Applied Microbiology and Biotechnology (2019) 103:1617–1626

https://doi.org/10.1007/s00253-018-9552-x

MINI-REVIEW

Expression of cry genes in Bacillus thuringiensis biotechnology

Qi Peng 1 & Qingyue Yu 1 & Fuping Song 1

Received: 28 October 2018 / Revised: 28 November 2018 / Accepted: 29 November 2018 / Published online: 7 January 2019
# Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract
Bacillus thuringiensis is a gram-positive, spore-forming bacterium that produces insecticidal crystal proteins during sporulation.
The production of these crystals results primarily from the expression of cry genes. In this review, we focus on the expression and
application of cry genes directed by both cry gene promoters and non-cry gene promoters in different hosts. However, not all cry
genes and niches are compatible with B. thuringiensis. New delivery systems offsetting the current limitations in B. thuringiensis
application are needed to improve Cry production, niche fitness, and persistence. This review examines currently available
research and highlights areas in need of further research and development for more effective production and utilization of Cry
insecticidal proteins.

Keywords cry gene promoters . Non-cry gene promoters . Bacterial insecticide . Bacillus thuringiensis biotechnology

Introduction and Lereclus 1995; Baum and Malvar 1995; Deng et al.
2014; Komano et al. 2000). Here, we primarily focus on
Bacillus thuringiensis (Bt) is a gram-positive, spore-forming the expression and application of cry genes directed by
bacterium that produces insecticidal crystal proteins (ICPs) cry gene promoters and non-cry gene promoters in differ-
during sporulation. ICPs are mainly classified into Cry and ent hosts, which include Bt microorganisms, non-Bt mi-
Cyt, and their crystals have various forms: bipyramidal croorganisms, and transgenic crops in order to provide an
(Cry1), cuboidal (Cry2), flat rectangular (Cry3A), irregular overview of current knowledge and to highlight areas that
(Cry3B), spherical (Cry4A and Cry4B), and bar shape would benefit from further research. As Bt has been con-
(Cry11A). As an insect pathogen, Bt insecticidal activity is sidered the most successful bioinsecticide of the last cen-
attributed to parasporal crystals, which are toxic to a wide tury (Jouzani et al. 2017), an overview of known expres-
variety of insect species among the orders Lepidoptera, sion mechanisms and methods to increase application and
Coleoptera, Hymenoptera, Diptera, Homoptera, Orthoptera, effectiveness will help to provide possible direction for
and Mallophaga, and against nematodes, mites, and protozoa further research.
(Schnepf et al. 1998). As of October 2018, approximately 846
cry and cyt genes have been discovered (http://www.lifesci.
sussex.ac.uk/home/Neil_Crickmore/Bt/). cry genes are Expression of cry genes directed by cry gene
expressed during the stationary phase, and their products promoters
generally accumulate in mother cell compartments to form a
crystal inclusion that can account for 20 to 30 % of the dry Transcriptional regulation mechanisms of cry genes have
weight of sporulating cells (Schnepf et al. 1998). been classified into two types: sporulation-dependent pro-
Several cry gene promoters have been identified, and moters are controlled by sporulation-specific sigma factors
their transcription has been extensively reviewed (Agaisse SigK and/or SigE, and sporulation-independent promoters
are under the control of the vegetative SigA factor.
Accessory factors also contribute to the transcriptional regu-
* Fuping Song lation of cry gene expression such as Spo0A, ORF2, and
[email protected]
CcpA (Deng et al. 2014). Recently, Peng et al. (2018) report-
1
State Key Laboratory for Biology of Plant Diseases and Insect Pests,
ed that expression of cry5Ba was silenced when YBT-1518
Institute of Plant Protection, Chinese Academy of Agricultural strain was outside of the host; however, when ingested by
Sciences, Beijing 100193, China Caenorhabditis elegans, Cry5Ba was synthesized in vivo by
1618 Appl Microbiol Biotechnol (2019) 103:1617–1626

YBT-1518. Cry5Ba silencing was due to sRNA BtsR1, to synthesize Cry1Ab, Cry1Ab F371A, or mutant
which bind to the ribosomal binding site of the cry5Ba tran- Cry1AbMod proteins to study the mechanism of pore-
script via direct base pairing and inhibited cry5Ba expres- forming toxins directed by cry1A promoter (Pacheco et al.
sion. Identification of these cry gene promoters and clarifi- 2009). Another study used cry1Ac promoter to direct co-
cation of their regulation mechanisms provide expression expression of cry64Ba and cry64Ca genes in acrystalliferous
elements for cry gene expression. Bt strain HD73−, which resulted in high insecticidal activity
The expression of many cry genes is under the control of against two important Hemipteran rice pests, Laodelphax
cry gene promoters in Bt used for studying novel gene activity, striatellus and Sogatella furcifera (Liu et al. 2018).
genetic insecticidal mechanisms, construction of engineered The transcription of cry3 gene is initiated during vegetative
strains, and other applications (Table 1). The transcription of growth, activated at the end of the exponential phase, and
cry1A promoter is controlled by both SigE and SigK (Aceves- continues for several hours during the stationary phase
Diez et al. 2007; Bravo et al. 1996; Buasri and Panbangred (Agaisse and Lereclus 1994b; Agaisse and Lereclus 1995;
2012; Perez-Garcia et al. 2010; Sedlak et al. 2000; Walter and Salamitou et al. 1996). Production of Cry1AbMod and
Aronson 1999; Yang et al. 2012) and is most commonly used Cry1AcMod (Garcia-Gomez et al. 2013), Cry1Ac (Chaoyin
for the expression of cry genes, including cry1Ac (Roh et al. et al. 2007), Cry1C (Sanchis et al. 1996), Cry3A (de Souza
2004; Sun et al. 2016; Xia et al. 2009a; Xia et al. 2005; Xia et al. 1993), and Cry8 (Amadio et al. 2013) were found to be
et al. 2009b; Yan et al. 2014), cry2Ab27 (Somwatcharajit et al. directed by the cry3A promoter. pSTK is an E.coli-Bt shuttle
2014), cry11A (Wu and Federici 1995), cry8 (Amadio et al. vector, used to produce Cry8Ga1 (Jia et al. 2014) and
2013), cry64Ba, and cry64Ca (Liu et al. 2018). Co-expression Cry69Aa1 (Guan et al. 2014), that carries cry3A promoter.
of cry1Ac and Av3 produced a neurotoxin of Anemonia viridis Cry1AbMod and Cry1AcMod-encoded genes were cloned
that improved insecticidal toxicity in the Bt acrystalliferous efficiently under the regulation of the cry3A promoter region
strain Cry−B (Yan et al. 2014). Gomez et al. (2014) used Bt to drive their expression in Bt but demonstrated no expression

Table 1 Expression of cry genes directed by cry gene promoters in Bt

Cry protein Promoters Vectors Bt strain Ref.

Cry1Ab cry1A promoter pHT315 4Q7− (Gomez et al. 2014; Pacheco et al. 2009)
Cry1Ac5 cry1Ac promoter pHT315 Cry−B (Sun et al. 2016)
−
Cry1Ac-av3 cry1Ac promoter pHT315 Cry B (Yan et al. 2014)
Cry1Ac cry1Ac promoter pHT3101 4Q7− (Roh et al. 2004; Xia et al. 2005)
Cry1AbMod/Cry1AcMod cry3A promoter pHT315 407− (Garcia-Gomez et al. 2013)
−
Cry1Ac cry1Ac promoter pHT315 Cry B (Xia et al. 2009b)
Cry1Ac cry1Ac promoter pHT304 XBU001 (Xia et al. 2009a)
Cry1Ac cry3A promoter pBMB31-304 BMB171 (Chaoyin et al. 2007)
Cry1Ba cry8E promoter pHT315 HD73− (Zhou et al. 2014)
Cry1C cry3A promoter pHT304-18Z 407− (Sanchis et al. 1996)
−
Cry2Ab27 cry1Ac promoter pHT304-18Z SP41 and 407 (Somwatcharajit et al. 2014)
Cry2Ab cry2Aa promoter pHT3101 4Q7− (Jain et al. 2006)
−
Cry3A cry3A promoter pHT304 HD1 (de Souza et al. 1993)
Cry4B/Cry4A cry4B promoter pHT3101/pHT315 4Q2-71 (Delecluse et al. 1993; Rodriguez-Almazan et al. 2012)
Cry5B cry5B promoter pHT304 BMB171 (Sajid et al. 2018)
Cry6A cry6A promoter pHT304 BMB171 (Dementiev et al. 2016)
Cry8 cry1Ac /cry3Aa promoters pHT3101 4Q7− (Amadio et al. 2013)
Cry8Ga1 cry3A promoter pSTK BIOT185 (Jia et al. 2014)
Cry8Kb3/Cry8Pa3 cry8Kb3/cry8Pa3 pHT3101 4Q7− (Navas et al. 2014)
promoters
Cry11A cry1Ac promoter pHT3101 4Q7− (Wu and Federici 1995)
Cry26Aa/Cry28Aa cry1Ca promoter pHT304 YBT-020 (Ji et al. 2009)
Cry64Ba/Cry64Ca cry1Ac promoter pHT315 HD73− (Liu et al. 2018)
Cry69Aa1 cry3A promoter pSTK HD73− (Guan et al. 2014)
Appl Microbiol Biotechnol (2019) 103:1617–1626 1619

in E. coli cells (Garcia-Gomez et al. 2013). Nevertheless, the promoters to drive expression. They found that the volume
Cry1AMod proteins produced from the cry3A promoter in Bt of Cry3A crystals produced with cyt1Aa promoters and the
were not soluble and showed low toxicity against Plutella STAB-SD sequence was 1.3-fold that of typical bipyramidal
xylostella larvae. Construction of site-directed mutagenesis Cry1 crystals toxic to Lepidopteran insects (Park et al. 1998).
of cysteine residues in the p3A-Cry1AbMod and p3A- The dual-promoter/STAB-SD system offers an additional
Cry1AcMod protoxin gene reestablished solubility and toxic- method for potentially improving the efficacy of insecticides
ity to P. xylostella. This suggested that the combination of the based on Bt. Krishnan et al. (2017) used cyt1Aa promoter to
cry3A promoter expression system with single cysteine muta- direct the expression of cry41Aa to study the action mecha-
tions is a useful system for efficient expression of Cry1AMod nism of certain protein toxins from the normally insecticidal
toxins in Bt (Garcia-Gomez et al. 2013). bacterium Bt in targeted human cell lines. An amylase pro-
Expression of other cry genes is directed by their own pro- moter fragment, amyE, was fused into the promoter region to
moters, including cry4B and cry4A (Delecluse et al. 1993), induce the expression of cry1Ac (Yang et al. 2003) and cry1C
cry5B (Sajid et al. 2018), cry8Kb3 and cry8Pa3 (Navas (Chak et al. 1994) in the early log phase instead of the Bt cry
et al. 2014), and cry2Ab (Jain et al. 2006). Dementiev et al. replicon, which was promoted only at the sporulation stage. A
(2016) used Cry6Aa to determine the Cry6Aa structure in recent report showed that there are also some highly active
protoxin and trypsin-activated forms as well as the pore- non-cry gene promoters for expression of cry genes. PexsY, a
forming mechanism of action, which was under the control strong activity promoter of the exosporium basal layer struc-
of its own promoter in Bt strain BMB171. Other studies have tural gene exsY in the late sporulation phase, was used to
used plasmid pHT618 to produce Cry4Ba and Cry11Aa crys- express cry1Ac genes in Bt in order to discover new elements
tals by their own promoters in an acrystalliferous Bt strain to for cry gene expression (Zheng et al. 2014). A SigE-dependent
compare the cadherin-binding affinity of Cry4Ba and strong promoter of a non-cry gene (HD73_5014) was used to
Cry11Aa (Delecluse et al. 1993; Rodriguez-Almazan et al. direct strong cry1Ac gene expression in Bt HD73 (Zhang et al.
2012). Zhou et al. (2014) compared the transcriptional activity 2018). The expression of the cry1Ac gene directed by the
of cry1Ac, cry3A, cry4A, and cry8E promoters and found that HD73_5014 gene promoter was the same level as that directed
cry8E promoter showed the highest transcriptional activity by the previous strongest known cry promoter, Pcry8E. The
among these promoters in Bt. The researchers constructed a expression of crystal proteins initiated by these high-
novel E. coli-Bt shuttle vector, pHT315-8E21b, for cry gene activation promoters in Bt could be used to develop safe
expression using the cry8E promoter and multiple cloning high-efficiency biological pesticides.
sites from vector pET21b (based on vector pHT315), then
produced Cry1Ba in the sigK mutant against Ostrinia
furnacalis and Plutella xylostella. The findings suggested that Expression of cry genes in non-Bt
cry8E promoter can be an efficient transcriptional element for microorganisms
cry gene expression and utilized for construction of a geneti-
cally engineered strain. E. coli expression systems are commonly used for the expres-
sion of cry genes, especially in terms of novel gene activity
and genetic insecticidal mechanisms. T7 and tac promoters are
Expression of cry genes directed by non-cry most commonly used to direct the expression of cry genes
gene promoters carried on pET or pGEX series vectors with His or GST tags.
For example, evaluation of Cry1A (Azizoglu et al. 2016;
Non-cry gene promoters are usually used to direct the expres- Huang et al. 2004; Khasdan et al. 2007; Reddy et al. 2013),
sion of cry genes (Table 2). The most commonly used pro- Cry1Ea11 (Huang et al. 2018), Cry2 (Ogunjimi et al. 2002;
moter is Pcyt1A, which is also a SigE- and SigK-controlled Pan et al. 2014; Reyaz and Arulselvi 2016; Reyaz et al. 2017;
promoter (Sakano et al. 2017). It has been used to direct many Saleem and Shakoori 2017; Yilmaz et al. 2017), Cry4
Cry protein production including Cry1C-t (Park et al. 2000), (Boonserm et al. 2004; Zhang et al. 2014), Cry6A (Wang
Cry2A and Cry2B (Crickmore et al. 1994), Cry2A and et al. 2017), Cry11A (AP et al. 2016), Cry21 (Iatsenko et al.
Cry11A (Park et al. 1999), Cry3A (Park et al. 1998), 2014), Cry46Ab (Hayakawa et al. 2017), and Cry78Aa toxic
Cry9Ec1 (Wasano et al. 2005), Cry11Aa and Cry11Ba (Sun activity (Wang et al. 2018a), as well as studying the insecti-
et al. 2014), Cry11B (Park et al. 2001), Cry19A (Barboza- cidal mechanisms of Cry1A (Adegawa et al. 2017; Martinez-
Corona et al. 2012), Cry20Aa (Lee and Gill 1997), Cry27A Solis et al. 2018; Tanaka et al. 2016) and Cry2 (Shu et al.
(Saitoh et al. 2000), Cry30Ca, Cry60Aa, Cry60Ba (Sun et al. 2017; Xu et al. 2016). Another study used pET21b vector to
2013), and Cry41Aa (Krishnan et al. 2017). One study con- express Cry9Aa and Vip3Aa toxins to study the specific in-
structed vectors that expressed cry3A with (pPFT3As) and teraction between two Bt toxins creating insecticidal syner-
without (pPFT3A) in the STAB-SD sequence, using cytA gism and unraveling the molecular basis of this interaction
1620 Appl Microbiol Biotechnol (2019) 103:1617–1626

Table 2 Expression of cry genes directed by non-cry gene promoters in Bt

Cry gene Promoters Vectors Bt strain Ref.

Cry1C-t cyt1A promoter pHT3101 4Q7− (Park et al. 2000)

Cry2A/Cry2B cry2A/cytA promoter pSV2 An acrystalliferous Bt strain (Crickmore et al. 1994)
Cry2A/Cry11A cyt1A promoter pHT3101 4Q7− (Park et al. 1999)
−
Cry3A cyt1Aa promoter pHT3101 4Q7 (Park et al. 1998)
Cry9Ec1 cyt1A2 promoter pHT3101 BFR1 (Wasano et al. 2005)
Cry11Aa/ Cry11Ba cyt1A promoter pHT304 4Q7− (Sun et al. 2014)
Cry11B cyt1A promoter pHT3101 Bt subsp. israelensis strain (Park et al. 2001)
Cry19A cyt1A promoter pHT3101 4Q7− (Barboza-Corona et al. 2012)
Cry20Aa cyt1Aa promoter pHT304 YG1 or LG101 (Lee and Gill 1997)
Cry27A cyt1A promoter pHT3101 BFR1 (Saitoh et al. 2000)
Cry30Ca/Cry60Aa/Cry60Ba cyt1A promoter pHT315 4Q7− (Sun et al. 2013)
Cry41Aa cyt1Aa promoter pSVP27a 4D7 (Krishnan et al. 2017)
Cry1Ac amyE promoter pHY300PLK Tt14 (Yang et al. 2003)
−
Cry1C amyE promoter pSB909.4 Cry B (Chak et al. 1994)
Cry1Ac PexsY pHT315 HD73− (Zheng et al. 2014)
Cry1Ac HD73_5014 promoter pHT315 HD73− and HDsigK− (Zhang et al. 2018)

(Wang et al. 2018b). Herrero et al. (2004) used pBD150 plas- Bt is a large family of recognized entomopathogens found
mid to produce wild-type Cry1Ca and mutant protoxins in in various habitats (Jouzani et al. 2017). Many Bt-targeted
E.coli to determine toxin-binding parameters for specific re- insect pests inhabit niches that Bt cannot survive or stabilize
ceptors in brush border membrane vesicles of Spodoptera in, such as the plant rhizosphere. Many Bt toxins have shown
exigua. insecticidal activity against underground pests including nem-
Other microorganisms have also been used to express cry atodes and white grubs (Bi et al. 2015; Ruan et al. 2015).
genes. Expressions of cry1Aa and cry1Ia under the control of However, the LC50 of Cry proteins/spores for nematodes in
the lac promoter in Photorhabdus temperata strain K122 most reports was quite low (Jouzani et al. 2017). This limits
against Prays oleae (Tounsi et al. 2006) resulted in a clear the application of Bt products that can kill underground pests.
improvement in oral toxicity. This demonstrates that the het- Thus, new stable microorganisms are required for cry gene
erologous expression of Bt cry genes in P. temperata can be expression as an alternative delivery system to develop new-
used to improve and broaden the host range for insect control. generation biopesticides with improved persistence.
Additionally, baculoviruses have been genetically modified to
express cry1Ab under polyhedrin promoters in order to accel-
erate their killing speed (El-Menofy et al. 2014). Durmaz et al. Expression of cry genes in Bt transgenic crops
(2015) used a strong constitutive promoter (P6 promoter) to
express cry5B in Lactococcus lactis for use as an Bt toxin proteins have been extensively used in plant genetic
anthelminthic, while co-expression of mosquitocidal toxins engineering to deter pests. Bt application has allowed agricul-
cyt1Aa and cry11Aa from Bt subsp. Israelensis under the con- ture to cater to human interest, for example, food growth free
trol of Asticcacaulis excentricus tac promoter enhanced tox- of chemical pesticides and reduced environmental damage
icity to the third instar larvae of Culex quinquefasciatus ex- due to avoidance of excessive chemical application by using
pressing only cry11Aa (Zheng et al. 2007). Cry34Ab1 and various promoters to produce toxin proteins (Fig. 1).
Cry35Ab1 binary insecticidal proteins were produced in re- Cauliflower mosaic virus (CaMV) promoter 35S has been
combinant Pseudomonas fluorescens to provide large quanti- shown to be active in most plant organs and is considered
ties of protein for safety-assessment studies associated with constitutively expressed throughout plant development; it is
the registration of transgenic corn plants (Huang et al. 2007). also considered a common strong promoter in activating Bt
Alberghini et al. (2005) also used Pseudomonas as a host for toxin genes. The cry3A genes driven by 35S expressed in
expression of cry9Aa. Agaisse and Lereclus (1994a) used potatoes were toxic to the first-instar, and the more resistant
Bacillus subtilis for cry3A expression to study the transcrip- third-instar Colorado potato beetle larvae (Adang et al. 1993);
tional regulation of the cry3A gene. in transgenic Norway spruce (Picea abies), cry3A genes
Appl Microbiol Biotechnol (2019) 103:1617–1626 1621

showed toxicity against the spruce bark beetle (Briza et al. et al. 1992), tobacco hornworm (Manduca sexta), and tobacco
2013). Diamondback moth (Plutella xylostella L.), cabbage budworm (Heliothis virescens) (Carozzi et al. 1992)
looper (Trichoplusia ni Hübner), and corn earworm respectively.
(Helicoverpa zea Boddie) were completely controlled in Additionally, there is a non-constitutive promoter reported
transgenic canola containing cry1Ac (Stewart et al. 1996). to be stronger than 35S when expressing Bt toxin genes.
Cry1Ab transgenic rice showed 100 % feeding mortality rates Chrysanthemum ribulose-1, 5-bisphosphate carboxylase/
for the yellow stem borer (Scirpophaga incertulas) and the oxygenase small subunit (Rubisco SSU) promoter expressed
striped stem borer (Chilo suppressalis), and feeding inhibition cry1Ca in shallot and provided resistance against beet army-
of Cnaphalocrocis medinalis and Marasmia patnalis (Wunn worm (Zheng et al. 2005). cry1Ac driven by Arabidopsis
et al. 1996). Cry9C transgenic maize delayed the development thaliana Rubisco small subunit ats1A promoter with its asso-
and increased mortality rates of Plodia interpunctella (Giles ciated transit peptide showed 10- to 20-fold mRNA and pro-
et al. 2000). Cry6A toxin protein produced by 35S can confer tein expression compared to expression driven by the 35S
tomato resistance to an endoparasitic nematode (Meloidogyne promoter (Wong et al. 1992).
incognita) (Li et al. 2007). cyt2Ca1 driven by 35S expressed Rice actin1 promoter and maize ubiquitin1 promoter are
in citrus roots not only resulted in regular growth but also common strong plant-derived promoters in Bt transgenic rice,
protected the roots from larval Diaprepes abbreviatus both enable high expression of cry genes and high insect re-
(Mahmoud et al. 2017). Transgenic Pigeon pea containing sistance. Cry1Ac under the control of maize ubiquitin1 pro-
Cry2Aa gained resistance to gram pod borer (Helicoverpa moter was confirmed as highly toxic to yellow stem borer
armigera) (Singh et al. 2018). Double CaMV 35S promoter larvae (Khanna and Raina 2002; Nayak et al. 1997). Under
expressed cry1Ac against Dendrolimus punctatus (Walker) the control of rice actin1 promoter, transgenic elite rice lines
and Crypyothelea formosicola (Staud) in transgenic Loblolly producing a Cry1Ab/Cry1Ac fusion protein showed high pro-
pine (Pinus taeda L.) (Tang and Tian 2003). 35S promoter and tection against leaffolder and yellow stem borer without re-
alfalfa mosaic virus translational enhancer (AMV coat protein duced yield (Tu et al. 2000), while producing Cry1Ac protein
5′ untranslated leader sequence) were used to express cry3A exhibited resistance to striped stem borer (Chilo suppressalis
and cry1Ab in tobacco against potato beetle larvae (Sutton (Walker) in the laboratory (Liu et al. 2016).

Fig 1 Expression of cry genes in Bt transgenic crops

1622 Appl Microbiol Biotechnol (2019) 103:1617–1626

Tissue-specific promoters can control Bt gene expression in Ethical approval No human or animal studies were performed for this
review.
a tissue-dependent manner; Bt toxin genes driven by tissue-
specific promoters will be expressed only in tissues where in- Publisher’s Note Springer Nature remains neutral with regard to jurisdic-
sect resistance is desired, leaving the rest of the plant tissues tional claims in published maps and institutional affiliations.
unmodified. Green-tissue-specific rbcS promoter from
Nicotiana plumbaginifolia is a strong promoter; by expressing
cry1C primarily in leaf and green tissues, transgenic haploid
tobacco gained resistance to Spodoptera litura (Christov et al. References
1999). Maize phosphoenolpyruvate carboxylase gene (PEPC)
Aceves-Diez AE, Robles-Burgueno R, de la Torre M (2007) SKPDT is a
promoter expressed a hybrid cry1Ab/1Ac gene in Jatropha signaling peptide that stimulates sporulation and cry1Aa expression
curcas that then displayed insecticidal activity to Archips in Bacillus thuringiensis but not in Bacillus subtilis. Appl Microbiol
micaceanus (Gu et al. 2014). cry1Ab driven by maize PEPC Biotechnol 76(1):203–209. https://doi.org/10.1007/s00253-007-
promoter expressed in maize (Koziel et al. 1993), rice (Datta 0982-0
Adang MJ, Brody MS, Cardineau G, Eagan N, Roush RT, Shewmaker
et al. 1998), and potato (Hagh et al. 2009) showed toxicity
CK, Jones A, Oakes JV, McBride KE (1993) The reconstruction and
against European corn borer, larvae of the yellow stem borer, expression of a Bacillus thuringiensis cryIIIA gene in protoplasts
and tuber moth (Phthorimaea operculella (Zeller) respectively. and potato plants. Plant Mol Biol 21(6):1131–1145
There is also an inducible promoter for Bt transgenic plants. Adegawa S, Nakama Y, Endo H, Shinkawa N, Kikuta S, Sato R (2017)
The PR-1a promoter is inducible by chemicals including The domain II loops of Bacillus thuringiensis Cry1Aa form an over-
lapping interaction site for two Bombyx mori larvae functional re-
salicylic acid, 2,6-dichloro-iso-nicotinic acid (INA), and 1,2,3- ceptors, ABC transporter C2 and cadherin-like receptor. Biochim
benzothiadiazole-7-carbothioic acid S-methyl ester (BTH), Biophys Acta 1865(2):220–231. https://doi.org/10.1016/j.bbapap.
which has been registered as an antifungal chemical for field 2016.11.011
application. cry1Ab driven by PR-1a promoter in transgenic Agaisse H, Lereclus D (1994a) Expression in Bacillus subtilis of the
Bacillus thuringiensis cryIIIA toxin gene is not dependent on a
broccoli can control diamondback moth (Plutella xylostella sporulation-specific sigma factor and is increased in a spo0A mutant.
L.) from damage under chemical regulation (Cao et al. 2006). J Bacteriol 176(15):4734–4741
Agaisse H, Lereclus D (1994b) Structural and functional analysis of the
promoter region involved in full expression of the cryIIIA toxin gene
of Bacillus thuringiensis. Mol Microbiol 13(1):97–107
Agaisse H, Lereclus D (1995) How does Bacillus thuringiensis produce
Conclusion and perspectives so much insecticidal crystal protein? J Bacteriol 177(21):6027–6032
Alberghini S, Filippini R, Marchetti E, Dindo ML, Shevelev AB, Battisti
In microorganisms, the Bt system is still very efficient for cry A, Squartini A (2005) Construction of a Pseudomonas sp. derivative
gene expression. Many available strong promoters can direct carrying the cry9Aa gene from Bacillus thuringiensis and a proposal
most cry gene expression in acrystalliferous Bt strains. for new standard criteria to assess entomocidal properties of bacte-
ria. Res Microbiol 156(5-6):690–699. https://doi.org/10.1016/j.
However, not all cry genes and niches are compatible with resmic.2005.02.003
Bt. New delivery systems including Bt subspecies with genet- Amadio AF, Navas LE, Sauka DH, Berretta MF, Benintende GB,
ically different backgrounds, other microorganisms, and even Zandomeni RO (2013) Identification, cloning and expression of an
strong promoters can improve Cry production, niche fitness, insecticide cry8 gene from Bacillus thuringiensis INTA Fr7-4. J Mol
Microbiol Biotechnol 23(6):401–409. https://doi.org/10.1159/
and persistence.
000353206
In transgenic plants including potato, tomato, tobacco, rice, AP DEL, Lorenzon LB, Vianna AM, Santos FD, Pinto LS, Aires Berne
maize, and broccoli, cry genes have been used extensively. ME, Leite FP (2016) Larvicidal activity of Bacillus thuringiensis
Constitutive promoters, such as 35S, have been frequently var. israelensis Cry11Aa toxin against Haemonchus contortus.
utilized for expressing cry genes to protect transgenic plants Parasitology 143(12):1665–1671. https://doi.org/10.1017/
S0031182016001451
from pests. However, pests often damage the plants in special
Azizoglu U, Ayvaz A, Yilmaz S, Karaborklu S, Temizgul R (2016)
tissues during growth phases. Thus, more efficient tissue- Expression of cry1Ab gene from a novel Bacillus thuringiensis
specific and inducible promoters need to be investigated to strain SY49-1 active on pest insects. Braz J Microbiol 47(3):597–
express cry genes in plants suffering from pest infestation. 602. https://doi.org/10.1016/j.bjm.2016.04.011
Barboza-Corona JE, Park HW, Bideshi DK, Federici BA (2012) The 60-
Funding This work was funded by the National Natural Science kilodalton protein encoded by orf2 in the cry19A operon of Bacillus
Foundation (31530095) and the National Key Research and thuringiensis subsp. jegathesan functions like a C-terminal crystal-
Development Program of China (2017YFD0200400). lization domain. Appl Environ Microbiol 78(6):2005–2012. https://
doi.org/10.1128/AEM.06750-11
Baum JA, Malvar T (1995) Regulation of insecticidal crystal protein
Compliance with ethical standards production in Bacillus thuringiensis. Mol Microbiol 18(1):1–12
Bi Y, Zhang Y, Shu C, Crickmore N, Wang Q, Du L, Song F, Zhang J
Conflict of interest The authors declare that they have no conflict of (2015) Genomic sequencing identifies novel Bacillus thuringiensis
interest. Vip1/Vip2 binary and Cry8 toxins that have high toxicity to
Appl Microbiol Biotechnol (2019) 103:1617–1626 1623

Scarabaeoidea larvae. Appl Microbiol Biotechnol 99(2):753–760. Durmaz E, Hu Y, Aroian RV, Klaenhammer TR (2015) Intracellular and
https://doi.org/10.1007/s00253-014-5966-2 extracellular expression of Bacillus thuringiensis crystal protein
B o o n s e r m P, P o r n w i r o o n W, K a t z e n m e i e r G , P a n y i m S , Cry5B in Lactococcus lactis for use as an anthelminthic. Appl
Angsuthanasombat C (2004) Optimised expression in Escherichia Environ Microbiol 82(4):1286–1294. https://doi.org/10.1128/
coli and purification of the functional form of the Bacillus AEM.02365-15
thuringiensis Cry4Aa delta-endotoxin. Protein Expr Purif 35(2): El-Menofy W, Osman G, Assaeedi A, Salama M (2014) A novel recom-
397–403. https://doi.org/10.1016/j.pep.2004.02.016 binant baculovirus overexpressing a Bacillus thuringiensis Cry1Ab
Bravo A, Agaisse H, Salamitou S, Lereclus D (1996) Analysis of cryIAa toxin enhances insecticidal activity. Biol Proced Online 16:7. https://
expression in sigE and sigK mutants of Bacillus thuringiensis. Mol doi.org/10.1186/1480-9222-16-7
Gen Genet 250(6):734–741 Garcia-Gomez BI, Sanchez J, Martinez de Castro DL, Ibarra JE, Bravo A,
Briza J, Pavingerova D, Vlasak J, Niedermeierova H (2013) Norway Soberon M (2013) Efficient production of Bacillus thuringiensis
spruce (Picea abies) genetic transformation with modified Cry3A Cry1AMod toxins under regulation of cry3Aa promoter and single
gene of Bacillus thuringiensis. Acta Biochim Pol 60(3):395–400 cysteine mutations in the protoxin region. Appl Environ Microbiol
Buasri W, Panbangred W (2012) Large crystal toxin formation in chro- 79(22):6969–6973. https://doi.org/10.1128/AEM.02546-13
mosomally engineered Bacillus thuringiensis subsp. aizawai due to Giles KL, Hellmich RL, Iverson CT, Lewis LC (2000) Effects of trans-
sigmaE accumulation. Appl Environ Microbiol 78(6):1682–1691. genic Bacillus thuringiensis maize grain on B. thuringiensis-suscep-
https://doi.org/10.1128/AEM.06505-11 tible Plodia interpunctella (Lepidoptera: Pyralidae). J Econ Entomol
Cao J, Bates SL, Zhao JZ, Shelton AM, Earle ED (2006) Bacillus 93(3):1011–1016
thuringiensis protein production, signal transduction, and insect Gomez I, Sanchez J, Munoz-Garay C, Matus V, Gill SS, Soberon M,
control in chemically inducible PR-1a/cry1Ab broccoli plants. Bravo A (2014) Bacillus thuringiensis Cry1A toxins are versatile
Plant Cell Rep 25(6):554–560. https://doi.org/10.1007/s00299- proteins with multiple modes of action: two distinct pre-pores are
005-0091-4 involved in toxicity. Biochem J 459(2):383–396. https://doi.org/10.
Carozzi NB, Warren GW, Desai N, Jayne SM, Lotstein R, Rice DA, 1042/BJ20131408
Evola S, Koziel MG (1992) Expression of a chimeric CaMV 35S Gu K, Mao H, Yin Z (2014) Production of marker-free transgenic
Bacillus thuringiensis insecticidal protein gene in transgenic tobac- Jatropha curcas expressing hybrid Bacillus thuringiensis delta-
co. Plant Mol Biol 20(3):539–548 endotoxin Cry1Ab/1Ac for resistance to larvae of tortrix moth
Chak KF, Tseng MY, Yamamoto T (1994) Expression of the crystal (Archips micaceanus). Biotechnol Biofuels 7:68. https://doi.org/10.
protein gene under the control of the alpha-amylase promoter in 1186/1754-6834-7-68
Bacillus thuringiensis strains. Appl Environ Microbiol 60(7):
Guan P, Dai X, Zhu J, Li Q, Li S, Wang S, Li P, Zheng A (2014) Bacillus
2304–2310
thuringiensis subsp. sichuansis strain MC28 produces a novel crys-
Chaoyin Y, Wei S, Sun M, Lin L, Faju C, Zhengquan H, Ziniu Y (2007)
tal protein with activity against Culex quinquefasciatus larvae.
Comparative study on effect of different promoters on expression of
World J Microbiol Biotechnol 30(4):1417–1421. https://doi.org/10.
cry1Ac in Bacillus thuringiensis chromosome. J Appl Microbiol
1007/s11274-013-1548-1
103(2):454–461. https://doi.org/10.1111/j.1365-2672.2006.03269.x
Hagh ZG, Rahnama H, Panahandeh J, Rouz BBK, Jafari KMA, Mahna N
Christov NK, Imaishi H, Ohkawa H (1999) Green-tissue-specific expres-
(2009) Green-tissue-specific, C-4-PEPC-promoter-driven expres-
sion of a reconstructed cry1C gene encoding the active fragment of
sion of Cry1Ab makes transgenic potato plants resistant to tuber
Bacillus thuringiensis delta-endotoxin in haploid tobacco plants
moth (Phthorimaea operculella, Zeller). Plant Cell Rep 28(12):
conferring resistance to Spodoptera litura. Biosci Biotechnol
1869–1879. https://doi.org/10.1007/s00299-009-0790-3
Biochem 63(8):1433–1444
Crickmore N, Wheeler VC, Ellar DJ (1994) Use of an operon fusion to Hayakawa T, Sakakibara A, Ueda S, Azuma Y, Ide T, Takebe S (2017)
induce expression and crystallisation of a Bacillus thuringiensis Cry46Ab from Bacillus thuringiensis TK-E6 is a new mosquitocidal
delta-endotoxin encoded by a cryptic gene. Mol Gen Genet toxin with aerolysin-type architecture. Insect Biochem Mol Biol 87:
242(3):365–368 100–106. https://doi.org/10.1016/j.ibmb.2017.06.015
Datta K, Vasquez A, Tu J, Torrizo L, Alam MF, Oliva N, Abrigo E, Khush Herrero S, Gonzalez-Cabrera J, Ferre J, Bakker PL, de Maagd RA (2004)
GS, Datta SK (1998) Constitutive and tissue-specific differential Mutations in the Bacillus thuringiensis Cry1Ca toxin demonstrate
expression of the cryIA(b) gene in transgenic rice plants conferring the role of domains II and III in specificity towards Spodoptera
resistance to rice insect pest. Theor Appl Genet 97(1-2):20–30. exigua larvae. Biochem J 384(Pt 3):507–513. https://doi.org/10.
https://doi.org/10.1007/s001220050862 1042/BJ20041094
de Souza MT, Lecadet MM, Lereclus D (1993) Full expression of the Huang Z, Guan C, Guan X (2004) Cloning, characterization and expres-
cryIIIA toxin gene of Bacillus thuringiensis requires a distant up- sion of a new cry1Ab gene from Bacillus thuringiensis WB9.
stream DNA sequence affecting transcription. J Bacteriol 175(10): Biotechnol Lett 26(20):1557–1561. https://doi.org/10.1023/B:
2952–2960 BILE.0000045652.00137.1f
Delecluse A, Poncet S, Klier A, Rapoport G (1993) Expression of cryIVA Huang KX, Badger M, Haney K, Evans SL (2007) Large scale produc-
and cryIVB genes, independently or in combination, in a crystal- tion of Bacillus thuringiensis PS149B1 insecticidal proteins
negative strain of Bacillus thuringiensis subsp. israelensis. Appl Cry34Ab1 and Cry35Ab1 from Pseudomonas fluorescens.
Environ Microbiol 59(11):3922–3927 Protein Expr Purif 53(2):325–330. https://doi.org/10.1016/j.pep.
Dementiev A, Board J, Sitaram A, Hey T, Kelker MS, Xu X, Hu Y, Vidal- 2007.01.010
Quist C, Chikwana V, Griffin S, McCaskill D, Wang NX, Hung SC, Huang T, Lin Q, Qian X, Zheng Y, Yao J, Wu H, Li M, Jin X, Pan X,
Chan MK, Lee MM, Hughes J, Wegener A, Aroian RV, Narva KE, Zhang L, Guan X (2018) Nematicidal activity of Cry1Ea11 from
Berry C (2016) The pesticidal Cry6Aa toxin from Bacillus Bacillus thuringiensis BRC-XQ12 against the pine wood nematode
thuringiensis is structurally similar to HlyE-family alpha pore- (Bursaphelenchus xylophilus). Phytopathology 108(1):44–51.
forming toxins. BMC Biol 14:71. https://doi.org/10.1186/s12915- https://doi.org/10.1094/PHYTO-05-17-0179-R
016-0295-9 Iatsenko I, Boichenko I, Sommer RJ (2014) Bacillus thuringiensis DB27
Deng C, Peng Q, Song F, Lereclus D (2014) Regulation of cry gene produces two novel protoxins, Cry21Fa1 and Cry21Ha1, which act
expression in Bacillus thuringiensis. Toxins (Basel) 6(7):2194– synergistically against nematodes. Appl Environ Microbiol 80(10):
2209. https://doi.org/10.3390/toxins6072194 3266–3275. https://doi.org/10.1128/AEM.00464-14
1624 Appl Microbiol Biotechnol (2019) 103:1617–1626

Jain D, Udayasuriyan V, Arulselvi PI, Dev SS, Sangeetha P (2006) Navas LE, Berretta MF, Perez MP, Amadio AF, Ortiz EM, Sauka DH,
Cloning, characterization, and expression of a new cry2Ab gene Benintende GB, Zandomeni RO (2014) Sequence and expression of
from Bacillus thuringiensis strain 14-1. Appl Biochem Biotechnol two cry8 genes from Bacillus thuringiensis INTA Fr7-4, a native
128(3):185–194 strain from Argentina. J Mol Microbiol Biotechnol 24(4):241–248.
Ji F, Zhu Y, Ju S, Zhang R, Yu Z, Sun M (2009) Promoters of crystal https://doi.org/10.1159/000365929
protein genes do not control crystal formation inside exosporium of Nayak P, Basu D, Das S, Basu A, Ghosh D, Ramakrishnan NA, Ghosh
Bacillus thuringiensis ssp. finitimus strain YBT-020. FEMS M, Sen SK (1997) Transgenic elite indica rice plants expressing
Microbiol Lett 300(1):11-17. https://doi.org/10.1111/j.1574-6968. CryIAc delta-endotoxin of Bacillus thuringiensis are resistant
2009.01743.x against yellow stem borer (Scirpophaga incertulas). Proc Natl
Jia Y, Zhao C, Wang Q, Shu C, Feng X, Song F, Zhang J (2014) A Acad Sci U S A 94(6):2111–2116
genetically modified broad-spectrum strain of Bacillus thuringiensis Ogunjimi AA, Chandler JM, Gbenle GO, Olukoya DK, Akinrimisi EO
toxic against Holotrichia parallela, Anomala corpulenta and (2002) Heterologous expression of cry2 gene from a local strain of
Holotrichia oblita. World J Microbiol Biotechnol 30(2):595–603. Bacillus thuringiensis isolated in Nigeria. Biotechnol Appl Biochem
https://doi.org/10.1007/s11274-013-1470-6 36(Pt 3):241–246. https://doi.org/10.1042/BA20020053
Jouzani GS, Valijanian E, Sharafi R (2017) Bacillus thuringiensis: a suc- Pacheco S, Gomez I, Arenas I, Saab-Rincon G, Rodriguez-Almazan C,
cessful insecticide with new environmental features and tidings. Gill SS, Bravo A, Soberon M (2009) Domain II loop 3 of Bacillus
Appl Microbiol Biotechnol 101(7):2691–2711. https://doi.org/10. thuringiensis Cry1Ab toxin is involved in a Bping pong^ binding
1007/s00253-017-8175-y mechanism with Manduca sexta aminopeptidase-N and cadherin
Khanna HK, Raina SK (2002) Elite indica transgenic rice plants express- receptors. J Biol Chem 284(47):32750–32757. https://doi.org/10.
ing modified Cry1Ac endotoxin of Bacillus thuringiensis show en- 1074/jbc.M109.024968
hanced resistance to yellow stem borer (Scirpophaga incertulas). Pan Z, Xu L, Zhu Y, Shi H, Chen Z, Chen M, Chen Q, Liu B (2014)
Transgenic Res 11(4):411–423. https://doi.org/10.1023/A: Characterization of a new cry2Ab gene of Bacillus thuringiensis
1016378606189 with high insecticidal activity against Plutella xylostella L. World J
Khasdan V, Sapojnik M, Zaritsky A, Horowitz AR, Boussiba S, Rippa M, Microbiol Biotechnol 30(10):2655–2662. https://doi.org/10.1007/
Manasherob R, Ben-Dov E (2007) Larvicidal activities against ag- s11274-014-1689-x
ricultural pests of transgenic Escherichia coli expressing combina- Park HW, Ge B, Bauer LS, Federici BA (1998) Optimization of Cry3A
tions of four genes from Bacillus thuringiensis. Arch Microbiol yields in Bacillus thuringiensis by use of sporulation-dependent
188(6):643–653. https://doi.org/10.1007/s00203-007-0285-y promoters in combination with the STAB-SD mRNA sequence.
Appl Environ Microbiol 64(10):3932–3938
Komano T, Takabe S, Sakai H (2000) Transcription of the insecticidal
crystal protein genes of Bacillus thuringiensis. Biotechnol Annu Park H, Bideshi DK, Johnson JJ, Federici BA (1999) Differential en-
Rev 5:131–154 hancement of Cry2A versus Cry11A yields in Bacillus thuringiensis
by use of the cry3A STAB mRNA sequence. FEMS Microbiol Lett
Koziel MG, Beland GL, Bowman C, Carozzi NB, Crenshaw R,
181(2):319–327
Crossland L, Dawson J, Desai N, Hill M, Kadwell S (1993) Field
Park HW, Bideshi DK, Federici BA (2000) Molecular genetic manipula-
performance of elite transgenic maize plants expressing an insecti-
tion of truncated Cry1C protein synthesis in Bacillus thuringiensis to
cidal protein derived from Bacillus thuringiensis. Nat Biotechnol
improve stability and yield. Appl Environ Microbiol 66(10):4449–
11(2):194–200
4455
Krishnan V, Domanska B, Elhigazi A, Afolabi F, West MJ, Crickmore N
Park HW, Delecluse A, Federici BA (2001) Construction and character-
(2017) The human cancer cell active toxin Cry41Aa from Bacillus
ization of a recombinant Bacillus thuringiensis subsp. israelensis
thuringiensis acts like its insecticidal counterparts. Biochem J
strain that produces Cry11B. J Invertebr Pathol 78(1):37–44.
474(10):1591–1602. https://doi.org/10.1042/BCJ20170122
https://doi.org/10.1006/jipa.2001.5038
Lee HK, Gill SS (1997) Molecular cloning and characterization of a novel Peng D, Luo X, Zhang N, Guo S, Zheng J, Chen L, Sun M (2018) Small
mosquitocidal protein gene from Bacillus thuringiensis subsp. RNA-mediated Cry toxin silencing allows Bacillus thuringiensis to
fukuokaensis. Appl Environ Microbiol 63(12):4664–4670 evade Caenorhabditis elegans avoidance behavioral defenses.
Li XQ, Wei JZ, Tan A, Aroian RV (2007) Resistance to root-knot nem- Nucleic Acids Res 46(1):159–173. https://doi.org/10.1093/nar/
atode in tomato roots expressing a nematicidal Bacillus gkx959
thuringiensis crystal protein. Plant Biotechnol J 5(4):455–464. Perez-Garcia G, Basurto-Rios R, Ibarra JE (2010) Potential effect of a
https://doi.org/10.1111/j.1467-7652.2007.00257.x putative sigma(H)-driven promoter on the over expression of the
Liu X, Zhang C, Li X, Tu J (2016) Pyramiding and evaluation of both a Cry1Ac toxin of Bacillus thuringiensis. J Invertebr Pathol 104(2):
foreign Bacillus thuringiensis and a Lysine-rich proteingene in the 140–146. https://doi.org/10.1016/j.jip.2010.02.010
eliteindicarice 9311. Breed Sci 66(4):591–598 Reddy VP, Rao NN, Devi PS, Sivaramakrishnan S, Narasu ML, Kumar
Liu Y, Wang Y, Shu C, Lin K, Song F, Bravo A, Soberon M, Zhang J VD (2013) Cloning, characterization, and expression of a new
(2018) Cry64Ba and Cry64Ca, two ETX/MTX2-type Bacillus cry1Ab gene from DOR Bt-1, an indigenous isolate of Bacillus
thuringiensis insecticidal proteins active against Hemipteran pests. thuringiensis. Mol Biotechnol 54(3):795–802. https://doi.org/10.
Appl Environ Microbiol 84(3). https://doi.org/10.1128/AEM. 1007/s12033-012-9627-3
01996-17 Reyaz AL, Arulselvi PI (2016) Cloning, characterization and expression
Mahmoud SB, Ramos JE, Shatters RG Jr, Hall DG, Lapointe SL, Niedz of a novel haplotype cry2A-type gene from Bacillus thuringiensis
RP, Rouge P, Cave RD, Borovsky D (2017) Expression of Bacillus strain SWK1, native to Himalayan valley Kashmir. J Invertebr
thuringiensis cytolytic toxin (Cyt2Ca1) in citrus roots to control Pathol 136:1–6. https://doi.org/10.1016/j.jip.2016.02.005
Diaprepes abbreviatus larvae. Pestic Biochem Physiol 136:1–11. Reyaz AL, Gunapriya L, Indra Arulselvi P (2017) Molecular characteri-
https://doi.org/10.1016/j.pestbp.2016.07.006 zation of indigenous Bacillus thuringiensis strains isolated from
Martinez-Solis M, Pinos D, Endo H, Portugal L, Sato R, Ferre J, Herrero Kashmir valley. 3 Biotech 7(2):143. https://doi.org/10.1007/
S, Hernandez-Martinez P (2018) Role of Bacillus thuringiensis s13205-017-0756-z
Cry1A toxins domains in the binding to the ABCC2 receptor from Rodriguez-Almazan C, Reyes EZ, Zuniga-Navarrete F, Munoz-Garay C,
Spodoptera exigua. Insect Biochem Mol Biol 101:47–56. https:// Gomez I, Evans AM, Likitvivatanavong S, Bravo A, Gill SS,
doi.org/10.1016/j.ibmb.2018.07.006 Soberon M (2012) Cadherin binding is not a limiting step for
Appl Microbiol Biotechnol (2019) 103:1617–1626 1625

Bacillus thuringiensis subsp. israelensis Cry4Ba toxicity to Aedes crystal proteins from Bacillus thuringiensis subsp. jegathesan. Appl
aegypti larvae. Biochem J 443(3):711–717. https://doi.org/10.1042/ Environ Microbiol 79(11):3364–3370. https://doi.org/10.1128/
BJ20111579 AEM.00078-13
Roh JY, Lee IH, Li MS, Chang JH, Choi JY, Boo KS, Je YH (2004) Sun Y, Zhao Q, Zheng D, Ding X, Wang J, Hu Q, Yuan Z, Park HW, Xia
Expression of a recombinant Cry1Ac crystal protein fused with a L (2014) Construction and characterization of the interdomain chi-
green fluorescent protein in Bacillus thuringiensis subsp. kurstaki meras using Cry11Aa and Cry11Ba from Bacillus thuringiensis and
Cry-B. J Microbiol 42(4):340–345 identification of a possible novel toxic chimera. Biotechnol Lett
Ruan L, Crickmore N, Peng D, Sun M (2015) Are nematodes a missing 36(1):105–111. https://doi.org/10.1007/s10529-013-1330-3
link in the confounded ecology of the entomopathogen Bacillus Sun Y, Fu Z, He X, Yuan C, Ding X, Xia L (2016) Enhancement of
thuringiensis? Trends Microbiol 23(6):341–346. https://doi.org/10. Bacillus thuringiensis insecticidal activity by combining Cry1Ac
1016/j.tim.2015.02.011 and bi-functional toxin HWTX-XI from spider. J Invertebr Pathol
Saitoh H, Hwang SH, Park YS, Higuchi K, Mizuki E, Ohba M (2000) 135:60–62. https://doi.org/10.1016/j.jip.2015.02.005
Cloning and characterization of a Bacillus thuringiensis serovar higo Sutton DW, Havstad PK, Kemp JD (1992) Synthetic cryIIIA gene from
gene encoding a novel class of the delta-endotoxin protein, Cry27A, Bacillus thuringiensis improved for high expression in plants.
specifically active on the Anopheles mosquito. Syst Appl Microbiol Transgenic Res 1(5):228–236
23(1):25–30 Tanaka S, Miyamoto K, Noda H, Endo H, Kikuta S, Sato R (2016) Single
Sajid M, Geng C, Li M, Wang Y, Liu H, Zheng J, Peng D, Sun M (2018) amino acid insertions in extracellular loop 2 of Bombyx mori
Whole-genome analysis of Bacillus thuringiensis revealing partial ABCC2 disrupt its receptor function for Bacillus thuringiensis
genes as a source of novel cry toxins. Appl Environ Microbiol Cry1Ab and Cry1Ac but not Cry1Aa toxins. Peptides 78:99–108.
84(14). https://doi.org/10.1128/AEM.00277-18 https://doi.org/10.1016/j.peptides.2016.01.006
Sakano Y, Park HW, Bideshi DK, Ge B, Federici BA (2017) Tang W, Tian YC (2003) Transgenic loblolly pine (Pinus taeda L.) plants
Contributions of 5'-UTR and 3'-UTR cis elements to Cyt1Aa syn- expressing a modified delta-endotoxin gene of Bacillus
thesis in Bacillus thuringiensis subsp. israelensis. J Invertebr Pathol thuringiensis with enhanced resistance to Dendrolimus punctatus
149:66–75. https://doi.org/10.1016/j.jip.2017.08.002 Walker and Crypyothelea formosicola Staud. J Exp Bot 54(383):
Salamitou S, Agaisse H, Bravo A, Lereclus D (1996) Genetic analysis 835–844. https://doi.org/10.1093/jxb/erg071
of cryIIIA gene expression in Bacillus thuringiensis. Tounsi S, Aoun AE, Blight M, Rebai A, Jaoua S (2006) Evidence of oral
Microbiology 142(Pt 8):2049–2055. https://doi.org/10.1099/ toxicity of Photorhabdus temperata strain K122 against Prays oleae
13500872-142-8-2049 and its improvement by heterologous expression of Bacillus
Saleem F, Shakoori AR (2017) The first Cry2Ac-type protein toxic to thuringiensis cry1Aa and cry1Ia genes. J Invertebr Pathol 91(2):
Helicoverpa armigera: cloning and overexpression of Cry2ac7 131–135. https://doi.org/10.1016/j.jip.2005.11.004
Gene from SBS-BT1 strain of Bacillus thuringiensis. Toxins Tu J, Zhang G, Datta K, Xu C, He Y, Zhang Q, Khush GS, Datta SK
(Basel) 9(11). https://doi.org/10.3390/toxins9110358 (2000) Field performance of transgenic elite commercial hybrid rice
Sanchis V, Agaisse H, Chaufaux J, Lereclus D (1996) Construction of expressing Bacillus thuringiensis delta-endotoxin. Nat Biotechnol
new insecticidal Bacillus thuringiensis recombinant strains by using 18(10):1101–1104. https://doi.org/10.1038/80310
the sporulation non-dependent expression system of cryIIIA and a Walter T, Aronson A (1999) Specific binding of the E2 subunit of pyru-
site specific recombination vector. J Biotechnol 48(1-2):81–96 vate dehydrogenase to the upstream region of Bacillus thuringiensis
Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, protoxin genes. J Biol Chem 274(12):7901–7906
Zeigler DR, Dean DH (1998) Bacillus thuringiensis and its pestici- Wang B, Wang H, Xiong J, Zhou Q, Wu H, Xia L, Li L, Yu Z (2017) A
dal crystal proteins. Microbiol Mol Biol Rev 62(3):775–806 proteomic analysis provides novel insights into the stress responses
Sedlak M, Walter T, Aronson A (2000) Regulation by overlapping pro- of Caenorhabditis elegans towards nematicidal Cry6A toxin from
moters of the rate of synthesis and deposition into crystalline inclu- Bacillus thuringiensis. Sci Rep 7(1):14170. https://doi.org/10.1038/
sions of Bacillus thuringiensis delta-endotoxins. J Bacteriol 182(3): s41598-017-14428-3
734–741 Wang Y, Liu Y, Zhang J, Crickmore N, Song F, Gao J, Shu C (2018a)
Shu C, Zhang F, Chen G, Joseph L, Barqawi A, Evans J, Song F, Li G, Cry78Aa, a novel Bacillus thuringiensis insecticidal protein with
Zhang J, Crickmore N (2017) A natural hybrid of a Bacillus activity against Laodelphax striatellus and Nilaparvata lugens. J
thuringiensis Cry2A toxin implicates Domain I in specificity deter- Invertebr Pathol 158:1–5. https://doi.org/10.1016/j.jip.2018.07.007
mination. J Invertebr Pathol 150:35–40. https://doi.org/10.1016/j. Wang Z, Fang L, Zhou Z, Pacheco S, Gomez I, Song F, Soberon M,
jip.2017.09.002 Zhang J, Bravo A (2018b) Specific binding between Bacillus
Singh S, Kumar NR, Maniraj R, Lakshmikanth R, Rao KYS, thuringiensis Cry9Aa and Vip3Aa toxins synergizes their toxicity
Muralimohan N, Arulprakash T, Karthik K, Shashibhushan NB, against Asiatic rice borer (Chilo suppressalis). J Biol Chem 293(29):
Vinutha T, Pattanayak D, Dash PK, Kumar PA, Sreevathsa R 11447–11,458. https://doi.org/10.1074/jbc.RA118.003490
(2018) Expression of Cry2Aa, a Bacillus thuringiensis insecticidal Wasano N, Saitoh H, Maeda M, Ohgushi A, Mizuki E, Ohba M (2005)
protein in transgenic pigeon pea confers resistance to gram pod Cloning and characterization of a novel gene cry9Ec1 encoding
borer, Helicoverpa armigera. Sci Rep 8(1):8820. https://doi.org/ lepidopteran-specific parasporal inclusion protein from a Bacillus
10.1038/s41598-018-26,358-9 thuringiensis serovar galleriae strain. Can J Microbiol 51(11):988–
Somwatcharajit R, Tiantad I, Panbangred W (2014) Coexpression of the 995. https://doi.org/10.1139/w05-084
silent cry2Ab27 together with cry1 genes in Bacillus thuringiensis Wong EY, Hironaka CM, Fischhoff DA (1992) Arabidopsis thaliana
subsp. aizawai SP41 leads to formation of amorphous crystal toxin small subunit leader and transit peptide enhance the expression of
and enhanced toxicity against Helicoverpa armigera. J Invertebr Bacillus thuringiensis proteins in transgenic plants. Plant Mol Biol
Pathol 116:48–55. https://doi.org/10.1016/j.jip.2013.12.008 20(1):81–93
Stewart CN Jr, Adang MJ, All JN, Raymer PL, Ramachandran S, Parrott Wu D, Federici BA (1995) Improved production of the insecticidal
WA (1996) Insect Control and Dosage Effects in Transgenic Canola CryIVD protein in Bacillus thuringiensis using cryIA(c) promoters
Containing a Synthetic Bacillus thuringiensis cryIAc Gene. Plant to express the gene for an associated 20-kDa protein. Appl
Physiol 112(1):115–120 Microbiol Biotechnol 42(5):697–702
Sun Y, Zhao Q, Xia L, Ding X, Hu Q, Federici BA, Park HW (2013) Wunn J, Kloti A, Burkhardt PK, Biswas GC, Launis K, Iglesias VA,
Identification and characterization of three previously undescribed Potrykus I (1996) Transgenic Indica rice breeding line IR58
1626 Appl Microbiol Biotechnol (2019) 103:1617–1626

expressing a synthetic cryIA(b) gene from Bacillus thuringiensis Yilmaz S, Azizoglu U, Ayvaz A, Temizgul R, Atciyurt ZB, Karaborklu S
provides effective insect pest control. Biotechnology (N Y) 14(2): (2017) Cloning and expression of cry2Aa from native Bacillus
171–176 thuringiensis strain SY49-1 and its insecticidal activity against
Xia L, Sun Y, Ding X, Fu Z, Mo X, Zhang H, Yuan Z (2005) Culex pipiens (Diptera: Culicidae). Microb Pathog 105:81–85.
Identification of cry-type genes on 20-kb DNA associated with https://doi.org/10.1016/j.micpath.2017.02.016
Cry1 crystal proteins from Bacillus thuringiensis. Curr Microbiol Zhang W, Zhang J, Crickmore N, Wu Z, Yang Y, Qian J, Wu H, Wang R,
51(1):53–58. https://doi.org/10.1007/s00284-005-4504-y Fang X (2014) Identification of a mosquitocidal toxin from Bacillus
Xia L, Long X, Ding X, Zhang Y (2009a) Increase in insecticidal toxicity thuringiensis using mass spectrometry. World J Microbiol
by fusion of the cry1Ac gene from Bacillus thuringiensis with the Biotechnol 30(12):3273–3277. https://doi.org/10.1007/s11274-
neurotoxin gene hwtx-I. Curr Microbiol 58(1):52–57. https://doi. 014-1744-7
org/10.1007/s00284-008-9265-y Zhang X, Gao T, Peng Q, Song L, Zhang J, Chai Y, Sun D, Song F (2018)
Xia L, Zeng Z, Ding X, Huang F (2009b) The expression of a recombi- A strong promoter of a non-cry gene directs expression of the
nant cry1Ac gene with subtilisin-like protease CDEP2 gene in cry1Ac gene in Bacillus thuringiensis. Appl Microbiol Biotechnol
acrystalliferous Bacillus thuringiensis by Red/ET homologous re- 102(8):3687–3699. https://doi.org/10.1007/s00253-018-8836-5
combination. Curr Microbiol 59(4):386–392. https://doi.org/10. Zheng SJ, Henken B, de Maagd RA, Purwito A, Krens FA, Kik C (2005)
1007/s00284-009-9449-0 Two different Bacillus thuringiensis toxin genes confer resistance to
Xu L, Pan ZZ, Zhang J, Liu B, Zhu YJ, Chen QX (2016) Proteolytic beet armyworm (Spodoptera exigua Hubner) in transgenic Bt-
activation of Bacillus thuringiensis Cry2Ab through a belt-and- shallots (Allium cepa L.). Transgenic Res 14(3):261–272
braces approach. J Agric Food Chem 64(38):7195–7200. https:// Zheng D, Valdez-Cruz NA, Armengol G, Sevrez C, Munoz-Olaya JM,
doi.org/10.1021/acs.jafc.6b03111 Yuan Z, Orduz S, Crickmore N (2007) Co-expression of the
Yan F, Cheng X, Ding X, Yao T, Chen H, Li W, Hu S, Yu Z, Sun Y, Zhang mosquitocidal toxins Cyt1Aa and Cry11Aa from Bacillus
Y, Xia L (2014) Improved insecticidal toxicity by fusing Cry1Ac of thuringiensis subsp. israelensis in Asticcacaulis excentricus. Curr
Bacillus thuringiensis with Av3 of Anemonia viridis. Curr Microbiol Microbiol 54(1):58–62. https://doi.org/10.1007/s00284-006-0352-7
68(5):604–609. https://doi.org/10.1007/s00284-013-0516-1 Zheng Q, Wang G, Zhang Z, Qu N, Zhang Q, Peng Q, Zhang J, Gao J,
Yang CY, Pang JC, Kao SS, Tsen HY (2003) Enterotoxigenicity and Song F (2014) Expression of cry1Ac gene directed by PexsY pro-
cytotoxicity of Bacillus thuringiensis strains and development of a moter of the exsY gene encoding component protein of exosporium
process for Cry1Ac production. J Agric Food Chem 51(1):100–105. basal layer in Bacillus thuringiensis. Wei Sheng Wu Xue Bao
https://doi.org/10.1021/jf025863l 54(10):1138–1145
Yang H, Wang P, Peng Q, Rong R, Liu C, Lereclus D, Zhang J, Song F, Zhou C, Zheng Q, Peng Q, Du L, Shu C, Zhang J, Song F (2014)
Huang D (2012) Weak transcription of the cry1Ac gene in Screening of cry-type promoters with strong activity and application
nonsporulating Bacillus thuringiensis cells. Appl Environ in Cry protein encapsulation in a sigK mutant. Appl Microbiol
Microbiol 78(18):6466–6474. https://doi.org/10.1128/AEM. Biotechnol 98(18):7901–7909. https://doi.org/10.1007/s00253-
01229-12 014-5874-5