Synthetic promoter libraries for Corynebacterium glutamicum.

The ability to modulate gene expression is an important genetic tool in systems biology and biotechnology. Here, we demonstrate that a previously published easy and fast PCR-based method for modulating gene expression in lactic acid bacteria is also applicable to Corynebacterium glutamicum. We constructed constitutive promoter libraries based on various combinations of a previously reported C. glutamicum -10 consensus sequence (gngnTA(c/t)aaTgg) and the Escherichia coli -35 consensus, either with or without an AT-rich region upstream. A promoter library based on consensus sequences frequently found in low-GC Gram-positive microorganisms was also included. The strongest promoters were found in the library with a -35 region and a C. glutamicum -10 consensus, and this library also represents the largest activity span. Using the alternative -10 consensus TATAAT, which can be found in many other prokaryotes, resulted in a weaker but still useful promoter library. The upstream AT-rich region did not appear to affect promoter strength in C. glutamicum. In addition to the constitutive promoters, a synthetic inducible promoter library, based on the E. coli lac-promoter, was constructed by randomizing the 17-bp spacer between -35 and -10 consensus sequences and the sequences surrounding these. The inducible promoter library was shown to result in ß-galactosidase activities ranging from 284 to 1,665 Miller units when induced by IPTG, and the induction fold ranged from 7-59. We find that the synthetic promoter library (SPL) technology is convenient for modulating gene expression in C. glutamicum and should have many future applications, within basic research as well as for optimizing industrial production organisms.

Physiological heterogeneities in microbial populations and implications for physical stress tolerance.

BACKGROUND: Traditionally average values of the whole population are considered when analysing microbial cell cultivations. However, a typical microbial population in a bioreactor is heterogeneous in most phenotypes measurable at a single-cell level. There are indications that such heterogeneity may be unfavourable on the one hand (reduces yields and productivities), but also beneficial on the other hand (facilitates quick adaptation to new conditions--i.e. increases the robustness of the fermentation process). Understanding and control of microbial population heterogeneity is thus of major importance for improving microbial cell factory processes. RESULTS: In this work, a dual reporter system was developed and applied to map growth and cell fitness heterogeneities within budding yeast populations during aerobic cultivation in well-mixed bioreactors. The reporter strain, which was based on the expression of green fluorescent protein (GFP) under the control of the ribosomal protein RPL22a promoter, made it possible to distinguish cell growth phases by the level of fluorescence intensity. Furthermore, by exploiting the strong correlation of intracellular GFP level and cell membrane integrity it was possible to distinguish subpopulations with high and low cell membrane robustness and hence ability to withstand freeze-thaw stress. A strong inverse correlation between growth and cell membrane robustness was observed, which further supports the hypothesis that cellular resources are limited and need to be distributed as a trade-off between two functions: growth and robustness. In addition, the trade-off was shown to vary within the population, and the occurrence of two distinct subpopulations shifting between these two antagonistic modes of cell operation could be distinguished. CONCLUSIONS: The reporter strain enabled mapping of population heterogeneities in growth and cell membrane robustness towards freeze-thaw stress at different phases of cell cultivation. The described reporter system is a valuable tool for understanding the effect of environmental conditions on population heterogeneity of microbial cells and thereby to understand cell responses during industrial process-like conditions. It may be applied to identify more robust subpopulations, and for developing novel strategies for strain improvement and process design for more effective bioprocessing.

Escherichia coli strains with promoter libraries constructed by Red/ET recombination pave the way for transcriptional fine-tuning.

System-oriented applications of genetic engineering, such as metabolic engineering, often require the serial optimization of enzymatic reaction steps, which can be achieved by transcriptional fine-tuning. However, approaches to changing gene expression are usually limited to deletion and/or strong overexpression and rarely match the desired optimal transcript levels. A solution to this all-or-nothing approach has been the use of a synthetic promoter library (SPL) that is based on randomized sequences flanking the consensus -10 and -35 promoter regions and allows for fine-tuning of bacterial gene expression. Red/ET recombination perfectly complements SPL technology, since it enables easy modification of the Escherichia coli genome and can be accomplished with linear DNA (i.e., the SPL). To demonstrate the synergistic use of Red/ET and SPL for metabolic engineering applications, we replaced the native promoter of a genomic localized phosphoglucose isomerase (pgi)-lacZ reporter construct by an SPL. Using these technologies together, we were able to rapidly identify synthetic promoter sequences that resulted in activity range of 25% to 570% of the native pgi-promoter.

Transformation of Leuconostoc carnosum 4010 and evidence for natural competence of the organism.

Plasmid transformation in Leuconostoc carnosum 4010 was analyzed. A successful transformation protocol for L. carnosum was established by modifying an existing protocol for Lactococcus lactis. Several parameters, including the number of generations that the cells had grown at the time of harvest, glycine concentration, the time of incubation for phenotypic expression, and the electrical field strength, were investigated and proved to have influence on the transformation frequency. Electrocompetence was found to be transient and to peak in the early exponential growth phase. Optimized conditions resulted in transformation frequencies of up to 6.7 x 10(5) transformants per microgram of plasmid DNA. A total of five plasmids in L. carnosum were successfully introduced and maintained. Interestingly, we discovered that DNA uptake was of a frequency of 3 x 10(-6) to 19 x 10(-6) transformants per CFU in the absence of an applied electrical field. We concluded that L. carnosum is naturally competent.