Dataset Information

L-Asparaginase from Penicillium sizovae Produced by a Recombinant Komagataella phaffii Strain.

ABSTRACT: L-asparaginase is an important enzyme in the pharmaceutical field used as treatment for acute lymphoblastic leukemia due to its ability to hydrolyze L-asparagine, an essential amino acid synthesized by normal cells, but not by neoplastic cells. Adverse effects of L-asparaginase formulations are associated with its glutaminase activity and bacterial origin; therefore, it is important to find new sources of L-asparaginase produced by eukaryotic microorganisms with low glutaminase activity. This work aimed to identify the L-asparaginase gene sequence from Penicillium sizovae, a filamentous fungus isolated from the Brazilian Savanna (Cerrado) soil with low glutaminase activity, and to biosynthesize higher yields of this enzyme in the yeast Komagataella phaffii. The L-asparaginase gene sequence of P. sizovae was identified by homology to L-asparaginases from species of Penicillium of the section Citrina: P. citrinum and P. steckii. Partial L-asparaginase from P. sizovae, lacking the periplasmic signaling sequence, was cloned, and expressed intracellularly with highest enzymatic activity achieved by a MUT⁺ clone cultured in BMM expression medium; a value 5-fold greater than that obtained by native L-asparaginase in P. sizovae cells. To the best of our knowledge, this is the first literature report of the heterologous production of an L-asparaginase from a filamentous fungus by a yeast.

SUBMITTER: Freitas M

PROVIDER: S-EPMC9227789 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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L-Asparaginase from <i>Penicillium sizovae</i> Produced by a Recombinant <i>Komagataella phaffii</i> Strain.

Freitas Marcela M Souza Paula P Homem-de-Mello Mauricio M Fonseca-Bazzo Yris M YM Silveira Damaris D Ferreira Filho Edivaldo X EX Pessoa Junior Adalberto A Sarker Dipak D Timson David D Inácio João J Magalhães Pérola O PO

Pharmaceuticals (Basel, Switzerland) 20220614 6

L-asparaginase is an important enzyme in the pharmaceutical field used as treatment for acute lymphoblastic leukemia due to its ability to hydrolyze L-asparagine, an essential amino acid synthesized by normal cells, but not by neoplastic cells. Adverse effects of L-asparaginase formulations are associated with its glutaminase activity and bacterial origin; therefore, it is important to find new sources of L-asparaginase produced by eukaryotic microorganisms with low glutaminase activity. This wo ...[more]

PMID: 35745665

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Project description:BackgroundThe β-galactosidase from Paenibacillus wynnii (β-gal-Pw) is a promising candidate for lactose hydrolysis in milk and dairy products, as it has a higher affinity for the substrate lactose (low KM value) compared to industrially used β-galactosidases and is not inhibited by the hydrolysis-generated product D-galactose. However, β-gal-Pw must firstly be produced cost-effectively for any potential industrial application. Accordingly, the yeast Komagataella phaffii was chosen to investigate its feasibility to recombinantly produce β-gal-Pw since it is approved for the regulated production of food enzymes. The aim of this study was to find the most suitable way to produce the β-gal-Pw in K. phaffii either extracellularly or intracellularly.ResultsFirstly, 11 different signal peptides were tested for extracellular production of β-gal-Pw by K. phaffii under the control of the constitutive GAP promoter. None of the signal peptides resulted in a secretion of β-gal-Pw, indicating problems within the secretory pathway of this enzyme. Therefore, intracellular β-gal-Pw production was investigated using the GAP or methanol-inducible AOX1 promoter. A four-fold higher volumetric β-galactosidase activity of 7537 ± 66 µkatoNPGal/Lculture was achieved by the K. phaffii clone 27 using the AOX1 promoter in fed-batch bioreactor cultivations, compared to the clone 5 using the GAP promoter. However, a two-fold higher specific productivity of 3.14 ± 0.05 µkatoNPGal/gDCW/h was achieved when using the GAP promoter for β-gal-Pw production compared to the AOX1 promoter. After partial purification, a β-gal-Pw enzyme preparation with a total β-galactosidase activity of 3082 ± 98 µkatoNPGal was obtained from 1 L of recombinant K. phaffii culture (using AOX1 promoter).ConclusionThis study showed that the β-gal-Pw was produced intracellularly by K. phaffii, but the secretion was not achieved with the signal peptides chosen. Nevertheless, a straightforward approach to improve the intracellular β-gal-Pw production with K. phaffii by using either the GAP or AOX1 promoter in bioreactor cultivations was demonstrated, offering insights into alternative production methods for this enzyme.

Project description:The yeast Komagataella phaffii has become a popular host strain among biotechnology start-up companies for producing recombinant proteins for food and adult nutrition applications. Komagataella phaffii is a host of choice due to its long history of safe use, open access to protocols and strains, a secretome free of host proteins and proteases, and contract manufacturing organizations with deep knowledge in bioprocess scale-up. However, a recent publication highlighted the abundance of an unknown polysaccharide that accumulates in the supernatant during fermentation. This poses a significant challenge in using K. phaffii as a production host. This polysaccharide leads to difficulties in achieving high purity products and requires specialized and costly downstream processing steps for removal. In this study, we describe the use of the common K. phaffii host strain YB-4290 for production of the bioactive milk protein lactoferrin. Upon purification of lactoferrin using membrane-based separation methods, significant amounts of carbohydrate were copurified with the protein. It was determined that the carbohydrate is mostly composed of mannose residues with minor amounts of glucose and glucosamine. The polysaccharide fraction has an average molecular weight of 50 kDa and consists mainly of mannan, galactomannan, and amylose. In addition, a large fraction of the carbohydrate has an unknown structure likely composed of oligosaccharides. Additional strains were tested in fermentation to further understand the source of the carbohydrates. The commonly used industrial hosts, BG10 and YB-4290, produce a basal level of exopolysaccharide; YB-4290 producing slightly more than BG10. Overexpression of recombinant protein stimulates exopolysaccharide production well above levels produced by the host strains alone. Overall, this study aims to provide a foundation for developing methods to improve the economics of recombinant protein production using K. phaffii as a production host.One-sentence summaryOverexpression of recombinant protein stimulates the hyperproduction of high-molecular-weight, mannose-based, exopolysaccharides by the industrial yeast Komagataella phaffii.

Project description:BackgroundSpecific productivity (qP) in yeast correlates with growth, typically peaking at intermediate or maximum specific growth rates (μ). Understanding the factors limiting productivity at extremely low μ might reveal decoupling strategies, but knowledge of production dynamics and physiology in such conditions is scarce. Retentostats, a type of continuous cultivation, enable the well-controlled transition to near-zero µ through the combined retention of biomass and limited substrate supply. Recombinant Komagataella phaffii (syn Pichia pastoris) secreting a bivalent single domain antibody (VHH) was cultivated in aerobic, glucose-limited retentostats to investigate recombinant protein production dynamics and broaden our understanding of relevant physiological adaptations at near-zero growth conditions.ResultsBy the end of the retentostat cultivation, doubling times of approx. two months were reached, corresponding to µ = 0.00047 h-1. Despite these extremely slow growth rates, the proportion of viable cells remained high, and de novo synthesis and secretion of the VHH were observed. The average qP at the end of the retentostat was estimated at 0.019 mg g-1 h-1. Transcriptomics indicated that genes involved in protein biosynthesis were only moderately downregulated towards zero growth, while secretory pathway genes were mostly regulated in a manner seemingly detrimental to protein secretion. Adaptation to near-zero growth conditions of recombinant K. phaffii resulted in significant changes in the total protein, RNA, DNA and lipid content, and lipidomics revealed a complex adaptation pattern regarding the lipid class composition. The higher abundance of storage lipids as well as storage carbohydrates indicates that the cells are preparing for long-term survival.ConclusionsIn conclusion, retentostat cultivation proved to be a valuable tool to identify potential engineering targets to decouple growth and protein production and gain important insights into the physiological adaptation of K. phaffii to near-zero growth conditions.

Dataset Information

L-Asparaginase from Penicillium sizovae Produced by a Recombinant Komagataella phaffii Strain.

Publications

L-Asparaginase from <i>Penicillium sizovae</i> Produced by a Recombinant <i>Komagataella phaffii</i> Strain.

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