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Autolysis to Bacillus subtilis Fermentation Bioprocess
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This entry is adapted from the peer-reviewed paper 10.3390/fermentation8120685

Bacillus subtilis is a gram-positive bacterium, a promising microorganism due to its strong extracellular protein secretion ability, non-toxic, and relatively mature industrial fermentation technology. With the fast advancement of molecular biology and genetic engineering technology, various advanced procedures and gene editing tools have been used to successfully construct autolysis-resistant B. subtilis chassis cells to manufacture various biological products. 

Bacillus subtilis cell autolysis gene editing

1. Introduction

B. subtilis is a generally recognized as safe (GRAS) microorganism [1], which is famous for its strong ability for heterologous expression, clear physiological and biochemical characteristics, and legacy relatively simple to operate. In addition, they have the advantages of being easy to cultivate and robust in industrial fermentations [2]. Using B. subtilis as the chassis cell, the exogenous introduction of the desired synthetic pathway and the systematic optimization of the strain’s own global metabolism and the coordination of the balance between the strain’s metabolic network and the exogenous pathway allow for the efficient production of a large number of products. As a crucial industrial biotechnology powerful chassis cell, B. subtilis produces valuable enzymes and biopolymers [3]. For example, xylanase can be fermented in a recombinant strain of Bacillus subtilis to obtain an enzymatic activity of 38 U/mL [4], and L-asparaginase (ASN) can reach a yield of 407.6 U/mL (2.5 g/L) by Bacillus subtilis fermenters [5]. Biopolymers are primarily represented by riboflavin, surface activator, ethylene coupling, phytase, xylanase, L-asparaginase, chondroitin, N-ethyleneglycolate phytase, xylanase, L-asparaginase, chondroitin, N-acetyl acyl glucosamine, etc. [6] (shown in Figure 1). The yield of riboflavin in Bacillus subtilis was ahead of other strains of the chassis. As early as 1999, Perkins et al. applied genetic engineering technology to construct a high-yielding strain of riboflavin using Bacillus subtilis as the substrate cells, and the yield of riboflavin could reach 15 g/L at 56 h of fermentation [7]. In the agriculture field, B. subtilis is not only used to treat plant and animal illnesses and to replace hazardous chemical fungicides to minimize the danger to other species and the environment [8]. Regarding probiotic foods, B. subtilis can enter animals in the form of spores [9]. After germination, it can produce many antibacterial substances to inhibit the growth of harmful intestinal microorganisms such as Escherichia coli to achieve the effect of gastrointestinal protection [10].
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Figure 1. Application of B. subtilis as industrial production chassis and factors related to autolysis of B. subtilis.
However, there is a problem with cell autolysis in fermentation cultures of B. subtilis. Cell autolysis is widespread in microorganisms, such as bacteria, actinomycetes, and fungi. The researchers defined the self-structural degradation process that bacteria undergo at the end of their life cycle as “autolysis” and found that “autolysins” such as cytosolic hydrolases and peptidoglycan hydrolases, which catalyze the hydrolysis of the cell wall peptidoglycan layer, are closely related to the autolysis process. Reduced cell autolysis in B. subtilis leads to a significant increase in cell secretion of recombinant proteins [11] and other productions. In a later stage of fermentation or under fermentation conditions unsuitable for cell growth, the cells will begin to autolyze. This effect leads to a substantial reduction in cell biomass, which seriously affects the expression of products and fermentation efficiency [12]. In particular, high-yield strains modified by metabolic engineering are more likely to occur during cell autolysis. For example, WB800 [13] (B. subtilis with eight extracellular proteases knocked out), most often applied to express extracellular proteases, has more severe cell autolysis [14]. Therefore, it is essential to study the autolysis of B. subtilis. Studies have shown that the root cause of B. subtilis autolysis is that the activity of autolysins is controlled by various conditions to perform their respective functions and thus hydrolyze the peptidoglycan of the cells. These autolysis enzymes include those closely related to the growth and development of B. subtilis and those from prophages.

2. B. subtilis Key Autolysins Enzymes

From the above studies, it is clear that autolysis in B. subtilis is ultimately caused by autolysins. The final results of various evoked autolytic factors are stimulated autolysins expression. Therefore, the research conducted on autolysins is the key to solving the autolysis problem. B. subtilis produces several hydrolases during the trophic growth phase [15][16][17]. Smith et al. [18] nicely summarized the autolysins associated with B. subtilis growth and development. Based on the specificity of autolysins’ hydrolysis of B. subtilis cell wall peptidoglycan and the specificity of the hydrolytic chemical bonds of the autolysins, autolysins and peptidoglycan hydrolases can be classified as muramidase, acetyl-glucosaminidase, acetylmuramoyl alanine amidase, and endopeptidase. The functions of several of the most critical enzymes and their mechanisms of action in autolysis are detailed in Figure 2.
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Figure 2. The role of key autolysis enzymes of B. subtilis in life processes and the factors affecting them.

2.1. Analysis of the Mechanism of Action of Related Enzymes

The respective functions of the life cycle of B. subtilis autolysis enzymes are demonstrated in Figure 2. Among these hydrolases, N-acetylmuramoyl-L-alanine amidase (amidase, LytC or CwIB) and N-acetyl-β-glucosaminidase (glucosaminidase, LytD or CwlG) are two of the significant autolysins [19][20][21]. During the nutritional development stage of bacteria, they carry the majority of the autolysis activity. Inactivation of LytC and LytD show that these two autolysins bear 95% of the autolytic activity of the cells [22][23]. In addition to the main autolysins described above, other less significant enzymes were discovered individually in nutritional cell surface extracts, including LytF (CwlE) and LytE (CwIF) [24][25][26]. Table 1 summarizes the peptidoglycan hydrolase-related genes. People are still researching the roles of various additional, less significant, but potentially vital autolysins. Different groups studying the functions of the above autolysins have come to different conclusions due to subtle differences in strain genetic background and experimental conditions. Still, they all have the same impression about these autolysins. There is a high degree of functional redundancy in these various multifunctional autolysins.

2.1.1. The Role of Autolysins in the Formation of Spores

Autolysins aid cell differentiation. Regarding B. subtilis differentiation activities, including the formation of spores and germination, which need substantial peptidoglycan rearrangements and alterations, almost all of them are affected by autolysins [27][28].
Even though spores have considerable resistance and dormancy, they possess a stress-sensitive system that reacts to germination agents. Maternal cell autolysis aids in the dispersion of mature spores to a more favorable environment. Foster et al. [29] found the presence of a 30 kDa sized protein associated with maternal cell autolysis in the formation of spores that are well expressed before maternal cell autolysis occurs. It is presumed to be primarily responsible for maternal cell autolysis. Then, Kuroda et al. [30] cloned and identified the gene responsible for encoding this protein and named it “cwlC.” They tested the function of the cwlC deletion mutant. They showed that the mutant did not undergo significant phenotypic changes in shoot cell formation, germination, and shoot resistance to heat and autolysins. Moreover, in the cwlC mutant, the mother cell still underwent cell autolysis. They also found that CwlC proteins hydrolyze both the nutrient cell wall peptidoglycan of B. subtilis and the peptidoglycan of spores. Mother cell autolysis required for the release of mature endosperms during the formation of spores is mainly dependent on two amidases of the LytC family, CwlC and LytC; CwlC and LytC play complementary roles in hydrolyzing the cell wall peptidoglycan of mother cells [17][29].
In addition, endospore formation in B. subtilis produces a germination-specific lyase (GSLE) that can only be isolated from germinating spores in its active form and can only hydrolyze the stress peptidoglycan of permeabilized, undamaged spores in vitro. GSLE is present in dormant spores as an inactive preprotein [31].

2.1.2. Role of Autolysins in the Digestion of the Asymmetric Septum, Cortical

Maturation, and Related Differentiation Processes

The B. subtilis autolysins play an essential role in asymmetric septum digestion, cortical maturation, and related differentiation processes [32]. spoIID is involved in the digestion of the asymmetric septum, as strains carrying mutations in this gene are found when the septum is only partially digested and remains in an encircled state around the cell. spoIID encodes a modified protein similar to the LytB sequence. Studies claim that spoIID may control the movement of an autolysin to digest peptidoglycan from asymmetric septa [33].

2.1.3. The Role of Autolysins during the Vegetative Phase

The main autolysins during the vegetative phase of B. subtilis are LytC and LytD; the lytC gene encodes LytC, and lytC is located within the operon element lytLABC [23]. lytC is active at the end of the logarithmic growth phase of B. subtilis, and this autolytic activity is maintained until late in the stabilization phase. It remains active even during the formation of spores. LytC and LytD are the main autolysins produced by B. subtilis during growth. Both of them are transcriptionally regulated by sigD. sigma D is expressed during vegetative growth and is associated with exponential growth and transcription of genes expressed during early stationary periods [34][35]. Furthermore, both proteins are required for bacterial motility. Knocking out the lytC gene in B. subtilis ATCC 6051 increased bacteriophage density and amylase activity by 1.6-fold. lytC and lytD deletion also inhibited cell lysis, but the inhibition was less than in the lytC-only mutant.
lytE and lytF encode peptide chain endopeptidases and are involved in bacterial segregation. Deleting lytE and lytF alone resulted in mutants with defective growth segregation (division or separation). Still, the inhibitory effect on cell autolysis was not significant. In addition, during cell growth, cells must expand covalently closed macromolecules of the cell membrane [36]. B. subtilis contains two functionally redundant D, L-endopeptidases (CwlO and LytE) that cleave peptide crosslinks to allow expansion of PG (the cell wall peptidoglycan) [37]. Double deletion of lytE with the cwlO mutant, which affects cell length increase, leads to a bacteriophage lethal effect [38].

2.2. Prophage Autolysins

In addition to the numerous autolysins involved in the growth and development, there are also autolysins from B. subtilis prophage. When the expression of autolysin encoded by prophages (PBSX) is induced, pores with auxiliary or unknown functions are produced in the peptidoglycan cell wall. The cytoplasmic membrane material protrudes through these openings and is released as a membrane sac (MV) [39]. The induced cells eventually die due to loss of membrane integrity. The vesicle-producing cell leads to cell autolysis through the enzymatic action of releasing autolysin, which induces the formation of MV in neighboring cells [40]. The study of prophage sequences has been studied since as early as 1997; Presecan [41] identified many potentially non-essential chromosomal motifs in the whole genome nucleotide sequence of B. subtilis, particularly 10 relatively large AT-rich regions representing known prophages and prophage-like regions scattered across the genome. Analog sequences (pro1-7) are obtained by horizontal gene transfer [42][43][44]. Prophage PBSX, spβ, and skin remain lysogenic [45][46][47]. Lysogenic phages usually encode peptidoglycan hydrolases, called prophage autolysins. Under certain conditions, it can lyse host cell walls and release phage particles after replication.

2.2.1. Prophage Autolysins PBSX

PBSX, a phage-like phibacin of B. subtilis 168, carried by bacteria can be induced to lyse DNA-damaging agents and produce PBSX particles; however, these particles cannot transmit the PBSX genome [48]. This suicide reaction produces particles that kill non-lysogenic strains of PBSX. The prophage PBSX includes xlyA, which encodes the peptidoglycan hydrolase XlyA mentioned above, xepA encodes an export protein XepA, xhIA encodes a membrane-associated protein XhlA, and xhIB encodes a perforin protein XhIB. The proteins encoded by the latter three genes play an assisting role in cell autolysis. In subsequent studies conducted by Krogh et al. [49], it was found that a late operon located within B. subtilis PBSX was used to express autolysins associated with the prophage PBSX, as shown in Figure 3. Four different genes on late operon encode these autolysins proteins [48].
Table 1. Summary of peptidoglycan hydrolase-related genes.
Table
  Gene Comments Function Ref
1 cwlC Amidase, mother cell-specific Cell separation, spore [16][28]
2 lytC Amidase, vegetative/sporulation expressed only during vegetative growth Cell separation, motility, cell lysis, mother cell lysis [19][20][21]
3 lytD Glucosaminidase, vegetative Motility [21][22]
4 lytE lytF Encoding peptide chain endopeptidase Participation in bacteriophage isolationCell separation, motility, cell lysis, mother cell lysis [19][20][21][23][24][25]
5 gslEgslE GSLE-related proteinsGSLE-related proteins Cortex hydrolysisCortex hydrolysis [30]
6 spoIIDlytE lytF Control of the activity of an autolysin to digest peptidoglycan from asymmetric septaEncoding peptide chain endopeptidase digestion of the asymmetric septumParticipation in bacteriophage isolation [23][24][25][33]
7 xlyAspoIID Amidase, mitomycin C-inducibleControl of the activity of an autolysin to digest peptidoglycan from asymmetric septa PBSX lysisdigestion of the asymmetric septum [33][50]
8 cwlAcwlC Amidase, silent gene in the skin elementAmidase, mother cell-specific Cryptic prophageCell separation, spore [16][28][51][52]

2.2.2. XlyA Amidase Family

As further research into it, Longchamp et al. [47] divided and identified two peptidoglycan hydrolases of the XlyA amidase family, XlyA and YomC, and found YomC is within the spβ, respectively, and belongs to the B. subtilis prophage autolysins. Another enzyme from the XlyA family, N-acetylmuramoyl-L-alanine amidase, is an active peptidoglycan hydrolase [50]. The protein N-acetylmuramoyl-L-alanine amidase (CwlA) is related to cell autolysis. Later studies showed that CwlA is an autolysin gene located within the skin component of the prophage [51]. Kunst et al. [52] also found that the two open reading frames yqxH and yqxG immediately adjacent to cwlA encode two other proteins, respectively, which they hypothesized to be related to the autolytic activity of CwlA and named them “Yqxh” (similar to holin) and “Yqxg” (similar to phage-related lytic exoenzyme), respectively.

2.2.3. Prophage Autolysins BlyA

Regamey and Karamata [45] identified and characterized a DNA fragment in B. subtilis and named it “blyA” gene, which encodes a protein with a molecular weight of 39.6 kDa and contains 367 amino acids (BlyA). BlyA exhibited N-acetylcytidyl-L-alanine amidase activity associated with spβ phage-mediated cell autolysis. They conclusively demonstrated that BlyA belongs to the prophage spβ autolysins and that heat stimulation of B. subtilis CU1147 (CU1147 is an Spβc2 lysogenic strain, and Spβc2 is a temperature-sensitive phage) would induce this Spβc2 to express BlyA and lead to cell autolysis.

2.3. The Effect of Phosphopiridic Acid on Peptidoglycan Hydrolase

It has been shown that the peptidoglycan hydrolase associated with B. subtilis autolysis is regulated by lipoteichoic acid in the cell wall and the action of extracellular proteases. Lipoteichoic acid (LTA) can absorb Mg2+ to support the activity of several synthetic enzymes in the cell membrane. It also functions as a storage element and regulates the activity of intracell autolysis enzymes [53]. In B. subtilis, LTA modification is controlled by the dltA-E operon. Previous studies have shown that deletion of the dltA-D gene disrupts LTA modification, which further alters the cell surface microenvironment and enhances cell autolysis [54][55][56].

2.4. Effect of Proteases on Cell Autolysis

B. subtilis produces at least eight extracellular proteases [57]. Some examples suggest that these extracellular proteases are associated with cell autolysis. Jolliffe et al. reported that B. subtilis protease mutants have autolysin levels in the cell wall equal to or higher than wild-type strains [58]. Extracellular proteases have been identified as involved in degrading peptidoglycan hydrolases in the cell walls. Extracellular protease deficient strains showed increased lysis, where the extracellular proteases AprE and NprE had a greater ability to stabilize autolysins than NprB, Bpr, Mpr, and Epr [59]. Coxon et al. [60] found that the B. subtilis protease deletion mutant had a higher peptidoglycan folding rate while increasing susceptibility to intracell autolysis. In his research, Stephenson found that B. subtilis strains with inactivated protease genes exhibited cell autolysis, and mutants lacking multiple extracellular proteases became more susceptible to autolysis [14].

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Kexin Ren
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Qiang Wang
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Mengkai Hu
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Yan Chen
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Rufan Xing
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Jiajia You
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Meijuan Xu
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Xian Zhang
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Zhiming Rao
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