Optogenetic Regulation of Tunable Gene Expression in Yeast Using Photo-Labile Caged Methionine.

Light-mediated gene expression enables the noninvasive regulation of cellular functions. Apart from their classical application of regulating single cells with high spatiotemporal resolution, we highlight the potential of light-mediated gene expression for biotechnological issues. Here, we demonstrate the first light-mediated gene regulation in Saccharomyces cerevisiae using the repressible pMET17 promoter and the photolabile NVOC methionine that releases methionine upon irradiation with UVA light. In this system, the expression can be repressed upon irradiation and is reactivated due to consumption of methionine. The photolytic release allows precise control over the methionine concentration and therefore over the repression duration. Using this light regulation mechanism, we were able to apply an in-house constructed 48-well cultivation system which allows parallelized and automated irradiation programs as well as online detection of fluorescence and growth. This system enables screening of multiple combinations of several repression/derepression intervals to realize complex expression programs (e.g., a stepwise increase of temporally constant expression levels, linear expression rates with variable slopes, and accurate control over the expression induction, although we used a repressible promoter.) Thus, we were able to control all general parameters of a gene expression experiment precisely, namely start, pause, and stop at desired time points, as well as the ongoing expression rate. Furthermore, we gained detailed insights into single-cell expression dynamics with spatiotemporal resolution by applying microfluidics cultivation technology combined with fluorescence time-lapse microscopy.

Comparative expression of lipase CAL-A in the yeasts Saccharomyces cerevisiae, Kluyveromyces lactis and Hansenula polymorpha to investigate a possible host influence.

Yeasts are an attractive expression platform, as they combine the ease of handling with the eukaryotic ability to process the produced protein. Important aspects of eukaryotic protein expression are posttranslational modifications, which can be required for functional expression of the protein of interest and can only be performed by eukaryotes. Each organism has its own modification pattern: for instance, the same protein produced in different hosts is subjected to various glycosylation pathways. It is amenable that these kinds of modifications can have an influence on the biochemical properties of the protein. To verify this thesis, the well-studied lipase CAL-A from Candida antarctica was chosen as a model enzyme. The codon bias of the gene sequence was uniformly optimized and expressed in three industrially relevant yeast hosts: Saccharomyces cerevisiae, Kluyveromyces lactis, and Hansenula polymorpha. The capacity of the expression systems to produce the enzyme was analyzed, as well as the biochemical properties of the produced and purified CAL-A. All hosts produced active enzyme; however, significant differences in the obtained yield were observed. H. polymorpha appeared to be the most productive host with a tenfold increase in productivity in comparison to S. cerevisiae. Studies on thermostability and activity of the purified enzymes towards various substrates showed a significant impact of the host on the biochemical properties of the produced protein. The most thermostable CAL-A from K. lactis retained 70% of its activity after incubation at 60°C, in comparison to 45% remaining activity for the enzyme purified from S. cerevisiae. In the screenings with different substrates, a fourfold increase in activity between the enzymes from H. polymorpha and S. cerevisiae was found. Altogether, we herein exemplify how the selection of the host even within one taxonomic family (Saccharomycetaceae) significantly affects the produced enzyme's characteristics.

Lights on and action! Controlling microbial gene expression by light.

Light-mediated control of gene expression and thus of any protein function and metabolic process in living microbes is a rapidly developing field of research in the areas of functional genomics, systems biology, and biotechnology. The unique physical properties of the environmental factor light allow for an independent photocontrol of various microbial processes in a noninvasive and spatiotemporal fashion. This mini review describes recently developed strategies to generate photo-sensitive expression systems in bacteria and yeast. Naturally occurring and artificial photoswitches consisting of light-sensitive input domains derived from different photoreceptors and regulatory output domains are presented and individual properties of light-controlled expression systems are discussed.

In vivo detection of molybdate-binding proteins using a competition assay with ModE in Escherichia coli.

Molybdenum is an important trace element as it forms the essential part of the active site in all molybdenum-containing enzymes. We have designed an assay for the in vivo detection of molybdate binding to proteins in Escherichia coli. The assay is based on (i). the molybdate-dependent transcriptional regulation of the moa operon by the ModE protein, and (ii). the competition for molybdate between ModE and other molybdate-binding proteins in the cytoplasm of E. coli. We were able to verify in vivo molybdate binding to three different bacterial proteins that are known to bind molybdate. This sensitive in vivo system allows the testing of different proteins for molybdate binding under in vivo conditions and will facilitate the identification of other cellular factors needed for molybdate binding. As a first example, we examined the eukaryotic protein Cnx1 that is involved in the last step of molybdenum cofactor biosynthesis in plants, and show that it is able to compete with ModE for molybdate in a molybdopterin-dependent fashion.