Kluyveromyces lactis: An emerging tool in biotechnology

Spohner, Sebastian C.; Schaum, Vivienne; Quitmann, Hendrich; Czermak, Peter

doi:10.1016/j.jbiotec.2016.02.023

Cited by 118 publications

(83 citation statements)

References 116 publications

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“…The pGKL plasmids could also help us better understand basic processes and host cell-virus interactions that are not easily studied in mammalian systems. Last but not least, K. lactis is one of the most important yeast for research and industrial biotechnology (129), and our results suggest that lsm1Δ strains can greatly improve the production of heterologous proteins from pGKL-based expression plasmids.…”

Section: Discussionmentioning

confidence: 71%

Messenger RNAs transcribed from yeast linear cytoplasmic plasmids possess unconventional 5’ and 3’ UTRs and suggest a novel mechanism of translation

Vopálenský

Sýkora

Mašek

et al. 2018

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Linear plasmids with almost identical compact genetic organization have been found in the cytoplasm of yeast species from nine genera. We employed pGKL1,2 plasmids from Kluyveromyces lactis as a model to investigate the previously unstudied transcriptome of yeast cytoplasmic linear plasmids. We performed 5' and 3' RACE analysis of all the pGKL1,2 mRNAs and found them not 3' polyadenylated and containing mostly uncapped 5' poly(A) leaders that are not complementary to the plasmid DNA. The degree of 5' capping and/or 5' polyadenylation is specific to each gene and is controlled by the corresponding promoter regions. We refined the description of the pGKL1,2 promoters and found new alternative promoters of several genes. We also provide evidence that K2ORF3 encodes an mRNA cap guanine-N 7 -methyltransferase and that 5' capped pGKL1,2 transcripts contain N 7 -methylated caps. Translation of pGKL1,2 transcripts is enhanced in lsm1Δ and pab1Δ strains and is independent of eIF4E and Pab1 translation factors. We suggested a model of a primitive regulation of pGKL1,2 plasmids gene expression where degree of 5' mRNA capping, degree of 5' non-template polyadenylation and presence of negative regulators as PAB1 and Lsm1 play an important role. Our data also suggest a close relationship between linear plasmids and poxviruses. doi: bioRxiv preprint 4 synthesized in a template-independent manner. We show that plasmid promoters directly determine the formation of the mRNA 5' end leaders and their capping, pointing to a possible role of the plasmid RNA polymerase in this unusual phenomenon. We also provide the first evidence that MTase is an essential part of the K2Orf3p enzyme and that capped pGKL mRNAs are methylated at the N 7 position of the terminal guanosine moiety. Using in vitro and in vivo approaches, we demonstrate that translation of plasmid-specific mRNAs is independent of major cellular cap-binding proteins, translation initiation factor 4E (eIF4E), and the Pab1 and Lsm1 proteins that were speculated to be possible translational enhancers and/or stabilizers of cellular and viral mRNAs bearing 5' poly(A) stretches (38,39). Our results thus suggest the existence of a novel translation initiation mechanism. Materials and Methods General Strategy for the Modification of pGKL Plasmids Using Homologous Recombination In VivoK. lactis IFO1267 was transformed with a PCR-generated fragment consisting of 5' and 3' ends homologous to the part of the pGKL plasmid to be modified, a non-homologous part that introduces mutation/s into a plasmid-specific protein coding sequence, and a gene encoding a resistance marker (typically against G418 or hygromycin B) whose transcription is driven by the UCR of K1ORF1 or K1ORF2. This type of construct was prepared by fusion PCR; for the organization of such a construct, see Fig. 2A. After transformation, yeast cells were plated onto selective agar media, and their DNA content was analyzed for the presence of the modified pGKL plasmid using agarose electrophoresis. Both modified and wild-type t...

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Section: Discussionmentioning

confidence: 71%

Messenger RNAs transcribed from yeast linear cytoplasmic plasmids possess unconventional 5’ and 3’ UTRs and suggest a novel mechanism of translation

Vopálenský

Sýkora

Mašek

et al. 2018

Preprint

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“…Kluyveromyces lactis, the Crabtree-negative yeast, is widely used in the food industry because it is considered as "generally recognized as safe" (GRAS) [27]. It is extensively studied for the host system to produce recombinant proteins.…”

Section: Introductionmentioning

confidence: 99%

Biopurification of Oligosaccharides by Immobilized Kluyveromyces Lactis

et al. 2019

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Oligosaccharides with diverse and complex structures such as milk oligosaccharides have physiological functions including modulating intestinal microbiota or stimulating immune cell responses. However, milk carbohydrates include about 40-50% of lactose which requires a cost-effective method to separate. We developed a new method to purify the oligosaccharides from carbohydrate mixtures such as human milk oligosaccharides (HMOs) and galactooligosaccharides (GOSs) by exploiting immobilized Kluyveromyces lactis as microbial catalysts. Evaluation of media components exhibited no significant differences in the lactose removal efficiency when nutrient-rich media, minimal salt media, and distilled water without any media components were used. With the immobilization on alginate beads, the lactose removal efficiency was increased 3.4 fold compared to that of suspension culture. When the immobilized cells were reused to design a continuous process, 4 h of pre-activation enhanced the lactose eliminating performance 2.5 fold. Finally, immobilized K. lactis was used as microbial catalysts for the biopurification of HMOs and GOSs, and lactose was effectively removed without altering the overall distribution of oligosaccharides.

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“…Kluyveromyces lactis and S. cerevisiae are two food-grade yeasts widely used in biotechnology [21, 22]. Apart from other advantages for heterologous protein production, K. lactis is able to use a greater variety of carbon sources than S. cerevisiae , being well documented the case of lactose, i.e., the ability to metabolize lactose of the former and the inability of the second species, while both can use sucrose [23, 24].…”

Section: Introductionmentioning

confidence: 99%

“…Apart from other advantages for heterologous protein production, K. lactis is able to use a greater variety of carbon sources than S. cerevisiae , being well documented the case of lactose, i.e., the ability to metabolize lactose of the former and the inability of the second species, while both can use sucrose [23, 24]. Due to its special features , K. lactis has been used successfully as a cell factory for recombinant protein secretion in large-scale cultures; however, the use of agro-industrial wastes such as whey-molasses mixed media as substrates has not been reported [21]. Mixtures of molasses or bagasse and milk or cheese whey have been used for ethanol production by Kluyveromyces sp.…”

Section: Introductionmentioning

confidence: 99%

Valuation of agro-industrial wastes as substrates for heterologous production of α-galactosidase

et al. 2018

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BackgroundThe recycling of agro-industrial wastes is at present limited by the availability of efficient and low-cost enzyme cocktails. The use of these materials as culture media to produce the enzymes can contribute to the profitability of the recycling process and to the circular economy. The aim of this work is the construction of a recombinant yeast strain efficient to grow in mixed whey (residue of cheese making) and beet molasses (residue of sugar manufacture) as culture medium, and to produce heterologous α-galactosidase, an enzyme with varied industrial applications and wide market.ResultsThe gene MEL1, encoding the α-galactosidase of Saccharomyces cerevisiae, was integrated (four copies) in the LAC4 locus of the Kluyveromyces lactis industrial strain GG799. The constructed recombinant strain produces high levels of extracellular α-galactosidase under the control of the LAC4 promoter, inducible by lactose and galactose, and the native MEL1 secretion signal peptide. K. lactis produces natively beta-galactosidase and invertase thus metabolizing the sugars of whey and molasses. A culture medium based on whey and molasses was statistically optimized, and then the cultures scaled-up at laboratory level, thus obtaining 19 U/mL of heterologous α-galactosidase with a productivity of 0.158 U/L h, which is the highest value reported hitherto from a cheap waste-based medium.ConclusionsA K. lactis recombinant strain was constructed and a sustainable culture medium, based on a mixture of cheese whey and beet molasses, was optimized for high productivity of S. cerevisiae α-galactosidase, thus contributing to the circular economy by producing a heterologous enzyme from two agro-industrial wastes.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0988-6) contains supplementary material, which is available to authorized users.

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Kluyveromyces lactis: An emerging tool in biotechnology

Cited by 118 publications

References 116 publications

Messenger RNAs transcribed from yeast linear cytoplasmic plasmids possess unconventional 5’ and 3’ UTRs and suggest a novel mechanism of translation

Messenger RNAs transcribed from yeast linear cytoplasmic plasmids possess unconventional 5’ and 3’ UTRs and suggest a novel mechanism of translation

Biopurification of Oligosaccharides by Immobilized Kluyveromyces Lactis

Valuation of agro-industrial wastes as substrates for heterologous production of α-galactosidase

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