Deletion of the trehalose tps1 gene in Kluyveromyces lactis does not impair growth in glucose

ABSTRACT Trehalose is a non-reducing disaccharide composed of two α-glucose molecules and synthesized by an enzyme complex containing four subunits TPS1 (EC 2.4.1.15), TPS2 (EC 3.1.3.12), TPS3 and TSL1. First reports about trehalose classified this sugar as an energy reserve compound like glycogen. However, lately, trehalose is known to assist yeast cells during heat, osmotic and starvation stresses. In Saccharomyces cerevisiae, the deletion of the tps1 encoding gene eliminated the yeast ability to grow on glucose as the sole carbon source. Kluyveromyces lactis is a yeast present in various dairy products and is currently utilized for the synthesis of more than 40 industrial heterologous products. In this study, the deletion of the tps1 gene in K. lactis showed that unlike S. cerevisiae, tps1 gene disruption does not cause growth failure in glucose, galactose, or fructose. The µMAX rate values of K. lactis tps1Δ strains were equal than the non-disrupted strains, showing that the gene deletion does not affect the yeast growth. After gene disruption, the absence of trehalose into the metabolism of K. lactis was also confirmed.

MYB21 interacts with MYC2 to control the expression of terpene synthase genes in flowers of Freesia hybrida and Arabidopsis thaliana

Previously, linalool was found to be the most abundant component among the cocktail of volatiles released from flowers of Freesia hybrida. Linalool formation is catalysed by monoterpene synthase TPS1. However, the regulatory network developmentally modulating the expression of the TPS1 gene in Freesia hybrida remains unexplored. In this study, three regulatory genes, FhMYB21L1, FhMYB21L2, and FhMYC2, were screened from 52 candidates. Two MYB transcription factor genes were synchronously expressed with FhTPS1 and could activate its expression significantly when overexpressed, and the binding of FhMYB21L2 to the MYBCORE sites in the FhTPS1 promoter was further confirmed, indicating a direct role in activation. FhMYC2 showed an inverse expression pattern compared with FhTPS1; its expression led to a decreased binding of FhMYB21 to the FhTPS1 promoter to reduce its activation capacity when co-expressed, suggesting a role for an MYB–bHLH complex in the regulation of the FhTPS1 gene. In Arabidopsis, both MYB21 and MYC2 regulators were shown to activate the expression of sesquiterpene synthase genes, and the regulatory roles of AtMYB21 and AtMYC2 in the expression of the linalool synthase gene were also confirmed, implying conserved functions of the MYB–bHLH complex in these two evolutionarily divergent plants. Moreover, the expression ratio between MYB21 and MYC2 orthologues might be a determinant factor in floral linalool emission.

Isolation of cDNA and upstream sequence of a gene encoding trehalose-6-phosphate synthase 1 from Beauveria bassiana and its functional identification in Pichia pastoris

Trehalose is an important molecule in fungal cells that helps to protect against various environmental stresses. In most fungi, trehalose-6-phosphate synthase 1 (TPS1) catalyzes the synthesis of trehalose-6-phosphate, and is the key enzyme for biosynthesis of this sugar. In this study, the full-length Beauveria bassiana tps1 gene sequence was determined. Full-length Bbtps1 (1,906 bp) included a 1,563 bp open reading frame that contained a 55 bp intron located between deduced amino acids 104 and 105. Bioinformatics analysis predicted that the BbTPS1 protein comprised 520 residues with a calculated pI value of 5.63 and a molecular weight of 58.3 kDa. Using DNA walking experiments, we determined the 2,963 bp upstream sequence that included several typical promoter elements and putative transcription factor binding sites, such as TATA-box, GC-box, Oct-1, CRE-BP, CdxA, and GATA. Stress-response and heat-shock elements were also found in this upstream sequence. Recombinant BbTPS1 was expressed in Pichia pastoris GS115 in order to probe the function of Bbtps1. SDS-PAGE analysis showed that the expressed protein had a molecular weight of approximately 60 kDa as expected. Enzymatic activity measurements revealed specific TPS1 activity that peaked at 1.38 U/mL at 96 h. This work provides a basis for further functional investigation of the mechanism of trehalose anabolism in B. bassiana. It could also assist the construction of engineered B. bassiana strains with enhanced stress tolerance.

Expression of Escherichia coli otsA in a Saccharomyces cerevisiae tps1 mutant restores trehalose 6-phosphate levels and partly restores growth and fermentation with glucose and control of glucose influx into glycolysis

The TPS1 gene, encoding trehalose-6-phosphate synthase (TPS), exerts an essential control on the influx of glucose into glycolysis in the yeast Saccharomyces cerevisiae. The deletion of TPS1 causes an inability to grow on glucose because of a hyperaccumulation of sugar phosphates and depletion of ATP and phosphate. We show that expression of the Escherichia coli homologue, otsA, in a yeast tps1 mutant results in high TPS activity. Although the trehalose 6-phosphate (Tre6P) level during exponential growth on glucose was at least as high as in a wild-type yeast strain, growth on glucose was only partly restored and the lag phase was much longer. Measurement of the glycolytic metabolites immediately after the addition of glucose showed that in spite of a normal Tre6P accumulation there was still a partial hyperaccumulation of sugar phosphates. Strong elevation of the Tre6P level by the additional deletion of the TPS2 gene, which encodes Tre6P phosphatase, was not able to cause a strong decrease in the sugar phosphate levels in comparison with the wild-type strain. In addition, in chemostat experiments the short-term response to a glucose pulse was delayed, but normal metabolism was regained over a longer period. These results show that Tre6P synthesis from a heterologous TPS enzyme can to some extent restore the control of glucose influx into glycolysis and growth on glucose in yeast. However, they also indicate that the yeast TPS enzyme, as opposed to the E. coli otsA gene product, is able to increase the efficiency of the Tre6P control on glucose influx into yeast glycolysis.