New Expression System Tightly Controlled by Zinc Availability in Lactococcus lactis

Here we developed the new expression system P_Zn zitR, based on the regulatory signals (P_Zn promoter and zitR repressor) of the Lactococcus lactis zit operon, involved in Zn²⁺ high-affinity uptake and regulation. A P_Zn zitR-controlled expression vector was constructed, and expression regulation was studied with two reporter genes, uspnuc and lacLM; these genes encode, respectively, a protein derived from Staphylococcus aureus secreted nuclease and Leuconostoc mesenteroides cytoplasmic β-galactosidase. Nuclease and β-galactosidase activities of L. lactis MG1363 cells expressing either uspnuc or lacLM under the control of P_Zn zitR were evaluated on plates and quantified from liquid cultures as a function of divalent metal ion, particularly Zn²⁺, availability in the environment. Our results demonstrate that P_Zn zitR is highly inducible upon divalent cation starvation, obtained either through EDTA addition or during growth in chemically defined medium, and is strongly repressed in the presence of excess Zn²⁺. The efficiency of the P_Zn zitR expression system was compared to that of the well-known nisin-controlled expression (NICE) system with the same reporter genes cloned under either P_Zn zitR or P_nisA nisRK control. lacLM induction levels reached with both systems were on the same order of magnitude, even though the NICE system is fivefold more efficient than the P_Zn zitR system. An even smaller difference or no difference was observed after 3 h of induction when nuclease was used as a reporter for Western blotting detection. P_Zn zitR proved to be a powerful expression system for L. lactis, as it is tightly controlled by the zinc concentration in the medium.     

Lactococcus lactis, an efficient cell factory for recombinant protein production and secretion

The use of Gram-positive bacteria for heterologous protein production proves to be a useful choice due to easy protein secretion and purification. The lactic acid bacterium Lactococcus lactis emerges as an attractive alternative to the Gram-positive model Bacillus subtilis. Here, we review recent work on the expression and secretion systems available for heterologous protein secretion in L. lactis, including promoters, signal peptides and mutant host strains known to overcome some bottlenecks of the process. Among the tools developed in our laboratory, inactivation of HtrA, the unique housekeeping protease at the cell surface, or complementation of the Sec machinery with B. subtilis SecDF accessory protein each result in the increase in heterologous protein yield. Furthermore, our lactococcal expression/secretion system, using both P(Zn)zitR, an expression cassette tightly controlled by environmental zinc, and a consensus signal peptide, SP(Exp4), allows efficient production and secretion of the staphylococcal nuclease, as evidenced by protein yields (protein amount/biomass) comparable to those obtained using NICE or P170 expression systems under similar laboratory conditions. Finally, the toolbox we are developing should contribute to enlarge the use of L. lactis as a protein cell factory.     

A new and efficient phosphate starvation inducible expression system for Lactococcus lactis

A new expression system for Lactococcus lactis was developed. The system is based on a phosphate starvation inducible pstF promoter of L. lactis MG1363. Intracellular beta-galactosidase and secreted alpha-amylase were produced using this tightly regulated system. No evidence of regulatory sites in regions of the 5'-end of the pstF coding sequence was found. High expression levels of the beta-galactosidase gene were obtained using the original pstF RBS in a phosphate-depleted medium. The results suggested that with the phosphate starvation inducible system, it is possible to achieve expression levels comparable to the ones obtained with the widely used nisin-controlled gene expression system (NICE). A specific beta-galactosidase activity of 670 microkat g(-1) using a phosphate-depleted medium and an alpha-amylase activity of 3.6 microkat l(-1) in a bioreactor cultivation were produced. The advantages of the current expression system include that no prior removal of phosphate from the medium in bioreactor scale is required, and no additions of inducing agents are needed. Furthermore, the system can be operated in L. lactis without introduction of regulatory genes into the host.     

Development of a PCR-based mating-type assay for Clavicipitaceae

A new controlled production system to target heterologous proteins to cytoplasm or extracellular medium is described for Lactococcus lactis NCDO2118. It is based on the use of a xylose-inducible lactococcal promoter, P(xylT). The capacities of this system to produce cytoplasmic and secreted proteins were tested using the Staphylococcus aureus nuclease gene (nuc) fused or not to the lactococcal Usp45 signal peptide. Xylose-inducible nuc expression is tightly controlled and resulted in high-level and long-term protein production, and correct targeting either to the cytoplasm or to the extracellular medium. Furthermore, this expression system is versatile and can be switched on or off easily by adding either xylose or glucose, respectively. These results confirm the potential of this expression system as an alternative and useful tool for the production of proteins of interest in L. lactis.     

Leuconostoc lactis beta-galactosidase is encoded by two overlapping genes.

A 16-kb BamHI fragment of the lactose plasmid pNZ63 from Leuconostoc lactis NZ6009 was cloned in Escherichia coli MC1061 by using pACYC184 and was found to express a functional beta-galactosidase. Deletion and complementation analysis showed that the coding region for beta-galactosidase was located on a 5.8-kb SalI-BamHI fragment. Nucleotide sequence analysis demonstrated that this fragment contained two partially overlapping genes, lacL (1,878 bp) and lacM (963 bp), that could encode proteins with calculated sizes of 72,113 and 35,389 Da, respectively. The L. lactis beta-galactosidase was overproduced in E. coli by using a lambda pL expression system. Two new proteins with M(r)s of 75,000 and 36,000 appeared upon induction of PL. The N-terminal sequences of these proteins corresponded to those deduced from the lacL and lacM gene sequences. Mutation and deletion analysis showed that lacL expression is essential for LacM production and that both the lacL and lacM genes are required for the production of a functional beta-galactosidase in E. coli. The deduced amino acid sequences of the LacL and LacM proteins showed considerable identity with the sequences of the N- and C-terminal parts, respectively, of beta-galactosidases from other lactic acid bacteria or E. coli. DNA and protein sequence alignments suggest that the L. lactis lacL and lacM genes have been generated by an internal deletion in an ancestral beta-galactosidase gene.     

Cloning and expression of the beta-galactosidase genes from Lactobacillus reuteri in Escherichia coli

Heterodimeric beta-galactosidase of Lactobacillus reuteri L103 is encoded by two overlapping genes, lacL and lacM. The lacL (1887bp) and lacM (960bp) genes encode polypeptides with calculated molecular masses of 73,620 and 35,682Da, respectively. The deduced amino acid sequences of lacL and lacM show significant identity with the sequences of beta-galactosidases from other lactobacilli and Escherichia coli. The coding regions of the lacLM genes were cloned and successfully overexpressed in E. coli using an expression system based on the T7 RNA polymerase promoter. Expression of lacL alone and coexpression of lacL and lacM as well as activity staining of both native and recombinant beta-galactosidases suggested a translational coupling between lacL and lacM, indicating that the formation of a functional beta-galactosidase requires both genes. Recombinant beta-galactosidase was purified to apparent homogeneity, characterized and compared with the native beta-galactosidase from L. reuteri L103.     

The lactose transporter in Leuconostoc lactis is a new member of the LacS subfamily of galactoside-pentose-hexuronide translocators.

The gene encoding the lactose transport protein (lacS) of Leuconostoc lactis NZ6009 has been cloned from its native lactose plasmid, pNZ63, by functional complementation of lactose permease-deficient Escherichia coli mutants. Nucleotide sequence analysis revealed an open reading frame with the capacity to encode a protein of 639 amino acids which had limited but significant identity to the lactose transport carriers (LacS) of Streptococcus thermophilus (34.5%) and Lactobacillus bulgaricus (35.6%). This similarity was present both in the amino-terminal hydrophobic carrier domain, which is homologous to the E. coli melibiose transporter, and in the carboxy-terminal enzyme IIA-like regulatory domain. The flanking regions of DNA surrounding lacS were also sequenced. Preceding the lacS gene was a small open reading frame in the same orientation encoding a deduced 95-amino-acid protein with a sequence similar to the amino-terminal portion of beta-galactosidase I from Bacillus stearothermophilus. The lacS gene was separated from the downstream beta-galactosidase genes (lacLM) by 2 kb of DNA containing an IS3-like insertion sequence, which is a novel arrangement for lac genes in comparison with that in other lactic acid bacteria. The lacS gene was cloned in an E. coli-Streptococcus shuttle vector and was expressed both in a lacS deletion derivative of S. thermophilus and in a pNZ63-cured strain, L. lactis NZ6091. The role of the LacS protein was confirmed by uptake assays in which substantial uptake of radiolabeled lactose or galactose was observed with L. lactis or S. thermophilus plasmids harboring an intact lacS gene. Furthermore, galactose uptake was observed in NZ6091, suggesting the presence of at least one more transport system for galactose in L. lactis.     

The development of TnNuc and its use for the isolation of novel secretion signals in Lactococcus lactis

We have previously used Tn917 for the identification and characterization of regulated promoters from Lactococcus lactis [Israelsen et al., Appl. Environ. Microbiol. 61 (1995) 2540-2547]. We describe here the construction of a new Tn917-transposon derivative, termed TnNuc, which includes the Staphylococcus aureus nuclease gene (nuc) as a reporter for secretion. Transposition of TnNuc into the L. lactis chromosome allows the generation of fusions in-frame with the nuc gene. TnNuc includes also lacZ, a reporter used for identification of relevant clones from the library, i.e. clones with Lac+ phenotype result from transposition of TnNuc into a functional gene on the L. lactis chromosome. The presence of a functional signal sequence at the upstream flanking region of the left repeat of the transposed element results in the detection of nuclease activity using a sensitive plate assay. TnNuc was used for the identification of novel secretion signals from L. lactis. The sequences identified included known and unknown lactococcal-secreted proteins containing either a signal peptidase-I or -II recognition sequence. In one case, the gene identified codes for a transmembrane protein. The sequences identified were used to study functionality when located in a plasmid under the control of the pH and growth phase-dependent promoter P170 [Madsen et al., Mol. Microbiol. 32 (1999) 75-87]. In all cases, concurrent secretion of nuclease was observed during induction of P170 in a fermentor.     

Expression of a beta-galactosidase gene from Clostridium acetobutylicum in Lactococcus lactis subsp. lactis

A beta-galactosidase gene from Clostridium acetobutylicum NCIB 2951 was expressed after cloning into pSA3 and electroporation into derivatives of Lactococcus lactis subsp. lactis strains H1 and 7962. When the clostridial gene was introduced into a plasmid-free derivative of the starter-type Lact. lactis subsp. lactis strain H1, the resulting construct had high beta-galactosidase activity but utilized lactose only slightly faster than the recipient. beta-galactosidase activity in the construct decreased by over 50% if the 63 kb Lac plasmid pDI21 was also present with the beta-galactosidase gene. Growth rates of Lac+ H1 and 7962 derivatives were not affected after introduction of the clostridial beta-galactosidase, even though beta-galactosidase activity in a 7962 construct was more than double that of the wild-type strain. When pDI21 was electroporated into a plasmid-free variant of strain 7962, the recombinant had high phospho-beta-galactosidase activity and a growth rate equal to that of the H1 wild-type strain. The H1 plasmid-free strain grew slowly in T5 complex medium, utilized lactose and contained low phospho-beta-galactosidase activity. We suggest that beta-galactosidase expression can be regulated by the lactose phosphotransferase system-tagatose pathway and that Lact. lactis subsp. lactis strain H1 has an inefficient permease for lactose and contains chromosomally-encoded phospho-beta-galactosidase genes.     

Adaptation of the nisin-controlled expression system in Lactobacillus plantarum: a tool to study in vivo biological effects

The potential of lactic acid bacteria as live vehicles for the production and delivery of therapeutic molecules is being actively investigated today. For future applications it is essential to be able to establish dose-response curves for the targeted biological effect and thus to control the production of a heterologous biopeptide by a live lactobacillus. We therefore implemented in Lactobacillus plantarum NCIMB8826 the powerful nisin-controlled expression (NICE) system based on the autoregulatory properties of the bacteriocin nisin, which is produced by Lactococcus lactis. The original two-plasmid NICE system turned out to be poorly suited to L. plantarum. In order to obtain a stable and reproducible nisin dose-dependent synthesis of a reporter protein (beta-glucuronidase) or a model antigen (the C subunit of the tetanus toxin, TTFC), the lactococcal nisRK regulatory genes were integrated into the chromosome of L. plantarum NCIMB8826. Moreover, recombinant L. plantarum producing increasing amounts of TTFC was used to establish a dose-response curve after subcutaneous administration to mice. The induced serum immunoglobulin G response was correlated with the dose of antigen delivered by the live lactobacilli.     

Development of a LAC4 promoter-based gratuitous induction system in Kluyveromyces lactis

A gratuitous induction system based on the strong, indigenous LAC4 promoter was developed for Kluyveromyces lactis. To prevent consumption of the inducer galactose, a strain with a gal1-209 mutation was employed; this mutation disables the galactokinase function but retains the regulatory function for induction. The Escherichia coli lacZ gene (encoding beta-galactosidase) is functional in K. lactis and was used as the reporter gene downstream of the LAC4 promoter on a multicopy plasmid. The gal1-209 strain exhibited several unexpected phenomena, including partial consumption of the inducer galactose (although at a much slower rate relative to GAL1 strains) and growth inhibition at high concentrations of galactose. These unusual characteristics, however, did not prevent the successful construction of a strong gratuitous induction system. Due to the low rate of inducer consumption for the gratuitous strain, very low concentrations of galactose (1:20 galactose:glucose) resulted in high-level induction. Under these conditions, beta-galactosidase specific and volumetric activities were 4.2- and 5.5-fold higher, respectively, than those for the "GAL1" nongratuitous strain. This research demonstrated the improved productivity possible via LAC4 promoter-based gratuitous induction (and thus a more stable inducer concentration). The effects of various carbon source concentrations on growth and induction were also determined.     

Identification of Nisin-Producing Strains by Nisin-Controlled Gene Expression System

A specific method to identify nisin-producing strains was developed based on Nisin-Controlled gene Expression (NICE) vector pSec:Nuc. The plasmid pSec:Nuc was transformed into non-nisin-producing strain Lactococcus lactis NZ9000, a host commonly used for the NICE system. The generating strain L. lactis NZ9000/pSec:Nuc could sense extracellular inducer nisin and efficiently secrete a reporter protein Nuc, the staphylococcal nuclease (Nuc) into the medium. Instead of using purified nisin, the culture supernatants of nisin-producing strains were also used as inducers. Therefore, the NICE system could be used to identify nisin-producing strains. With this principle, 4 among 56 lactococci strains isolated from raw milk were identified as nisin producers. The results were further confirmed by polymerase chain reaction amplification with their genomic DNA as templates, and nucleotide sequencing revealed that three of them produced nisin A, and the others produced nisin Z. Those results made it possible to isolate and identify nisin-producing strains specifically and rapidly using NICE system.     

Development and application of a microtiter plate-based autoinduction bioassay for detection of the lantibiotic subtilin

Production of the lantibiotic subtilin in Bacillus subtilis ATCC 6633 is regulated in a quorum sensing-like mechanism with subtilin acting as autoinducer and signal transduction via the subtilin-specific two-component regulation system SpaRK. Here, we report the construction and application of a subtilin reporter strain in which subtilin induced lacZ gene expression in a B. subtilis ATCC 6633 spaS gene deletion mutant is monitored and visualized by the beta-galactosidase in a chromogenic plate assay. A quantitative microtiter plate subtilin bioassay was developed and optimized. Maximal sensitivity of the system was achieved after 6 h of incubation of the reporter strain together with subtilin in a medium containing 300 mM NaCl. This sensitive and unsusceptible method was applied to identify subtilin producing B. subtilis wild type strains from both, culture collections and soil samples. The B. subtilis lantibiotic ericin S with four amino acid exchanges compared to subtilin induces the subtilin reporter strain, in contrast to the structurally closely related Lactococcus lactis lantibiotic nisin. These observations suggest a certain substrate specificity of the histidine kinase SpaK, which however, also would allow the identification of subtilin-isoform producing microorganisms.     

Regulation of beta-galactosidase synthesis in Escherichia coli by exogenous cyclic 3',5'-adenosine monophosphate]

The effect of cyclic 3',5'-adenosine monophosphate (cAMP) on the rate of beta-galactosidase biosynthesis was studied in the cells of Escherichia coli M-17 growing in MPB and mineral media with glucose and maltose, i.e. under the conditions of various catabolite repression, as well as upon lac-operon induction by isopropyl-beta-D-galactopyranoside (IPGP). The stimulating action of exogenous cAMP was found only in a medium with salts and glucose. The induction by IPGP was highest during the growth in a medium with glucose and maltose. When the medium contained IPGP, cAMP accelerated the enzyme synthesis in all media, but only at the early growth phases, while cAMP eliminated the effect of IPGP at the stationary phase of growth. The regulation of beta-galactosidase biosynthesis by cAMP demonstrated for the first time that this effect depended on the physiological state of E. coli: the expression of catabolite-sensitive E. coli genes was subject to both positive and negative regulation in one and the same inducible system. The effect exerted by cAMP depended on the nature of a carbon source in the growth medium.