The synthesis of ribosomes in Saccharomyces cerevisiae consumes a prodigious amount of the cell's resources and, consequently, is tightly regulated. The rate of ribosome synthesis responds not only to nutritional cues but also to signals dependent on other macromolecular pathways of the cell, e.g., a defect in the secretory pathway leads to severe repression of transcription of both rRNA and ribosomal protein genes. A search for mutants that interrupted this repression revealed, surprisingly, that inactivation of RPL1B, one of a pair of genes encoding the 60S ribosomal protein L1, almost completely blocked the repression of rRNA and ribosomal protein gene transcription that usually follows a defect in the secretory pathway. Further experiments showed that almost any mutation leading to a defect in 60S subunit synthesis had the same effect, whereas mutations affecting 40S subunit synthesis did not. Although one might suspect that this effect would be due to a decrease in the initiation of translation or to the presence of half-mers, i.e., polyribosomes awaiting a 60S subunit, our data show that this is not the case. Rather, a variety of experiments suggest that some aspect of the production of defective 60S particles or, more likely, their breakdown suppresses the signal generated by a defect in the secretory pathway that represses ribosome synthesis.

Saccharomyces boulardii is a probiotic yeast that has been used for promoting gut health as well as preventing diarrheal diseases. This yeast not only exhibits beneficial phenotypes for gut health but also can stay longer in the gut than Saccharomyces cerevisiae. Therefore, S. boulardii is an attractive host for metabolic engineering to produce biomolecules of interest in the gut. However, the lack of auxotrophic strains with defined genetic backgrounds has hampered the use of this strain for metabolic engineering. Here, we report the development of well-defined auxotrophic mutants (leu2, ura3, his3, and trp1) through clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-based genome editing. The resulting auxotrophic mutants can be used as a host for introducing various genetic perturbations, such as overexpression or deletion of a target gene, using existing genetic tools for S. cerevisiae. We demonstrated the overexpression of a heterologous gene (lacZ), the correct localization of a target protein (red fluorescent protein) into mitochondria by using a protein localization signal, and the introduction of a heterologous metabolic pathway (xylose-assimilating pathway) in the genome of S. boulardii. We further demonstrated that human lysozyme, which is beneficial for human gut health, could be secreted by S. boulardii. Our results suggest that more sophisticated genetic perturbations to improve S. boulardii can be performed without using a drug resistance marker, which is a prerequisite for in vivo applications using engineered S. boulardii.

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To optimize the secretory expression of recombinant human serum albumin (HSA) under the control of methanol oxidase (MOX) promoter in the methylotrophic yeast Hansenula polymorpha DL-1, we analyzed several parameters affecting the expression of HSA from the MOX promoter. Removal of the 5'-untranslated region derived from HSA cDNA in the expression cassette led to at least a fivefold improvement of HSA expression efficiency at the translational level. With the optimized expression cassette, the gene dosage effect on HSA expression was abolished and thus, a single copy of the expression vector integrated into the MOX locus became sufficient for the maximal expression of HSA. Northern blot analysis revealed that the levels of HSA transcript did not increase any further upon increasing copy number. The mox-disrupted (mox Delta) transformant was constructed, in which the genomic MOX gene was transplaced with the HSA expression cassette, to examine the effect of the methanol oxidase-deficient phenotype of the host on HSA expression. The mox Delta transformant showed higher levels of HSA production in shake-flask cultures than the MOX wild-type transformant, especially at low concentrations of methanol and a twofold higher specific HSA production rate in fed-batch fermentation with an abrupt induction mode. The native prepro signal sequence of HSA secreted in H. polymorpha was correctly processed and the mature recombinant protein had a pI value identical to that of the authentic HSA. Our results suggest that the H. polymorpha expression systems developed in this study are suitable for large-scale production of recombinant albumin.

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