The genus Bacillus represents a diverse group of Gram-positive, endospore-forming bacterial species with the well-deserved fame of being potent, versatile, and one of the most promising industrial microorganisms yet discovered. They have an average genome size between 3.4 and 6.0 Mbp. Genetically modified B. subtilis and, to a lesser extent, B. licheniformis, B. amyloliquefaciens, and B. megaterium have been used for the heterologous expression of numerous proteins (enzymes, vaccine components, growth factors), platform chemicals, and other organic compounds of industrial importance.
1. Introduction
The genus
Bacillus represents a diverse group of Gram-positive, endospore-forming bacterial species with the well-deserved fame of being potent, versatile, and one of the most promising industrial microorganisms yet discovered. They have an average genome size between 3.4 and 6.0 Mbp
[1] and a low GC% content ranging from ~35% in
B. thuringiensis to 43.5–46.4% in
B. subtilis,
B. licheniformis, and
B. velezensis [2,3,4,5][2][3][4][5]. The distinct advantages of
Bacillus spp. used as microbial cell factories include a short fermentation cycle (around 48 h), ease of cultivation, and robust growth; non-pathogenic Generally Regarded as Safe (GRAS) status; the ability to secrete recombinant proteins in the medium; and the lack of external and endotoxins
[1,6][1][6]. In recent decades, genetically modified
B. subtilis and other
Bacillus spp., notably
B. licheniformis,
B. amyloliquefaciens, and
B. megaterium, have been used prodigiously for the heterologous production of anything from pharmaceutical proteins (antibody fragments, interferons and interleukins, growth factors, hormone precursors, and antimicrobial peptides) to industrial and food-grade enzymes. Large-scale genetic engineering has made possible the redirection of whole metabolic pathways toward valuable non-protein products such as organic acids, alcohols, and vitamins. Compared to
Escherichia coli, its chief rival among recombinant bacteria
[7],
Bacillus spp. used to have some limitations in the past associated with the relatively smaller number of suitable regulatory regions for gene expression, peculiarities in the secretion of recombinant proteins, and the need for the selection of domesticated host strains
[8]. This situation has been rapidly changed with the development of increasingly diverse and versatile vectors, novel genome editing tools such as the CRISPR/Cas9 system, and the construction of convenient strains deficient in multiple proteases.
An enormous variety of enzymes, growth factors, vitamins, peptides, amino acids, and low-MW compounds have been expressed in recombinant
Bacillus spp. (
Figure 1), often on a scale with industrial promise. Through the optimization of expression systems and developments in the field of bioengineering and the use of recombinant
Bacillus strains, the highest values of industrially important target products have been achieved (
Table 1).
Figure 1.
Biotechnological versatility of
Bacillus
spp.
2. Enzymes
Enzymes with applications in the food industry have predictably been in the spotlight. Genetic improvement of bacilli for the production of α-amylase leads to a gradual increase in the yields obtained (
Table 1). The goal of enhanced extracellular expression is achieved through signal peptide optimization and chaperone overexpression
[55][9], the prevention of extracellular degradation by improving the folding environment
[56][10], as well as by complex balancing of the entire secretion process
[57][11]. Thus, the recombinant strain
B. subtilis WHS9GSAB produced 35,779.5 U/mL α-amylase for 93 h, reaching a productivity of 384.7 U/mL/h
[58][12].
Table 1.
Application of recombinant
Bacillus
spp. with the highest production of industrially important compounds.
Strain |
Vector |
Compound |
Genetic Source |
Yield |
Reference |
B. subtilis WHS11YSA |
pHYYamySA |
α-amylase |
B. stearothermophilus |
9201.1 U/mL |
[55][9] |
Brevibacillus choshinensis (B. brevis) BCPPSQ |
pNCamyS-prsQ |
α-amylase |
B. stearothermophilus |
17,925.6 U/mL |
[56][10] |
B. subtilis WHS9GSAB |
pHYGamySAsecYEG |
α-amylase |
B. stearothermophilus |
35,779.5 U/mL |
[57][11] |
Br. choshinensis (B. brevis) |
pNCMO2 |
β-amylase |
B. aryabhattai CCTCC M2017320 |
5371.8 U/mL |
[58][12] |
B. subtilis WS9PUL |
pHYcas9 |
pullulanase |
B. deramificans |
5951.8 U/mL |
[59][13] |
B. subtilis WB600 |
pMA5 |
lipase A |
B. subtilis |
1164.9 U/mL |
[60][14] |
B. subtilis DB10 |
pSKE194 |
xylanase |
B. subtilis |
1296 U/mg |
[61][15] |
B. licheniformis MW3 |
pKVM1 |
2,3-butanediol |
B. licheniformis |
123.7 g/L |
[62][16] |
B. amyloliquefaciens B 10-127 |
pMA5 |
2,3-butanediol |
B. amyloliquefaciens |
132.9 g/L |
[63][17] |
B. subtilis 168 |
pMA5 |
acetoin |
B. subtilis |
91.8 g/L |
[64][18] |
B. subtilis KH2 |
pKVM1 pMA5 |
poly-γ-glutamic acid |
B. subtilis, B. licheniformis |
23.28 g/L |
[65][19] |
B. subtilis G600 |
T7-BOOST * |
GABA † |
B. subtilis |
109.8 g/L |
[66][20] |