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Characterization of putative mannoprotein in Kluyveromyces lactis for lactase production.

ABSTRACT: Lactase is a member of the β-galactosidase family of enzymes that can hydrolyze lactose into galactose and glucose. However, extracellular lactase production was still restricted to the process of cell lysis. In this study, lactase-producing Kluyveromyces lactis JNXR-2101 was obtained using a rapid and sensitive method based on the fluorescent substrate 4-methylumbelliferyl-β-d-galactopyranoside. The purified enzyme was identified as a neutral lactase with an optimum pH of 9. To facilitate extracellular production of lactase, a putative mannoprotein KLLA0_E01057g of K. lactis was knocked out. It could effectively promote cell wall degradation and lactase production after lyticase treatment, which showed potential on other extracellular enzyme preparation. After optimizing the fermentation conditions, the lactase yield from mannoprotein-deficient K. lactis JNXR-2101ΔE01057g reached 159.62 U/mL in a 5-L fed-batch bioreactor.

SUBMITTER: Shen X

PROVIDER: S-EPMC9880975 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Characterization of putative mannoprotein in <i>Kluyveromyces lactis</i> for lactase production.

Shen Xiuru X Liao Lingtong L Zhang Guoqiang G Zhou Jingwen J Li Jianghua J Du Guocheng G

Synthetic and systems biotechnology 20230105 1

Lactase is a member of the β-galactosidase family of enzymes that can hydrolyze lactose into galactose and glucose. However, extracellular lactase production was still restricted to the process of cell lysis. In this study, lactase-producing <i>Kluyveromyces lactis</i> JNXR-2101 was obtained using a rapid and sensitive method based on the fluorescent substrate 4-methylumbelliferyl-β-d-galactopyranoside. The purified enzyme was identified as a neutral lactase with an optimum pH of 9. To facilitat ...[more]

PMID: 36733311

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Project description:BackgroundThe Crabtree-negative yeast species Kluyveromyces lactis has been established as an attractive microbial expression system for recombinant proteins at industrial scale. Its LAC genes allow for utilization of the inexpensive sugar lactose as a sole source of carbon and energy. Lactose efficiently induces the LAC4 promoter, which can be used to drive regulated expression of heterologous genes. So far, strain manipulation of K. lactis by homologous recombination was hampered by the high rate of non-homologous end-joining.ResultsSelection for growth on lactose was applied to target the insertion of heterologous genes downstream of the LAC4 promoter into the K. lactis genome and found to yield high numbers of positive transformants. Concurrent reconstitution of the β-galactosidase gene indicated the desired integration event of the expression cassette, and β-galactosidase activity measurements were used to monitor gene expression for strain improvement and fermentation optimization. The system was particularly improved by usage of a cell lysis resistant strain, VAK367-D4, which allowed for protein accumulation in long-term fermentation. Further optimization was achieved by increased gene dosage of KlGAL4 encoding the activator of lactose and galactose metabolic genes that led to elevated transcription rates. Pilot experiments were performed with strains expressing a single-chain antibody fragment (scFvox) and a viral envelope protein (BVDV-E2), respectively. scFvox was shown to be secreted into the culture medium in an active, epitope-binding form indicating correct processing and protein folding; the E2 protein could be expressed intracellularly. Further data on the influence of protein toxicity on batch fermentation and potential post-transcriptional bottlenecks in protein accumulation were obtained.ConclusionsA novel Kluyveromyces lactis host-vector system was developed that places heterologous genes under the control of the chromosomal LAC4 promoter and that allows monitoring of its transcription rates by β-galactosidase measurement. The procedure is rapid and efficient, and the resulting recombinant strains contain no foreign genes other than the gene of interest. The recombinant strains can be grown non-selectively in rich medium and stably maintained even when the gene product exerts protein toxicity.

Project description:BackgroundEven before having its genome sequence published in 2004, Kluyveromyces lactis had long been considered a model organism for studies in genetics and physiology. Research on Kluyveromyces lactis is quite advanced and this yeast species is one of the few with which it is possible to perform formal genetic analysis. Nevertheless, until now, no complete metabolic functional annotation has been performed to the proteins encoded in the Kluyveromyces lactis genome.ResultsIn this work, a new metabolic genome-wide functional re-annotation of the proteins encoded in the Kluyveromyces lactis genome was performed, resulting in the annotation of 1759 genes with metabolic functions, and the development of a methodology supported by merlin (software developed in-house). The new annotation includes novelties, such as the assignment of transporter superfamily numbers to genes identified as transporter proteins. Thus, the genes annotated with metabolic functions could be exclusively enzymatic (1410 genes), transporter proteins encoding genes (301 genes) or have both metabolic activities (48 genes). The new annotation produced by this work largely surpassed the Kluyveromyces lactis currently available annotations. A comparison with KEGG's annotation revealed a match with 844 (~90%) of the genes annotated by KEGG, while adding 850 new gene annotations. Moreover, there are 32 genes with annotations different from KEGG.ConclusionsThe methodology developed throughout this work can be used to re-annotate any yeast or, with a little tweak of the reference organism, the proteins encoded in any sequenced genome. The new annotation provided by this study offers basic knowledge which might be useful for the scientific community working on this model yeast, because new functions have been identified for the so-called metabolic genes. Furthermore, it served as the basis for the reconstruction of a compartmentalized, genome-scale metabolic model of Kluyveromyces lactis, which is currently being finished.

Dataset Information

Characterization of putative mannoprotein in Kluyveromyces lactis for lactase production.

Publications

Characterization of putative mannoprotein in <i>Kluyveromyces lactis</i> for lactase production.

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