Applied Microbiology and Biotechnology (2020) 104:3797–3805

https://doi.org/10.1007/s00253-020-10426-0

MINI-REVIEW

Natural and engineered promoters for gene expression

in Lactobacillus species
Ángela Peirotén 1 & José M. Landete 1

Received: 30 September 2019 / Revised: 20 January 2020 / Accepted: 3 February 2020 / Published online: 4 March 2020
# Springer-Verlag GmbH Germany, part of Springer Nature 2020

Abstract
Lactobacillus species are attractive hosts for the expression of heterologous proteins, antigens, vaccines, and drugs due to their
GRAS (generally recognized as safe) status. The bioengineering techniques open new possibilities of improving Lactobacillus
strains. In this regard, the control of the gene expression in Lactobacillus strains through the adequate native or engineered
promoters acquires a key role in the development of biotechnological applications and for their function as probiotic bacteria.
Depending on the objective sought, the protein produced and the strain used, inducible or constitutive promoters can be chosen.
Whereas, when a fine-tuning of gene expression is required, the development of synthetic promoter libraries could be the best
approach. In this work, we revise the main constitutive and inducible natural promoters from Lactobacillus strains or from other
genus that have been applied in Lactobacillus, as well as the few engineered promoters developed for these bacteria.

Keywords Lactobacillus . Promoter . Constitutive . Inducible

Introduction Lactobacilli are therefore an important target for the genetic

modification in order to widen and improve their multiple
Lactobacillus is a genus of Gram-positive bacteria, facultative applications. Therefore, there has been an effort in exploring
anaerobic, bacilliform, non-spore-producing bacteria. the optimization of the expression systems for lactobacilli. A
Lactobacillus species are part of lactic acid bacteria (LAB) key element of the expression vectors is the promoter, which
due to their ability to produce lactic acid from sugars. regulates the timing and levels of expression of the introduced
Lactobacilli are used as starters to manufacture cheeses, yo- gene. The election of a suitable promoter is determined by
ghurt, sourdough breads, silage, table olives, sauerkraut, several factors, including the compatibility with the host
fermented fish, and sausages, and have been proposed as nat- Lactobacillus strain, the desired pattern of expression, and
ural biopreservatives in non-fermented vegetables (Leroy and the nature of the transcriptional product of the gene
Vuyst 2004; Wiernasz et al. 2017). Moreover, lactobacilli are (McCracken et al. 2000; Jensen and Hammer 1998a). Given
natural inhabitants of the human intestinal tract and some the importance of host compatibility, most of the promoters
strains have probiotic functions (Saarela et al. 2000). The wide used in Lactobacillus strains have been obtained from
utilization of many Lactobacillus species has granted them the lactobacilli (Table 1 and Table 2), because the expression
GRAS (generally recognized as safe) status. Moreover, can drop dramatically when a promoter from another micro-
lactobacilli are good candidates to be used as microbial cell organism is used (Scheirlinck et al. 1989; Jensen and Hammer
factories producing recombinant proteins, chemicals, or 1998a). Nevertheless, there are some examples of heterolo-
biofuels (Heiss et al. 2016; Bosma et al. 2017). gous genes expressed in lactobacilli under the control of pro-
moters from bacteria belonging to different genus such as the
constitutive promoters P32 and Plac and the inducible promoter
PnisA from Lactococcus lactis (Table 3).
* José M. Landete Regarding how they regulate the genetic expression, the
josem@inia.es
promoters present in the genome of organisms (natural pro-
1
Departamento de Tecnología de Alimentos, Instituto Nacional de moters) can be differentiated into constitutive promoters and
Investigación y Tecnología Agraria y Alimentaria (INIA), Carretera inducible promoters. The techniques for the determination of
de La Coruña Km 7.5, 28040 Madrid, Spain the gene expression such as microarray, RNAseq, and
3798 Appl Microbiol Biotechnol (2020) 104:3797–3805

Table 1 Examples of applications of constitutive promoters from Lactobacillus strains

Constitutive promoter Heterologous gene or protein Host species/strain Function/characteristic Reference

(gene and organism of origin)

Ptu (elongation factor Tu; evoglow-Pp1 of Lactobacillus strains Molecular markers for Landete et al. (2015),
L. reuteri CECT925) Pseudomonas putida monitoring LAB in food Landete et al. (2017)
and fecal environments
Ptu (elongation factor Tu; β-Glucosidase of L. mucosae L. fermentum INIA Hydrolysis of glucose from Unpublished data
L. reuteri CECT925) INIA P508 P584, L. plantarum flavonoids and lignans
WCFS1
Pldh (lactate dehydrogenase Glucosamine-6-phosphate L. plantarum WCFS1 Food-grade vector Chen et al. (2018)
gene; L. plantarum WCFS1) synthase gene (glmS1) of
L. plantarum WCFS1
Pldh (lactate dehydrogenase Oxalate decarboxylase gene L. plantarum NC8 Degraded oxalate Anbazhagan
gene; L. plantarum (oxdC) of Bacillus subtilis efficiently et al. (2013)
NCIMB8826) under in vitro conditions.
Pldh (lactate dehydrogenase Single-chain antibody against L. paracasei ATCC 393 Passive immunization Krüger et al. (2005)
gene; L. plantarum 80 SA I/II adhesin of
Streptococcus mutans
Pldh (lactate dehydrogenase Carboxymethyl cellulose of B. subtilis RM125 Development of an efficient Baek et al. (1997)
gene; L. casei ATCC393) B. subtilis BSE616 expression and secretion
system
Ppgm (phosphoglycerate Oxalate degradation pathway L. gasseri ATCC Overexpression of oxalate Duong et al. (2010)
mutase gene; L. acidophilus of L. acidophilus NCFM 33323, L. acidophilus degradation pathway.
NCFM) Complementation of
oxalate-deficient mutant
Ppgm (phosphoglycerate Mannanase gene (manB) of L. plantarum WCFS1 Constitutive expression and Nguyen et al. (2019)
mutase gene (pgm); Bacillus licheniformis cell-surface display of a
L. acidophilus NCFM) and DSM13 bacterial β-mannanase
PslpA (S-layer protein A gene;
L. acidophilus ATCC 4356)
PslpA (slpA; L. brevis) Cytokine IL-10 L. casei IGM393 Immunization Kajikawa et al. (2010)
PslpA (slpA; L. brevis) β-Glucuronidase (gusA), L. plantarum, L. Develop of an efficient Kahala and Palva (1999)
luciferase (luc) and gasseri, expression system
aminopeptidase N (pepN) as Lc. lactis
reporter genes
Pcbh (conjugated bile α-Amylase gene (amyL) of L. plantarum Integration for α-amylase Hols et al. (1994)
acid hydrolase gene; B. licheniformis and levanase NCIB8826 and levanase expression
L. plantarum Lp8O) gene (sac) from B. subtilis
Papf (aggregation-promoting Prolyl endopeptidase gene L. casei BL23 Prevention of celiac Alvarez-Sieiro et al.
factor gene; L. crispatus) (pep) of Myxococcus xanthus disease. (2014)
Food-grade vector
Pacc (acetyl coenzyme Cholesterol oxidase gene L. plantarum NCL21 Expression of heterologous Kiatpapan et al. (2001)
A carboxylase gene; (choA) of Streptomyces cholesterol oxidase
L. plantarum L137)

proteomic approach allow the functional study of the pro- promoter activity and is an emerging strategy for genetic mod-
moters. As an example, microarray analysis of the genome ification of lactobacilli.
of Lactobacillus acidophilus found operons that were differ-
entially expressed in response to the available carbohydrate
source, and operons constitutively expressed regardless of Constitutive promoters of Lactobacillus
carbohydrate source (Duong et al. 2010). The correspondent
inducible and constitutive promoters were used by these au- A constitutive promoter is an unregulated promoter that allows
thors to construct a series of expression vectors for use in the continual transcription of its associated gene. The most com-
lactobacilli. Moreover, promoters influence the yield of pro- mon strategies to identify constitutive promoters consist on
tein expressed, being usually classified into strong or weak screening random chromosomal DNA fragments by cloning
promoters. Strength of the promoters is an important trait them in vectors harboring reporter genes or genes that comple-
when choosing recombinant vectors and is a target of the ment auxotroph phenotypes (Bron et al. 2004). Moreover, con-
promoter engineering, which pursues the optimization of stitutive promoters can be identified easier from housekeeping
Appl Microbiol Biotechnol (2020) 104:3797–3805 3799

Table 2 Examples of applications of inducible promoters from Lactobacillus strains

Inducible promoter (gene and Heterologous gene or protein Host species/strain Function/characteristic Reference
organism of origin)

PsppQ (sakacin P cluster; β-Galactosidase of L. reuteri L. plantarum WCFS1 Inducible pSIP expression Nguyen et al. (2015)
L. sakei) system by the inducing
pheromone (IP-673)
PsppA and PsppQ (sakacin β-Galactosidase of L. reuteri L. plantarum WCFS1 Inducible pSIP expression Nguyen et al. (2011)
P cluster; L. sakei) L103 and L. plantarum WCFS1 system by SppIP pheromone.
Food-grade gene expression
systems for lactic acid
bacteria are useful for
applications in the food
industry
PorfX (sakacin P cluster; Chloramphenicol acetyltransferase L. plantarum C11 Expression induced by peptide Mathiesen
L. sakei LTH673) of Bacillus pumilus, pheromone (IP-C11). et al. (2004)
aminopeptidase N of Lc. lactis,
and chitinase B of Serratia
marcescens
PorfX (sakacin P cluster; Heterologous oxalate L. plantarum NC8 Expression of the ability to Kolandaswamy
L. sakei LTH673) decarboxylase gene (oxdc) degrade intestinal dietary et al. (2009)
from B. subtilis oxalate
Pα-amylase (α-amylase gene Single-chain antibody (scFv) L. paracasei ATCC393 Inducible by mannitol Krüger et al. (2005)
of L. amylovorus) against the SA I/II adhesin
of St. mutans
Plac (lac operon, L. casei) ilvBN genes of Lc. lactis L. casei BL23 Repressed by glucose and Gosalbes et al. (2000)
induced by lactose.
Integrative expression
vector for the obtaining of
stable food-grade integrants
Pα-amylase (α-amylase gene Phytase gene (phyC) from L. plantarum strain 755 Inducible by cellobiose Kerovuo and
of L. amylovorus) B. subtilis VTT E-68013 Tynkkynen (2000)
Pxyl (xylose operon of L. casei) Porcine parvovirus (PPV) L. casei ATCC393 Inducible by xylose mucosal Yigang and Yijing
major structural polypeptide vaccine against PPV infection (2008)
VP2.
Plp_0775 (argininosuccinate Cytokine IL-10 L. gasseri ATTC33323 Stress-inducible promoter Allain et al. (2016)
synthase of L. plantarum
WCFS1)

genes, which, since required for the maintenance of basal cellular and the L-lactate dehydrogenase (Anbazhagan et al. 2013),
functions, are expressed irrespective of the developmental stage, which has been used in a lot of applications, even as promoter
cell cycle state, or environmental factors. The rRNA promoter of for the expression of the chimeric single guide RNA of a
any strain of Lactobacillus is a good candidate for constitutive CRISPR-Cas9 system developed for L. casei (Song et al. 2017).
promoter (Rud et al. 2006). Other constitutive promoters are the
promoters of factors of initiation or elongation such as the pro-
moters of elongation factor Tu from Lactobacillus plantarum
CD033 (Ptuf33), Lactobacillus buchneri CD034 (Ptuf34) (Tauer Inducible promoters of Lactobacillus
et al. 2014), and Lactobacillus reuteri CECT925 (PtufR). The last
one was used in place of PnisA in pNZ8048 vector, with good Gene expression is regulated by different cellular mecha-
results in the expression of the reporter green fluorescent protein nisms, starting with the control of transcription at the promoter
for the traceability of Lactobacillus and other LAB strains level. Hence, many genes and operons are not constitutively
(Landete et al. 2015). Other constitutive promoter are the pro- expressed but rather their expression is regulated in response
moter of elongation factor P from L. buchneri CD034 (Pefp) to activator agents; and thus, the correspondent promoters can
(Tauer et al. 2014) and the promoter of elongation factor G and be used for controlling the time of the expression in
P and the promoter of initiation factor IF-2 from Lactobacillus Lactobacillus. Inducible promoters are often regulated by a
casei BL23 (Landete et al. 2017). two-component regulatory system (Sørvig et al. 2005;
Other constitutive promoters of Lactobacillus strains are the Pfeiler et al. 2007), whose encoding genes must be present
promoters of the phosphoglycerate mutase (Duong et al. 2010) in the bacteria in order to exert their regulatory function.
3800 Appl Microbiol Biotechnol (2020) 104:3797–3805

Table 3 Examples of promoters from other organisms used in Lactobacillus strains

Promoter (gene and Heterologous gene or protein Host species/strain Function/characteristic Reference
organism
of origin)

Pspa (protein A gen of Gene fusion SEZZ-VD4 Lactobacillus strains Live vaccine vectors Rush et al. (1997)
Streptococcus aureus) (St. aureus-Chlamydia psittaci)
P25 (Streptococcus M6-gp41E of Streptococcus L. plantarum NCIMB Heterologous secretion of antigens Hols et al. (1997)
thermophilus) pyogenes 8826
PnisA (nisinA of Lc. lactis) Betaine uptake system (BetL) L. salivarius UCC118 Enhancement of stress tolerance Sheehan et al.
of Listeria (2006)
PlacA (lactose operon Rumen microbial fibrolytic L. reuteri Pg4 Acquired ability to secrete fibrolytic Liu et al. (2005)
of Lc. lactis) enzyme genes of Neocallimastix enzymes, adherence to mucin
patriciarum, Fibrobacter and tolerance of acid and bile salts
succinogenes and Piromyces
rhizinflata
PnisA (nisinA of Lc. lactis) Amylase (B. licheniformis) L. reuteri DSM20016 Expression of amylase under Wu et al. (2006)
nisin induction
P32 (Lc. lactis) Alcohol dehydrogenase L. casei 686 Production of ethanol Gold et al. (1996)
(Zymomonas mobilis)
P32 (Lc. lactis) Exendin-4 (artificial sequence, L. paracasei L14 Expression of the therapeutic Zeng et al. (2016)
originally isolated from peptide drug for type 2 diabetes
Heloderma suspectum)
P32 (Lc. lactis) ctsR of Oenococcus oeni L. plantarum WCFS1 Study of the acid-ethanol stress re- Zhao et al. (2019)
sponse
P32 (Lc. lactis) Bile salt hydrolase (bsh) of L. casei LC2W Improve of the BSH activity Xiong et al. (2017)
L. plantarum AR113

Bacteriocin promoters for gene expression Carbon catabolism pathways–controlled expression

systems
One of the most used bacteriocin-inducible promoters,
the nisin-controlled gene expression system (NICE) has In lactobacilli, the regulation of gene expression has been
been successfully adapted to several LAB; nevertheless, studied mainly for carbon catabolism pathways, such as those
it was fo und to be le ss ap propr ia te for some of fructooligosaccharides, lactose, trehalose, xylose, ribose,
Lactobacillus species (Wu et al. 2006). In lactobacilli, maltose, malic acid, sorbitol, myo-inositol, and arginine
promoters from operons of the bacteriocins sakacin A (Duong et al. 2010; Zúñiga et al. 1998; Yebra et al. 2007;
and P, found in Lactobacillus sakei, have been used Alcántara et al. 2008; Monedero et al. 2008; Landete et al.
together with the correspondent regulatory system to 2010). Moreover, promoters from L. plantarum WCFS1
construct vector for inducible gene expression in L. lactose/galactose-inducible have been identified recently
sakei and L. plantarum (Sørvig et al. 2005). Other pro- (Zhao et al. 2019). Genes involved in transport and catabolism
moters from well-known bacteriocin genes have been of carbohydrates are usually organized into strongly expressed
those of plantaricin NC8 (Maldonado et al. 2003), the operons, which are controlled by the catabolite control protein
class IIb bacteriocins (salivaricinT, salivaricin P, and A (CcpA) (Muscariello et al. 2001). The promoters present in
ABP-118), and bactofencin A (Guinane et al. 2015). these operons have two regulatory mechanisms. On one hand,
Similar to NICE, the activity of those promoters is the catabolite repression element (cre) sequence produces the
controlled via a three-component signal transduction repression of the system in the presence of an easily assimila-
system, which responds to an externally added peptide ble carbon source such as glucose. On the other hand, the
pheromone (Maldonado et al. 2004). Once the required absence of glucose and the presence of the activator produce
inducing peptide level is reached, the signal is proc- the induction of expression. Two of those cre sites have been
essed by the regulatory system, which interacts with identified in the operon of the arginine deiminase, which is
the promoter of the bacteriocin genes to allow bacteri- induced by arginine and repressed by glucose through the
ocin production (Maldonado et al. 2003, 2004). These PTS-CcpA signal transduction pathway (Zúñiga et al. 1998).
promoters and peptide pheromone can be used to ex- Thus, consensus sequences have been suggested for the iden-
press genes of interest using expression vectors or even tification of these cre sites in the genome of some bacteria
after insertion in the bacterial genome. (Miwa et al. 2000). These regulatory systems rely on the
Appl Microbiol Biotechnol (2020) 104:3797–3805 3801

carbohydrates available in the media and have a more feasible concentrations (Böhmer et al. 2013). This expression system
application in industrial fermentations compared with pro- does not need the addition of an external inducing agent.
moters induced by peptides.
These promoters can be used for the regulated heterologous
expression of genes of interest in vectors or in integrative Strong and weak promoters of Lactobacillus
food-grade expression systems. An interesting strategy has
been described for the integration of foreign genes into the Constitutive and inducible promoters are classified as strong or
lactose operon of L. casei, putting the heterologous gene un- weak according to its affinity for the RNA polymerase, which is
der the same glucose repression and substrate induction than one of the most influencing factors defining the amount of
that of the lactose operon (Gosalbes et al. 2000). protein finally produced. That affinity is related to the sequence
architecture of the promoter. In Lactobacillus, consensus
Stress-inducible promoters hexamers appear at − 35 (TTGACA) and − 10 (TATAAT) with
respect to the transcription initiation site (Fig. 1), similarly to
The addition of a compound as activator of gene expression is other prokaryotes (Pouwels and Leer 1993), and are the loca-
not always desirable, economical, or even feasible. In those tion where the bacterial RNA polymerase binds. How closely
scenarios, the use of environmental stimuli-based expression the promoter sequence resembles the ideal consensus sequence
systems may be of interest. Therefore, gene expression in- of the − 35 and − 10 hexamers, alongside with the sequence and
duced by environmental stresses (SICE for stress-induced length of the spacer region connecting the two hexamers, influ-
controllable expression) such as low pH, temperature, bile ences greatly the strength of the promoter (Matern et al. 1994).
salts, or NaCl are a good option (Derzelle et al. 2002; The presence of the TG motif appears to be of considerable
Martínez-Fernández et al. 2019). importance in Gram-positive organisms, where introduction
The dnaK operon of L. sakei encodes several heat shock or deletion of the motif can influence promoter activity substan-
proteins and is heat induced. Its promoter region has been tially (Voskuil and Chambliss 1998; McCracken and Timms
probed to respond with a similar heat shocking transcription 1999). Additionally, the UP element, an AT-rich sequence up-
induction when included in an expression plasmid (Schmidt stream of the − 35 hexamer which is contacted by the C-
et al. 1999). Many promoters from Lactobacillus strains are terminal domain of the RNA polymerase α-subunit has been
also regulated in response to oxidative stress (Serrano et al. described to influence the transcription as well (Ross et al.
2007). Hertel et al. (1998) showed that the promoter of the 1993). Other elements adjacent to the promoter have also influ-
KatA, which encodes the true catalase of L. sakei LTH677, is ence in the regulation of the transcription, such as the sequence
regulated by the addition of H2O2 to anaerobic cultures, as of the ribosome binding site (RBS) (Salis et al. 2009) and length
well as by a switch to aerobic conditions, resulting in a strong of the space between it and the start codon (Tauer et al. 2014).
increase in the induction of the gene. A strategy for the determination of strong or weak promoters
Temperature conditions also can influence the gene expres- is usually the utilization of reporter genes. So, we demonstrated
sion. PcspL and PcspP from L. plantarum are induced in re- that the elongation factor Tu promoter from L. reuteri
sponse to cold shock (Mayo et al. 1997; Derzelle et al. CECT925 and the elongation factor P promoter from L. casei
2002). Binishofer et al. (2002) isolated a thermoinducible BL23 are strong and constitutive promoters and they could be
promoter-repressor cassette from the temperate L. casei phage expressed in different Gram-positive bacteria (Landete et al.
φFSW-TI, which is repressed at 28 °C and expressed at 42 °C. 2015, 2017). Likewise, other constitutive promoters corre-
Regarding engineering of probiotic lactobacilli, the control sponding to housekeeping genes are also strong promoters,
of the gene expression under gastrointestinal conditions could such as the rRNA promoters and elongation factors already
allow obtaining their improved effects once the probiotic is in mentioned in the constitutive promoters section.
the intestine. The promoter P16090 from L. casei BL23 was
selected and its bile induction confirmed by means of a gene
reporter in L. casei BL23, L. plantarum WCFS1, Engineered promoters for Lactobacillus
Lactobacillus rhamnosus INIA P232, L. rhamnosus INIA
P426, and L. reuteri INIA P572. The developed vector, Natural promoters do not encompass all the possibilities of
pNZ:16090-aFP, constitutes a promising tool suitable for the transcription regulation, and thus, several strategies have been
expression of genes of interest under intestinal conditions in developed to obtain new synthetic promoters, which would
probiotic Lactobacillus (Martínez-Fernández et al. 2019). allow the fine-tuning of gene regulation, of special interest
Finally, a novel system is based on the manganese in metabolic engineering in order to optimize production
starvation-inducible promoter from a specific manganese trans- (Blazeck and Alper 2013; Jensen and Hammer 1998a).
porter of L. plantarum NC8. The induction of expression was Promoter engineering is an evolving field that has developed
achieved by cultivating L. plantarum NC8 at low manganese diverse technologies for the manipulation of the promoter
3802 Appl Microbiol Biotechnol (2020) 104:3797–3805

Fig. 1 Consensus sequence of 16S rRNA promoters from L. plantarum WCFS1 (Rud et al. 2006). Semi-conserved bases: R = A or G; W = A or T; D =
A, G, or T; N = A, G, T, or C

DNA sequence, aimed towards generating a wide range of promoter, which allows the stable production of a high level
gene transcription levels. In this regard, new promoters can of protein in large-scale fermentations without the need for the
be obtained by constructing hybrid promoters or by altering addition of inducing compounds, avoiding the consequent ad-
the sequence of a natural promoter (Blazeck and Alper 2013). ditional cost. On its part, the libraries of engineered constitu-
Promoter engineering for Lactobacillus should take into ac- tive promoters could offer a wide range of activities of these
count the knowledge about promoters and their structure. So promoters, allowing the fine adjustment of gene expression
far, there are just a few examples of engineered promoters for and conferring advantages over other promoters in metabolic
lactobacilli. Rud et al. (2006) constructed a synthetic promoter engineering (Jensen and Hammer 1998a).
library for Lactobacillus strains by randomizing the non- However, many times, an unregulated promoter does not
consensus spacer sequence of the rRNA constitutive promoters provide the desired effect because, while the gene of interest is
of L. plantarum WCFS1. The resulting promoter library was being expressed at a high level, resources for the rest of met-
tested in L. plantarum and L. sakei obtaining a wide range of abolic routes of the cell are also being subtracted, hindering
promoter activities, evidencing the influence of the spacer se- the bacterial growth. Moreover, the heterologous protein may
quence in the promoter strength. Within the spacer, the TG have a toxic effect on the host cell. Therefore, it is advisable to
motif located upstream of the − 10 hexamer has shown to in- use inducible promoters, allowing the activation of expression
fluence the transcription in Lactobacillus. Hence, the introduc- only when it is necessary or viable. Inducible expression can
tion of consensus sequences − 35 and − 10 and a TG motif into be preferable in cases where the aim is to overproduce a re-
the L. acidophilus ATCC 4356 ribosomA promoter resulted in a combinant protein at high levels in a specific moment, while
increment in transcriptional activity in L. fermentum BR11, avoiding deleterious effects during growth phase (Terpe
although not in L. rhamnosus GG, showing that both strain 2006). The toxicity of the heterologous protein can also take
and context-dependent effects are critical factors influencing to choose a weak promoter, especially if high levels of expres-
transcription in Lactobacillus (McCracken and Timms 1999). sion are not required.
A different approach was used for optimizing two weak It is also necessary to take into account that inducible pro-
lactose/galactose-inducible promoters of L. plantarum moters usually only work within the same genus, as the case of
WCFS1 (Zhang et al. 2019). The sequences on − 35, − 10 re- the promoter inducible by bile (Martínez-Fernández et al.
gions, and RBSs were replace with consensus sequences, in 2019), and in many cases, they only work within the same
different combinations, obtaining strength increases in almost species or the same strain. This is caused many times by the
all the cases compared with their original promoters. Similarly, need of the adequate two-component systems. Therefore, in-
the mutagenesis of P15, a promoter-like sequence from ducible promoters are of much more restricted use, whereas
L. acidophilus ATCC 4359, resulted in the generation of the constitutive promoters usually have a wider application.
hexamers in − 35 and − 10 identical to the consensus se- Nevertheless, exceptions can be found, such as the promoter
quences, causing an increment of the promoter strength of Lc. lactis ilvBN genes, which also work in L. casei
(Arsenijevic and Topisirovic 2000). This optimized promoter (Gosalbes et al. 2000). Even so, inducible promoters can be
caused an increment in chloramphenicol resistance when intro- applied in other bacteria, if the genes involved in its regulation
duced, together with the correspondent gene, in L. reuteri and are also transferred, an example is the transfer of the NICE
L. plantarum, but a decrease of the resistance in L. acidophilus. system of Lc. lactis to strains of Lactobacillus that allows the
The development of engineered promoters for lactobacilli induction by nisin in Lactobacillus strains when the promoter
is still a field to explore. In addition, regulatory sequences, as of nisin is present (Wu et al. 2006).
the cre elements described above, can be a target for modifi- Conversely, constitutive promoters have many times a
cation or elimination in order to change the promoter activity wide range of suitable hosts. Constitutive promoters of
(Krüger and Hecker 1995). Lactobacillus have been used in other LAB such as
Lactococcus, Enterococcus, or Streptococcus, other Gram-
positive bacteria such as Bifidobacterium and Listeria
Key elements for choosing promoters (Landete et al. 2017), and even in E. coli (Klein et al. 1994).
In the same way, constitutive promoters of other species or
The straightforward approach for expressing a gene with high genus have been applied in Lactobacillus (Table 3).
production yield could be choosing a strong constitutive Regarding engineered promoters, Rud et al. (2006) observed
Appl Microbiol Biotechnol (2020) 104:3797–3805 3803

similar levels of expression in both L. plantarum and L. sakei biotechnological and probiotic properties. Although pro-
for the synthetic promoters developed. Nevertheless, a consti- moters from Lactobacillus are adequate for food-grade vec-
tutive promoter does not necessarily have the same activity in tors, most studies used non-food-grade vectors. Therefore,
different organisms (Jensen and Hammer 1998b). Even developing food-grade vectors and integrative food-grade ex-
among Lactobacillus, some promoters have been reported to pression system has a great potential in the development of
be species dependent (Chen and Steele 2005). food and in the production of different enzymes used in food,
human, or animals.
An interesting field is the identification of inducible pro-
Applications of promoters from Lactobacillus moters for the creation of biosensors. Those promoters regu-
strains late the increase of the reporter signal level according to the
concentration of the effector molecule, such as metals, con-
The main objective of searching for promoters is the expres- taminants, or specific molecules from microorganisms.
sion of genes of interest under the regulation of these pro- Finally, engineered promoters allow for the fine-tuning of
moters, through replicative vectors or chromosomal integra- gene expression, which is important for biotechnological ap-
tion. Tables 1 and 2 show examples of the different applica- plications. Therefore, more efforts should be made in the de-
tions of constitutive and inducible promoters from velopment of new engineered promoters generated for
Lactobacillus strains. Lactobacillus strains have the potential Lactobacillus and other GRAS bacteria.
as delivery systems for valuable proteins like antibodies and
antigens. Numerous promoters from Lactobacillus strains Funding information This work was supported by project RTA2017-
00002-00-00 from the Spanish Ministry of Economy and
have been used in oral vaccines to deliver different types of
Competitiveness.
antigens (Tables 1 and 2). In the same way, IL-10 has been
successfully expressed using different recombinant
Compliance with ethical standards
Lactobacillus using constitutive and inducible promoters.
Both tables show the use of promoters in vectors, as well as Conflict of interest The authors declare that they have no conflict of
promoters that have been integrated into the bacterial chromo- interest.
some. The use of promoters in the development of food-grade
vectors or integrative food-grade expression system is also Ethical statement This article does not contain any studies with human
encompassed. participants or animals performed by any of the authors.
There are constitutive promoters that have been used for
the expression of various proteins of interest, such as the pro-
moter of the elongation factor Tu of L. reuteri CECT925. This
and other constitutive promoters of Lactobacillus have been
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