Green Process: Improved Semi-Continuous Fermentation of Pichia pastoris Based on the Principle of Vitality Cell Separation

Wang, Denggang; Li, Wenjie; Zhang, Xinying; Liang, Shuli; Lin, Ying

doi:10.3389/fbioe.2021.777774

Cited by 3 publications

(2 citation statements)

References 46 publications

(55 reference statements)

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“…The method of PI staining refers to the previous studies in our laboratory [ 21 ]. The cells were collected at 6,000 rpm for 1 min, washed three times in 10 mM phosphate-buffered saline (PBS, pH 7.4).…”

Section: Methodsmentioning

confidence: 99%

Metabolic engineering of Komagataella phaffii for the efficient utilization of methanol

Wang,

Li,

Zhao

et al. 2024

Microb Cell Fact

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Background Komagataella phaffii, a type of methanotrophic yeast, can use methanol, a favorable non-sugar substrate in eco-friendly bio-manufacturing. The dissimilation pathway in K. phaffii leads to the loss of carbon atoms in the form of CO2. However, the ΔFLD strain, engineered to lack formaldehyde dehydrogenase—an essential enzyme in the dissimilation pathway—displayed growth defects when exposed to a methanol-containing medium. Results Inhibiting the dissimilation pathway triggers an excessive accumulation of formaldehyde and a decline in the intracellular NAD+/NADH ratio. Here, we designed dual-enzyme complex with the alcohol oxidase1/dihydroxyacetone synthase1 (Aox1/Das1), enhancing the regeneration of the formaldehyde receptor xylulose-5-phosphate (Xu5P). This strategy mitigated the harmful effects of formaldehyde accumulation and associated toxicity to cells. Concurrently, we elevated the NAD+/NADH ratio by overexpressing isocitrate dehydrogenase in the TCA cycle, promoting intracellular redox homeostasis. The OD600 of the optimized combination of the above strategies, strain DF02-1, was 4.28 times higher than that of the control strain DF00 (ΔFLD, HIS4+) under 1% methanol. Subsequently, the heterologous expression of methanol oxidase Mox from Hansenula polymorpha in strain DF02-1 resulted in the recombinant strain DF02-4, which displayed a growth at an OD600 4.08 times higher than that the control strain DF00 in medium containing 3% methanol. Conclusions The reduction of formaldehyde accumulation, the increase of NAD+/NADH ratio, and the enhancement of methanol oxidation effectively improved the efficient utilization of a high methanol concentration by strain ΔFLD strain lacking formaldehyde dehydrogenase. The modification strategies implemented in this study collectively serve as a foundational framework for advancing the efficient utilization of methanol in K. phaffii.

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Section: Methodsmentioning

confidence: 99%

Metabolic engineering of Komagataella phaffii for the efficient utilization of methanol

Wang,

Li,

Zhao

et al. 2024

Microb Cell Fact

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“…Among eukaryotes, Pichia pastoris has proven to be an ideal host to produce recombinant proteins. So far, P. pastoris has yielded almost 5000 recombinant proteins, [ 1,2 ] and current studies are targeted to better understand the cellular behavior of the organism. The capacity to grow on low‐cost media, ease of genetic manipulation, and the ability for post‐translational modification are some of the reasons for the prominence of this host in bioprocesses.…”

Section: Introductionmentioning

confidence: 99%

Macroscopic modeling of the growth and substrate consumption of wild type and genetically modified Pichia pastoris

Joseph,

Akkermans,

Van Impe

2023

Biotechnology Journal

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Pichia pastoris is a popular yeast platform to generate several industrially relevant products which have applications in a wide range of sectors. The complexities in the processes due to the addition of a foreign gene are not widely explored. Since these complexities can be dependent on the strain characteristics, promoter, and type of protein produced, it is vital to investigate the growth and substrate consumption patterns of the host to facilitate customized process optimization. In this study, the growth rates of P. pastoris GS115 wild type (WT) and genetically modified (GM) strains grown on glycerol and methanol in batch cultivation mode were estimated and the model providing the best representation of the true growth kinetics based on substrate consumption was identified. It was observed that the growth of P. pastoris exhibits Haldane kinetics on glycerol rather than the most commonly used Monod kinetics due to the inability of the latter to describe growth inhibition at high concentrations of glycerol. Whereas, the cardinal parameter model, a newly proposed model for this application, was found to be the best fitting to describe the growth of P. pastoris on methanol due to its ability to describe methanol toxicity. Interestingly, the findings from this study concluded that in both substrates, the genetically engineered strain exhibited a higher growth rate compared to the WT strain. Such an observation has not been established yet in other published works, indicating an opportunity to further optimize the carbon source feeding strategies when the host is grown in fed‐batch mode.

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Continuous cell recycling in methylotrophic yeast Pichia pastoris to enhance product yields: a case study with Yarrowia lipolytica lipase Lip2

Mohanty,

Babbal,

Chauhan

et al. 2024

Biotechnol Bioproc E

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Green Process: Improved Semi-Continuous Fermentation of Pichia pastoris Based on the Principle of Vitality Cell Separation

Cited by 3 publications

References 46 publications

Metabolic engineering of Komagataella phaffii for the efficient utilization of methanol

Metabolic engineering of Komagataella phaffii for the efficient utilization of methanol

Macroscopic modeling of the growth and substrate consumption of wild type and genetically modified Pichia pastoris

Continuous cell recycling in methylotrophic yeast Pichia pastoris to enhance product yields: a case study with Yarrowia lipolytica lipase Lip2

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