The Sequence of Spacers between the Consensus Sequences Modulates the Strength of Prokaryotic Promoters

Abstract

We constructed a library of synthetic promoters forLactococcus lactis in which the known consensus sequences were kept constant while the sequences of the separating spacers were randomized. The library consists of 38 promoters which differ in strength from 0.3 up to more than 2,000 relative units, the latter among the strongest promoters known for this organism. The ranking of the promoter activities was somewhat different when assayed inEscherichia coli, but the promoters are efficient for modulating gene expression in this bacterium as well. DNA sequencing revealed that the weaker promoters (which had activities below 5 relative units) all had changes either in the consensus sequences or in the length of the spacer between the −35 and −10 sequences. The promoters in which those features were conserved had activities from 5 to 2,050 U, which shows that by randomizing the spacers, at least a 400-fold change in activity can be obtained. Interestingly, the entire range of promoter activities is covered in small steps of activity increase, which makes these promoters very suitable for quantitative physiological studies and for fine-tuning of gene expression in industrial bioreactors and cell factories.

Lactate dehydrogenase has no control on lactate production but has a strong negative control on formate production in Lactococcus lactis

“…The last cloning step resulted in pHWA202. The ldh gene was equipped with synthetic promoters by digesting the plasmids pCP2, pCP7, pCP29, and pCP25 [22] with Xho I and Xba I, and cloning the promoter fragments in pHWA202 digested with the Sal I and Xba I. This cloning step resulted in the plasmids pMBP34, pHWA227, pHWA230, and pHWA231.…”

“…The experimental approach chosen to access the control exerted by lactate dehydrogenase was to modulate the expression of ldh using site-specific chromosomal integration of an additional ldh gene transcribed from synthetic promoters [22]. The integration was made in the wild-type and in a strain deficient in lactate dehydrogenase and hence, expression levels both above and below the wild-type were obtained.…”

Strain engineering to reduce acetate accumulation during microaerobic growth conditions in Escherichia coli

“…The gene coding for the anaplerotic enzyme phosphoenolpyruvate carboxylase (ppc gene product) was overexpressed by swapping its native promoter with a strong constitutive artificial promoter CP25. 51 Another anaplerotic enzyme, pyruvate carboxylase (pyc gene product) 56 from R. etli, was introduced into E. coli and CP25 promoter was used to drive the expression of pyc gene. The gene coding for acetyl-CoA synthase (acs gene product), an enzyme involved in acetate assimilation, was overexpressed using CP25 promoter.…”

“…The pS1080-gltA(R163L) plasmid was constructed by mutagenizing the plasmid pS1080-gltA using the QuikChange kit (Stratagene) and the following set of primers: QC-gltA(R163L)-F and QC-gltA(R163L)-R. The plasmid pS1080-gltA(R163L) was used to introduce R163L mutation in chromosomal gltA gene using allele exchange to obtain strain 67E3. A 1091 bp fragment containing a portion of the C-terminal region of argE gene, an artificial constitutive promoter (CP25) from a library designed for Lactococcus lactis, 51 an optimized ribosome binding site (RBS) for E. coli 52 and a portion of Nterminal region of ppc gene was synthesized (Blue Heron Bio, Bothell, WA). The resultant fragment was cloned into pS1080 digested with SacI and SpeI to obtain the plasmid pS1080-P CP25 -ppc.…”

Using the cre–lox system to randomize target gene expression states and generate diverse phenotypes

“…Titratable promoters allow rapid sampling of different combinations of gene expression using small molecules, but such promoters are limited in number and inducible signals may have pleiotropic effects (Cox et al, 2007;Guo et al, 2008;Maya et al, 2008). Library approaches can also be employed to fine-tune expression levels, often in the context of optimizing metabolic flux through pathways (Alper et al, 2005a;Jensen and Hammer, 1998;Pfleger et al, 2006), but they are limited to a single gene or operon. Two techniques that generate diverse libraries by rapidly modifying multiple genes are multiplex automated genome engineering (MAGE) (Wang et al, 2009) and random ligation of yeast artificial chromosomes (YACS) (Naesby et al, 2009).…”