promoters for methanol‐free protein expression without any requirement for induction by shifting the culture from one carbon source to another .…”

Section: Introductionmentioning

confidence: 99%

“…2,3 From the industrial perspective, however, the main drawbacks of this type of strain are the high amount of heat production and oxygen requirement as well as the presence of methanol as a toxic and flammable substance in the upstream and sometimes downstream stages. [11][12][13][14] Glyceraldehyde 3-phosphate dehydrogenase (GAP) promoter is one of the alternative natural promoters for methanol-free protein expression without any requirement for induction by shifting the culture from one carbon source to another. 14 Apart from natural promoters, since the last decade, there has also been growing interest in using other synthetic-engineered promoters to increase the production rate without the major challenges related to P AOX1.…”

Section: Introductionmentioning

confidence: 99%

Comparative study of μ‐stat methanol feeding control in fed‐batch fermentation of Pichia pastoris producing HBsAg: an open‐loop control versus recurrent artificial neural network‐based feedback control

Beiroti

Hosseini

Aghasadeghi

et al. 2019

J of Chemical Tech & Biotech

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BACKGROUND In recent decades, artificial neural network (ANN) has been shown to be a robust and promising tool in monitoring and controlling bioprocess systems. In a previous study, the authors designed a highly accurate and precise recurrent neural network (RNN) for predicting the biomass amount of recombinant Pichia pastoris Mut+ producing intracellular hepatitis B surface antigen (HBsAg) during fed‐batch methanol fermentation. In the current work, the aim was to compare the production efficiency of HBsAg between conventional predefined μ‐stat methanol feeding control (open‐loop control) – already established in large‐scale production – and methanol feeding based on a μ‐stat feedback control system using RNN. For this purpose, for each methanol feeding strategy, bench‐scale, fed‐batch fermentation processes were carried out twice. RESULTS According to the results, in contrast to the established μ‐stat predefined feeding strategy (open‐loop), the deviation of specific growth rate and biomass in μ‐stat feedback control based on RNN was negligible. Also, in the suggested methanol feeding control strategy, the HBsAg titer, specific productivity and yield between the performed fed‐batch fermentations – unlike the conventional method – were approximately identical, with average values of 110.8 μg mL−1, 1.52 μg g−1 dry biomass and 0.34 μg g−1 MeOH respectively. CONCLUSION Comparing the biomass growth pattern and HBsAg production efficiency with the conventional μ‐stat predefined feeding in open‐loop control, the new proposed feeding control system illustrated significantly high process efficiency. This reliable control system based on ANN can have many applications in the biopharmaceutical industry for the control of process key parameters as well as for enhancing process efficiency. © 2019 Society of Chemical Industry

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Towards systems metabolic engineering in Pichia pastoris

Cited by 124 publications

References 292 publications

Engineered bidirectional promoters enable rapid multi-gene co-expression optimization

Engineered bidirectional promoters enable rapid multi-gene co-expression optimization

Pichia pastoris as a Versatile Cell Factory for the Production of Industrial Enzymes and Chemicals: Current Status and Future Perspectives

Comparative study of μ‐stat methanol feeding control in fed‐batch fermentation of Pichia pastoris producing HBsAg: an open‐loop control versus recurrent artificial neural network‐based feedback control

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