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Genetic and physiological basis for antibody production by Kluyveromyces marxianus.

ABSTRACT: Kluyveromyces marxianus is a thermotolerant, crabtree-negative yeast, which preferentially directs metabolism (e.g., from the tricarboxylic acid cycle) to aerobic alcoholic fermentation. Thus K. marxianus has great potential for engineering to produce various materials under aerobic cultivation conditions. In this study, we engineered K. marxianus to produce and secrete a single-chain antibody (scFv), a product that is highly valuable but has historically proven difficult to generate at large scale. scFv production was obtained with strains carrying either plasmid-borne or genomically integrated constructs using various combinations of promoters (P MDH1 or P ACO1 ) and secretion signal peptides (KmINUss or Sc?-MFss). As the wild-type K. marxianus secretes endogenous inulinase predominantly, the corresponding INU1 gene was disrupted using a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated protein (CRISPR-Cas9) system to re-direct resources to scFv production. Genomic integration was used to replace INU1 with sequences encoding a fusion of the INU1 signal peptide to scFv; the resulting construct yielded the highest scFv production among the strains tested. Optimization of growth conditions revealed that scFv production by this strain was enhanced by incubation at 30 °C in xylose medium containing 200 mM MgSO4. These results together demonstrate that K. marxianus has the potential to serve as a host strain for antibody production.

SUBMITTER: Nambu-Nishida Y

PROVIDER: S-EPMC5897269 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Genetic and physiological basis for antibody production by Kluyveromyces marxianus.

Nambu-Nishida Yumiko Y Nishida Keiji K Hasunuma Tomohisa T Kondo Akihiko A Kondo Akihiko A

AMB Express 20180412 1

Kluyveromyces marxianus is a thermotolerant, crabtree-negative yeast, which preferentially directs metabolism (e.g., from the tricarboxylic acid cycle) to aerobic alcoholic fermentation. Thus K. marxianus has great potential for engineering to produce various materials under aerobic cultivation conditions. In this study, we engineered K. marxianus to produce and secrete a single-chain antibody (scFv), a product that is highly valuable but has historically proven difficult to generate at large sc ...[more]

PMID: 29651657

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Project description:BackgroundLow ethanol tolerance of Kluyveromyces marxianus limits its application in high-temperature ethanol fermentation. As a complex phenotype, ethanol tolerance involves synergistic actions of many genes that are widely distributed throughout the genome, thereby being difficult to engineer. TATA-binding protein is the most common target of global transcription machinery engineering for improvement of complex phenotypes.ResultsA random mutagenesis library of K. marxianus TATA-binding protein Spt15 was constructed and subjected to screening under ethanol stress. Two mutant strains with improved ethanol tolerance were identified, one of which (denoted as M2) exhibited increased ethanol productivity. The mutant of Spt15 in strain M2 (denoted as Spt15-M2) has a single amino acid substitution at position 31 (Lys → Glu). RNA-Seq-based transcriptomic analysis revealed cellular transcription profile changes resulting from Spt15-M2. Spt15-M2 caused changes in transcriptional level of most of the genes in the central carbon metabolism network. Compared with control strain, 444 differentially expressed genes (DEGs) were identified in strain M2 (fold change > 2, Padj < 0.05), including 48 up-regulated and 396 down-regulated. The up-regulated DEGs are involved in amino acid transport, long-chain fatty acid biosynthesis and MAPK signaling pathway, while the down-regulated DEGs are related to ribosome biogenesis, translation and protein synthesis. Five candidate genes (GAP1, GNP1, FAR1, STE2 and TEC1), which were found to be up-regulated in M2 strain, were overexpressed for a gain-of-function assay. However, the overexpression of no single gene helped improve ethanol tolerance as SPT15-M2 did.ConclusionsThis work demonstrates that ethanol tolerance of K. marxianus can be improved by engineering its TATA-binding protein. A single amino acid substitution (K31E) of TATA-binding protein Spt15 is able to bring differential expression of hundreds of genes that acted as an interconnected network for the phenotype of ethanol tolerance. Future perspectives of this technique in K. marxianus were discussed.

Dataset Information

Genetic and physiological basis for antibody production by Kluyveromyces marxianus.

Publications

Genetic and physiological basis for antibody production by Kluyveromyces marxianus.

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