Regulation of dctA and DctA by cAMP-CRP and EIIAGlc at the transcriptional and post-translational levels in E. coli: Consequences for aerobic uptake and metabolism of C4-dicarboxylates

The expression of dctA, encoding the aerobic C4-dicarboxylate (C4-DC) transporter DctA of Escherichia coli, and its use in the presence of alternative carbon sources was characterized. dctA is regulated by cAMP-CRP and substrates that control cAMP levels, either through the phosphotransferase system (PTS), or through their metabolic link to PEP synthesis. The data indicates that phosphorylation of the regulator EIIAGlc of the glucose-specific PTS represents the mediator for regulation. The dctA promotor region contains a class I CRP-binding site (position -81.5) and a DcuR-binding site (position -105.5). The response regulator DcuR of the C4-DC-activated DcuS-DcuR two-component system is known to stimulate expression of dctA, and cAMP-CRP is known to stimulate expression of dcuS-dcuR. Thus, activation of dctA expression by cAMP-CRP and DcuR is organized in a coherent feed-forward loop (FFL) where cAMP-CRP positively regulates the expression of dctA by direct stimulation and by stimulating the expression of dcuR. Stimulation by DcuR is presumed to require DNA bending by cAMP-CRP. In this way, CRP-FFL integrates carbon catabolite control and C4-DC-specific regulation. Moreover, EIIAGlc of the glucose-specific PTS strongly interacts with DctA, which could lead to substrate exclusion of C4-DCs when preferred carbon substrates such as sugars are present. Since C4-DCs are perceived in the periplasmic space by the sensor DcuS, the substrate exclusion is not linked to inducer exclusion, contrasting classical inducer exclusion known for the lactose permease LacY. Thus, aerobic C4-DC metabolism is tightly regulated at the transcriptional and post-translational levels, whereas uptake of L-aspartate by DcuA is essentially unaffected. Overall, transcriptional and post-translational regulation of dctA expression and DctA function efficiently fine-tunes C4-DC catabolism in response to other preferred carbon sources.

Inter-Strain Hybridization of Kluyveromyces lactis for creating efficient lactose-fermenting Yeast

A molecular genetic study of Kluyveromyces lactis yeasts isolated from various dairy products in the countries of the former Soviet Union and other regions of the world has been carried out. Based on physiological tests, four strains were selected that carry different LAC loci and are characterized by good fermentation intensity: VKM Y-1339 (LAC3), VKM Y-1333 (LAC3), NRRL Y-1118 (LAC1), and NRRL Y-1140 (LAC2). Eleven hybrids of the selected strains with different rates of lactose fermentation were obtained. No correlation was found between the intensity of lactose fermentation and the amino acid sequences of the LAC12 lactose permease gene of the LAC1, LAC2, and LAC3 loci. Apparently, a specific combination of genotypes of crossed strains has a more significant effect on the fermentation activity. The results obtained showed that inter-strain hybridization of K. lactis dairy yeast is an effective method for creating new strains with high fermentation capacity. Hybrids H2-3 (NRRL Y-1118 × VKM Y-1333) and H3-3 (NRRL Y-1140 × VKM Y-1333) with the highest ability to ferment lactose are of interest for further molecular genetic research and breeding programs. Key words: Kluyveromyces lactis, β-galactosidase, lactose permease, LAC4, LAC12, LAC1 locus, LAC2 locus, LAC3 locus, inter-strain hybridization, lactose fermentation, heterosis Acknowledgment - The authors are grateful to the Genomic Center of the Kurchatov Institute SRC---GosNIIgentika for sequencing the nucleotide sequences of the LAC12 genes for lactose permease on the Applied Biosystems 3730 automated analyzer. Funding - This work was supported by an internal grant from the National Research Center Kurchatov Institute (order of the National Research Center Kurchatov Institute No. 1779).

Improvement of Pseudoalteromonas haloplanktis TAC125 as a Cell Factory: IPTG-Inducible Plasmid Construction and Strain Engineering

Our group has used the marine bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) as a platform for the successful recombinant production of “difficult” proteins, including eukaryotic proteins, at low temperatures. However, there is still room for improvement both in the refinement of PhTAC125 expression plasmids and in the bacterium’s intrinsic ability to accumulate and handle heterologous products. Here, we present an integrated approach of plasmid design and strain engineering finalized to increment the recombinant expression and optimize the inducer uptake in PhTAC125. To this aim, we developed the IPTG-inducible plasmid pP79 and an engineered PhTAC125 strain called KrPL LacY+. This mutant was designed to express the E. coli lactose permease and to produce only a truncated version of the endogenous Lon protease through an integration-deletion strategy. In the wild-type strain, pP79 assured a significantly better production of two reporters in comparison to the most recent expression vector employed in PhTAC125. Nevertheless, the use of KrPL LacY+ was crucial to achieving satisfying production levels using reasonable IPTG concentrations, even at 0 °C. Both the wild-type and the mutant recombinant strains are characterized by an average graded response upon IPTG induction and they will find different future applications depending on the desired levels of expression.

Modular pathway engineering of key precursor supply pathways for lacto-N-neotetraose production in Bacillus subtilis

Background Lacto-N-neotetraose (LNnT) is one of the important ingredients of human milk oligosaccharides, which can enhance immunity, regulate intestinal bacteria and promote cell maturation. Results In this study, the synthetic pathway of LNnT was constructed by co-expressing the lactose permease (LacY) β-1,3-N-acetylglucosaminyltransferase (LgtA) and β-1,4-galactostltransferase (LgtB) in Bacillus subtilis, resulting in an LNnT titer of 0.61 g/L. Then, by fine-tuning the expression level of LgtB, the growth inhibition was reduced and the LNnT titer was increased to 1.31 g/L. In addition, by modular pathway engineering, the positive-acting enzymes of the UDP-GlcNAc and UDP-Gal pathways were strengthened to balance the two key precursors supply, and the LNnT titer was improved to 1.95 g/L. Finally, the LNnT titer reached 4.52 g/L in a 3-L bioreactor with an optimal glucose and lactose feeding strategy. Conclusions In general, this study showed that the LNnT biosynthesis could be significantly increased by optimizing enzymes expression levels and modular pathway engineering for balancing the precursors supply in B. subtilis.

Deconstructing glucose-mediated catabolite repression of the lac operon of Escherichia coli: I. Inducer exclusion, by itself, cannot account for the repression

The lac operon of Escherichia coli is repressed several 100-fold in the presence of glucose. This repression has been attributed to CRP-mediated transcriptional inhibition and EIIAGlc-mediated inducer exclusion. The growing evidence against the first mechanism has led to the postulate that the repression is driven by inducer exclusion. The literature shows that in fully induced cells, inducer exclusion reduces the permease activity only 2-fold. However, it is conceivable that inducer exclusion drastically reduces the permease activity in partially induced cells. We measured the decline of lactose permease activity due to inducer exclusion in partially induced cells, but found that the permease activity decreased no more than 6-fold. We show that the repression is small because these experiments are performed in the presence of chloramphenicol. Indeed, when glucose is added to a culture growing on glycerol and TMG, but no chloramphenicol, lac is repressed 900-fold. This repression is primarily due to reversal of the positive feedback loop, i.e., the decline of the intracellular TMG level leads to a lower permease level, which reduces the intracellular TMG level even further. The repression in the absence of chloramphenicol is therefore primarily due to positive feedback, which does not exist during measurements of inducer exclusion.

It takes two to tango: The dance of the permease

The lactose permease (LacY) of Escherichia coli is the prototype of the major facilitator superfamily, one of the largest families of membrane transport proteins. Structurally, two pseudo-symmetrical six-helix bundles surround a large internal aqueous cavity. Single binding sites for galactoside and H+ are positioned at the approximate center of LacY halfway through the membrane at the apex of the internal cavity. These features enable LacY to function by an alternating-access mechanism that can catalyze galactoside/H+ symport in either direction across the cytoplasmic membrane. The H+-binding site is fully protonated under physiological conditions, and subsequent sugar binding causes transition of the ternary complex to an occluded intermediate that can open to either side of the membrane. We review the structural and functional evidence that has provided new insight into the mechanism by which LacY achieves active transport against a concentration gradient.

Arg302 governs the pKa of Glu325 in LacY

Lactose permease is a paradigm for the major facilitator superfamily, the largest family of ion-coupled membrane transport proteins known at present. LacY carries out the coupled stoichiometric symport of a galactoside with an H+, using the free energy released from downhill translocation of H+ to drive accumulation of galactosides against a concentration gradient. In neutrophilic Escherichia coli, internal pH is kept at ∼7.6 over the physiological range, but the apparent pK (pKapp) for galactoside binding is 10.5. Surface-enhanced infrared absorption spectroscopy (SEIRAS) demonstrates that the high pKa is due to Glu325 (helix X), which must be protonated for LacY to bind galactoside effectively. Deprotonation is also obligatory for turnover, however. Here, we utilize SEIRAS to study the effect of mutating residues in the immediate vicinity of Glu325 on its pKa. The results are consistent with the idea that Arg302 (helix IX) is important for deprotonation of Glu325.