Modulation of gene expression made easy

Abstract

A new approach for modulating gene expression, based on randomization of promoter (spacer) sequences, was developed. The method was applied to chromosomal genes in Lactococcus lactis and shown to generate libraries of clones with broad ranges of expression levels of target genes. In one example, overexpression was achieved by introducing an additional gene copy into a phage attachment site on the chromosome. This resulted in a series of strains with phosphofructokinase activities from 1.4 to 11 times the wild-type activity level. In this example, the pfk gene was cloned upstream of a gusA gene encoding beta-glucuronidase, resulting in an operon structure in which both genes are transcribed from a common promoter. We show that there is a linear correlation between the expressions of the two genes, which facilitates screening for mutants with suitable enzyme activities. In a second example, we show that the method can be applied to modulating the expression of native genes on the chromosome. We constructed a series of strains in which the expression of the las operon, containing the genes pfk, pyk, and ldh, was modulated by integrating a truncated copy of the pfk gene. Importantly, the modulation affected the activities of all three enzymes to the same extent, and enzyme activities ranging from 0.5 to 3.5 times the wild-type level were obtained.

FIG. 1.

Overview of the approach used for modulating enzyme activities. (A) Chromosome with an arbitrary gene (geneX) and its upstream region (upsX). The promoter primer (N can be any of the four bases, while R is 50% A and 50% G) and two reverse primers are shown. (B) Two possible PCR products, the first of which contains a truncated geneX and the second of which contains a full-length geneX. (C) The resulting plasmids obtained after cloning of the PCR fragments. If a chromosomal promoter is to be replaced by a synthetic one, then strategy I is used. If the resulting plasmid is to be used in a simple integration event, then strategy Ib is used. If the resulting plasmid is to take part in a double-crossover event, then strategy Ia is used. If the plan is to introduce an additional gene copy into the chromosome, then strategy II is used.

FIG. 2.

Correlation between β-glucuronidase activity and relative PFK activity in strains containing an additional gene copy of pfk transcribed from synthetic promoters.

FIG. 3.

Relative LDH activity measured in strains with synthetic promoters in front of the las operon. Overnight cultures were used for these measurements, and these activities are generally somewhat higher than those measured in extracts from exponentially grown cells.

FIG. 4.

Relative activities of PFK, PK, and LDH measured in extracts from strains with synthetic promoters in front of the las operon during exponential-phase growth. The selected strains cover the entire activity range. MG1363 is the wild-type strain.

FIG. 5.

Strategy used for replacement of the native las promoter with the synthetic promoter obtained from CS8. The plasmid has two regions homologous to the las locus and allowed for the construction of a strain, CS1006, in which the vector has been excised (see text for further details).

FIG. 6.

Primer extension using RNAs from the wild-type strain MG1363 and from strain CS1006, in which a synthetic promoter has replaced the las promoter. Arrows indicate the transcript start. Note the alternative transcript start that was observed in both strains, also indicated by arrows.