Induced gene expression is an important trait in yeast metabolic engineering, but current regulations prevent the use of conventional expression systems, such as galactose and copper, in food and beverage fermentations. This article examines the suitability of temperature-inducible native promoters for use in the industrial yeast strain Saccharomyces pastorianus var. carlsbergensis TUM 34/70 under brewing conditions. Ten different promoters were cloned and characterized under varying temperature shifts and ethanol concentrations using a green fluorescent protein reporter. The activities of these promoters varied depending upon the stress conditions applied. A temperature shift to 4°C led to the highest fold changes of PSSA3, PUBI4 and PHSP104 by 5.4, 4.5 and 5.0, respectively. Ethanol shock at 24°C showed marked, concentration-dependent induction of the promoters. Here, PHSP104 showed its highest induction at ethanol concentrations between 4% (v/v) and 6% (v/v). The highest fold changes of PSSA3 and PUBI4 were found at 10% (v/v) ethanol. In comparison, the ethanol shock at a typical fermentation temperature (12°C) leads to lower induction patterns of these promoters. Taken together, the data show that three promoters (PHSP104, PUBI4 and PSSA3) have high potential for targeted gene expression in self-cloning brewing yeast using temperature shifts.

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Male sterile lines play important roles in plant breeding to obtain hybrid vigour. The male sterility Lembke (MSL) system is a thermosensitive genic male sterility system of Brassica napus and is one of the main systems used in European rapeseed breeding. Interestingly the MSL system shows high similarity to the 9012AB breeding system from China including the ability to revert to fertile in high temperature conditions. Here we demonstrate that the MSL system is regulated by the same restorer of fertility gene BnaC9-Tic40 as the 9012AB system, which is related to the translocon at the inner envelope membrane of chloroplasts 40 (TIC40) from Arabidopsis. The male sterility gene of the MSL system was also identified to be a chloroplast localised protein which we call BnChimera, this gene shows high sequence similarity to the sterility gene previously described for the 9012AB system. For the first time a direct protein interaction between the BnaC9-Tic40 and the BnChimera could be demonstrated. In addition, the corresponding amino acids that mediate this interaction were identified and possibly determine how BnaC9-Tic40 acts as the restorer of fertility. Using an RNA-seq approach, the effects of heat treatment on the male fertility restoration of an MSL system line C545 were investigated. This data demonstrated that many pollen developmental pathways were affected by the higher temperatures. It is hypothesised that heat stress reverses the male sterility via a combination of slower production of cell wall precursors in plastids and a slower flower development, which ultimately results in fertile pollen. The potential breeding applications of these results are discussed regarding the capacity of using the MSL system in producing thermotolerant fertile plants.