Polyhydroxyalkanote(s) (PHAs) are natural biopolymers. Many prokaryotic organisms accumulate PHAs as reserve material when carbon (C) source is available in excess in the environment and there is a limitation of nutrients essential for growth. It serves as a food source, which is mobilized by PHA depolymerase under stressed environmental conditions 1. Although PHAs may generally account upto 90% of the dry cell weight (DCW) of the microbes 2, however their production on industrial scale is still very costly in comparison to petrochemical-based plastics 34. The other basic drawbacks which hinder their exploitation on industrial scale are the highly crystalline nature and very low strength of poly(3-hydroxybutyrate) (PHB), the most well studied PHA. In contrast to homopolymers of 3HB (PHBs), copolymers of small chain length C3-C5 hydroxy acids (scl-HA) and medium chain length C6-C14 mcl-HA of PHA are more ductile, easier to mold and tough 5. These copolymers have better film forming and mechanical properties quite similar to low-density polyethylene. These features improve their strength and processability 67. Certain microbes can even produce a natural-synthetic hybrid block copolymer of polyhydroxyoctanoate-diethylene glycol, which results in significant changes in their physiochemical and material characteristics 89. Efforts are thus needed towards improving product quality 10 and efficiency of the recovery process, which will result in optimization of yields. In order to reduce the cost of production, efforts are being made to search 1112 or develop (genetically engineered) strains 13 capable of producing PHAs from inexpensive renewable sources 51415, or even develop transgenic plants for this purpose 16. The purpose of this review is primarily to consolidate the features of Bacillus subtilis as a potential candidate for producing biopolymers – PHAs.

The classical PHB biosynthetic pathway consists of reactions catalyzed by three distinct enzymes. The initial reaction involves condensation of two acetyl-CoA molecules to form acetoacetyl-CoA, which is catalyzed by β-ketothiolase (encoded by phaA). The second step is the reduction of acetoacetyl-CoA by an NADPH-dependent acetoacetyl-CoA dehydrogenase (encoded by phaB). Lastly, the (R)-3-hydroxybutyryl-CoA monomers are polymerized into P(3HB) by P(3HB) synthase (encoded by phaC) 3. In addition, some PHA producers use secondary pathways: i) methylmalonyl CoA pathway, ii) the de novo fatty acid synthetic pathway and iii) via a five-step metabolic pathway assisted by two stereospecific 2-enoyl-CoA hydratases prior to polymerization 5.

Of the three genes generally reported to be involved in PHA biosynthesis, phaA and phaB genes are also involved in glyoxylate regeneration in certain α-Proteobacteria and Actinobacterium. In addition, phaA gene is also involved in the synthesis of mevalonate, which is ubiquitously present in plants. The transfer of phaC gene into an organism gives a new dimension to the functioning of phaA and phaB genes, which occur together at a much higher frequency in different organisms than with phaC gene 17. PHA synthases belonging to the α/β hydrolase superfamily from 45 different bacteria show an overall identity of 8 to 96%. These could be grouped into 4 different classes 1: Class I and II PhaC, made up of one subunit (61 to 73 kDa) could produce scl-PHAs (in Ralstonia eutropha) and mcl-PHAs (in Pseudomonas aeruginosa), respectively. Class III PHA synthases reported from Allochromatium vinosum have two types of subunits: i) PhaC and ii) PhaE (approximately 40 kDa each) which preferably synthesize scl-PHAs. Class IV PHA synthases, which resemble Class III PHA synthases (PhaE is replaced by a 20 kDa PhaR) have been reported only in Bacillus spp. 1.

A perusal of the capacities of different microbes to produce PHAs reveals that certain Gram-negative bacterial species belonging to Alcaligenes, Ralstonia and Pseudomonas lead this group. Alcaligenes and Ralstonia are versatile organisms with well established abilities to utilize pure substrates, agricultural wastes, oily wastes, dairy products and carbon dioxide (CO₂) for PHA production 5181920. Pseudomonas can normally synthesize mcl-PHA on various aliphatic alkenes or fatty acids, agricultural and oily wastes 2021. Pseudomonas sp. can however, simultaneously produce scl-mcl PHAs 2223242526. Among the Gram-negative bacteria, certain archaeal strains of Haloferax, Halobacterium, Haloarcula and Haloquadratum have been reported for their abilities to synthesize PHA from inexpensive C sources as feed material 2728. Although Gram-positive bacteria have not been widely studied, a few genera reported to produce PHB and certain copolymers include Bacillus, Clostridium, Corynebacterium, Nocardia, Rhodococcus, Streptomyces and Staphylococcus 29.

B. subtilis is generally regarded as safe (GRAS) organism by Food and Drug Administration (FDA) 3031 and thus offers additional benefits. B. subtilis has been accorded the designation of industrial workhorse for being among the most widely used microbes for large scale production of recombinant proteins, amino acids and fine chemicals 3233. It will not be inappropriate to call them as "cell factory" 34 since B. subtilis is already known for production of valuable metabolites, bioremediation and generation of bioenergy 353637 but much attention has not been paid to them as PHA producers. Among the Bacillus spp. reported to be PHA producers, the PHA yields vary from 11 to 69% (w/w of DCW – upto 70 g/L): B. amyloliquefaciens DSM7, B. laterosporus, B. licheniformis, B. macerans, B. cereus, B. circulans, 6, B. firmus G2, B. subtilis K8,B. sphaericus X3, B. megaterium Y6 38, B. coagulans, B. brevis, B. sphaericus ATCC 14577 39, B. thuringiensis 37, B. mycoides RLJ B-017 40 and Bacillus sp. JMa5 41. Bacillus sp. INT005 and B. cereus UW85 could produce PHA with a wide range of compositions varying from PHB, P(3HB-co-3HV: copolymer of butyrate and valerate), P(3HB-co-3HHx: copolymer of butyrate and hexanoate), P(3HB-co-4HB-co-3HHx: copolymer of 3-hydroxy-, 4-hydroxy- butyrates and hexanoate) to P(3HB-co-6HHx-co-3HHx: terpolymer of butyrate, 6-hydroxy- and 3-hydroxy hexanoates) depending upon the substrate 4243. Various Bacillus spp. have been shown by different researchers to synthesize copolymers when co-fed with various substrates. Using B. cereus UW85, the production of terpolymer of 3HB, 3HV and 6HHx was recorded with ε-caprolactone as sole C source in mineral salts medium without any glucose. However, addition of glucose along with ε-caprolactone seemed to suppress copolymer synthesis and the result was the production of PHB 42. Bacillus sp. INT005 could accumulate PHB when glucose was used alone as C substrate in the medium. However, addition of various C sources along with very low glucose concentration resulted in copolymers of 3HB and 3HHx on octanoate and decanoate, copolymers of 3HB-4HB-4HHx on 4-hydroxybutanoate and 3HB-3HHx-6HHx on supplementation with ε-caprolactone 44. Recent studies have produced still more interesting information. B. cereus SPV when grown on structurally unrelated C sources such as fructose, sucrose and gluconate resulted in the incorporation of 4HB with the first two substrates and 4HB and 3HV with gluconate in the medium 45. Although limitation of nitrogen (N), phosphorous (P) and oxygen in the culture conditions are known to influence PHB production, however, potassium limiting media led to the production of a copolymer containing 3HB and 3HV monomers in contrast to only PHB under sulphur, P or N limitation 43. B. megaterium yielded PHB on cane molasses 46, B. cereus CFR06 on starch 47 and other Bacillus spp. could also produce PHB from industrial food waste water, soya waste and malt waste from beer brewery plant and pea-shell slurry 48. Bacillus sp. 256 utilize an unrefined natural substrate – mahua (Madhuca sp.) flower as C source (containing 57% w/w as sugars) to produce copolymers (90:10 mol% P(HB-co-HV)) 49.

There are a few reports which have shown B. subtilis to be natural producers 37383950. These results need to be viewed more cautiously since B. subtilis is a very heterogeneous group. Recent studies have shown that B. subtilis can be subdivided into two subspecies namely B. subtilis subsp.subtilis and B. subtilis subsp.spizizenii 5152, such that only one of them may possess members which are PHA producers, where as those belonging to B. subtilis subsp. subtilis (B. subtilis 168 belongs to this group) may be naturally non-producers types. This results in a very interesting situation with respect to B. subtilis, which can thus be "labeled" as non-producers just like Escherichia coli which has been widely used for expression of many genes including those of PHA biosynthesis 3553545556.

B. subtilis has been recently used as a host for over expression of phaCAB genes from P. aeruginosa and R. eutropha. Expression of phaC1 from P. aeruginosa and phaAB genes from R. eutropha in B. subtilis resulted in the production of copolymers P(HD-co-HDD, hydroxydecanoate-co-hydroxydodecanoate) and P(HB-co-HD-co-HDD) from malt waste 57. Since B. subtilis is not a human pathogen, further supporting its usage as a host for expression of foreign genes 58. B. megaterium phaPQRBC genes were cloned into B. subtilis 1A304(Φ105MU331). This report of stable plasmid integration into the B. subtilis chromosome paved the way for large scale fermentation process for production of PHA which eliminated the use of antibiotics, a desirable feature for reducing production cost 58. Recombinant B. subtilis could utilize malt waste as a C source, further raising the hopes for producing PHA at cheaper rates. This study showed that phaPQ of B. megaterium were essential for PHA production along with phaRBC, although they could not observe any sequence homology in NCBI database. It has been previously shown that phaP in R. eutrophus might play a role in determining the shape and size of the inclusion bodies 59.

Although B. subtilis has the potential to compete with E. coli, the most commonly used host for heterologous expression of genes 34 and it may even be superior in certain aspects, however, the time pressure limits the change of host organism in later stages of development of process optimization 60. Westers et al., 60 listed four drawbacks in using Bacillus as host organism – a) lack of suitable expression vectors, b) plasmid instability, c) presence of protease and d) presence of malfolded proteins. Recent efforts have circumvented certain issues such as the generation of suitable expression vectors 61 and stable plasmids particularly for PHA production 58. B. subtilis WB800 strain lacking eight extracellular proteases enabled the production of homologous proteins, including those which were otherwise susceptible to rapid degradation 34. Certain other features which further support B. subtilis as potential PHA producer have been discussed here.

In the post genomic era, 22 Bacillus strains have been sequenced and 32 projects are in progress 62. Among all Bacillus spp., B. subtilis is one of the most heterogeneous representative 526364. The concerted efforts of European Union funded consortia 656667 lead B. subtilis 68 to be the first Gram-positive, soil microorganism to be completely sequenced and opened the era of functional analysis of Gram-positive bacteria. The major resources made available as a results of these efforts in B. subtilis include datasets on transcriptome, proteome, secretome and metabolome. These also include a collection of more than 3000 mutants, vectors, tools and techniques for rapid production of heterologous proteins 356970.

Screening of metabolic (KEGG) and genomic (NCBI) databases for the presence of enzymes 71 involved in PHA biosynthesis reveals that genes for PhaA and PhaB are observed to be present in almost all the sequenced genomes of Bacillus except certain strains of B. megaterium 2972, B. thuringiensis, B. subtilis and Bacillus spp. (Table 1). However, quite a bit of variation is recorded in the case of phaC gene and phaR. PhaC is frequently observed largely in the members of B. cereus group, however, it's conserved domain is absent or partially present in most other Bacillus spp. The presence of conserved domain of PhaR was largely partial or absent. In general B. subtilis lacks genes related to PHA biosynthesis 1 providing opportunity to circumvent the need to eliminate or reduce the background effect caused due to homologous genes of the host during heterologous gene expression. The concomitant presence of PHA biosynthesis and depolymerization system has proved beneficial for efficient production of PHA 35. Incidentally, B. subtilis does contain phaZ encoding for PHA depolymerase.

The basic quest in biotechnology is to construct microbial strains capable of accomplishing the rapidly expanding array of desired biotransformations 34. The initial steps towards enhancing product efficiency and recovery process are to look for the presence of pathways that divert fluxes towards undesirable products or compete for the utilization of precursors and cofactors 73. Genome scale reconstruction of B. subtilis was converted into an in silico model to trace the metabolic pathways which can be verified through experimental works in B. subtilis 7374. This can be executed with relatively high confidence with the availability of databases on transcriptomes, proteomes and metabolomes 7374. Biolog's Phenotype Microarray™ technology enables phenotype analysis 74 of B. subtilis for a wide range of C, N, P and sulphur sources. A wide variety of C sources and their corresponding transporters present in B. subtilis reflected on its high adaptability to environmental conditions. The interesting part was its inability to utilize glycine probably because of production of H₂O₂, a toxic byproduct of glycine oxidase activity. The absence of glycoxylate shunt in B. subtilis justifies the observation that no growth is observed on acetate as sole C source although NADH and ATP could be produced by the metabolic network 74. The phenotypes showing the utilization of various substrates are indicative of the presence of corresponding pathways. In the work of Oh et al., 74 350 intracellular metabolites were identified but only 160 were present in the model, which implies that a large portion of B. subtilis metabolism is yet to be elucidated.

The urge to exploit microorganisms for maximum benefits to human beings has emerged in creation of cells with predictable behaviour. Genome reduction process for building collection of knockout mutants has been initiated for a wide range of bacteria such as Gram-positives – B. subtilis (Firmicutes), Corynebacterium glutamicum (Actinobacteria), and Gram-negatives – E. coli K-12, Haemophilus influenzae, P. aeruginosa, P. aeruginosa strain PA14 and Acinetobacter baylyi ADP1 (Gamma Proteobacteria) 73. Efforts in this direction have been made with E. coli and B. subtilis as model organisms (Table 2), for studying their diverse biological features 3275. Since the most daunting task is to predict genes to be deleted without detrimental effect(s), Hoshimoto et al., began with constructing and characterizing a series of large scale chromosomal deletion mutants of E. coli 76. The 16 mutants of E. coli K-12 strain MG1655 lacked 2.4–29.7% of the parental chromosome. These deletions resulted in aberrant cell shape, slow growth rate and modified nucleoid organization. Mutants of E. coli MG1655 and W3110 with the removal of mobile DNA, virulence genes and genes for amino acids, lipopolysaccharides, transporters etc. exhibited high electroporation efficiency 77 and higher threonine secretion 75 compared to wild type strain. B. subtilis the Gram-positive counter-part of the industrially important E. coli has only 271 essential genes out of a 4.2 Mb genome 78. The optimization of Bacillus as cell factory by deleting prophages, prophage-like regions and polyketide synthase operon of B. subtilis amounted to 7.7% reduction in genome size equivalent to elimination of 332 protein encoding genes 32. Subsequent efforts by Ara et al., 2007 lead to DNA deletion to the extent of 24.7% without any apparent benefits 79. However, Morimoto et al., 2008 61 have demonstrated that genome reduction may create bacterial cells with practical utility to industry – enhanced extracellular cellulase and protease and improved efficiency to utilize a wide range of C sources.

B. subtilis Δ6 has a few major advantages compared to conventional B. subtilis strains. B. subtilis Δ6 lacks the BsuM restriction-modification system. It confers advantages to Δ6, since BsuM restriction reduces transformation efficiency of B. subtilis with recombinant plasmids and is also responsible for structural plasmid instability. It consequently affects the application potential of plasmids for high-level protein production 32. This system can be assisted by highly stable expression vectors developed for the rapid purification of recombinant protein in B. subtilis 80. However, this cell factory engineering can be used to re-insert genes of importance into the chromosome of B. subtilis.

HGT plays an important role in the diversity and evolution of B. subtilis. Phage integration accounts for 16% of the HGT regions in B. subtilis 168 81. Extensive HGT in Bacillus sp. increases the chance of "easier" and extensive manipulation. HGT from A+T rich islands can be accomplished by transposes. B. subtilis has comparatively more stable chromosomal structure compared to other Bacillus spp. particularly those of B. cereus group. B. subtilis genome shows 10 transposons and related rearrangements compared to 112 such events in B. halodurans C-125 chromosome 82. In B. subtilis the ability to survive in a wide range of environmental niches can be because of large number of genes (586 equivalent to 10% of the ORFs) related to SpoA, regulatory protein important for initiating sporulation, genes encoding proteins involving antibiotics and cell wall synthesis 63.

In view of the fact that feedstock being a significant component of manufacturing costs of PHA, maximizing the C yield and reduced byproduct formation becomes a priority. The metabolic bottleneck for maximum yield of all products is either stoichiometrically constrained or limited by energy. Sauer et al., 83 suggested that to improve yields, it will be better to look for microorganisms and processes that may provide the necessary energetic efficiency 83. They recommended either to use alternative C substrates such as glycerol or sucrose (instead of glucose) which have reduced or non-existing stoichiometric limitations or go in for metabolic engineering 83. For maximum efficiency and regulation of metabolic processes in B. subtilis, an alternative C catabolite repression was suggested since it doesn't possess cAMP 84. A CcpA defective mutant of B. subtilis was found to grow at a slower rate in minimal medium with glucose and ammonia as C and N sources, in comparison to the wild type cells 84. CcpA represses the expression of citZ gene encoding citrate synthase. This reaction involves condensation (utilization) of acetyl-CoA with oxaloacetate 84. Manipulation of CcpA can enable larger availability of acetyl-CoA for PHA biosynthesis. Further, B. subtilis sporulates in response to depletion of nutrients 64 and glucose 79. Incidentally, there is a conflict between PHA production and spore formation, since both get induced under similar environmental conditions. A non-spore forming strain (wild or mutant) of Bacillus holds promise of better performance 2. So a knockout will enable it to grow vegetative and make sporulation genes redundant. In fact, this mechanism lead to increased cellulase activity in B. subtilis 168 79. Under laboratory conditions, it will be worth the effort to knockout sporulations genes and make room for re-introducing relevant genes, if necessary. Gram-positive bacteria can produce copolymers of PHA from simple, inexpensive and structurally un-related C sources. In contrast, Gram-negative bacteria depend upon expensive and structurally related substrates for producing copolymers of PHA 29.

Since PHA accumulates intracellularly, cell disruption is indispensable to recover it. On an industrial scale, the PHA recovery process involves the usage of a large amount of chemical reagents and/or enzymes 2. An efficient recovery system implies reduced cost of production 85. An optimum time to recover PHAs is immediately after the C source gets exhausted and before the commencement of degradation by depolymerase enzyme 86. A novel self disruption cell system has been developed in B. megaterium. In this system, a gene cassette carrying the cell lysis system (holin and endolysin of B. amyloliquefaciens phage) 87 was inserted into the E. coli – B. subtilis shuttle vector pX. In this expression system, xylR-xylA' target genes are induced by xylose but inhibited by glucose, which acts as an anti-inducer 86. It synchronizes the processes of spontaneous cell lysis and substrate exhaustion, which thus results in the release of accumulated PHAs 86. The efficiency of this regulatory process can be enhanced by manipulating the YoeB, a cell wall-associated protein, which gets induced in response to antibiotics stress. The expression of yoeB in B. subtilis is under a xylose-inducible promoter 88. yoeB mutants display an increased rate of autolysis in response to nutrient depletion and various cell envelope stress conditions 88. The process of autolysis in B. subtilis 168 can be aided by mutating yoeB gene and in the process making it independent of xylose regulation.

Gram-negative bacteria such as Ralstonia, A. latus and recombinant E. coli are among those which have been exploited for industrial scale PHB production. The outer cell membrane of most Gram-negative bacteria including E. coli contains lipo-polysaccharides (LPS), which are endotoxins 89 and are pryogenic in human beings 60. The purification of byproducts including PHAs is complicated due to the presence of endotoxins 60. Since LPS induces a strong immunogenic reaction, this feature is undesirable for biomedical applications of the PHAs 790. A review on the toxic nature of LPS reveals that cyanobacterial (Gram-positive bacteria) LPS are less toxic compared to those produced by members of Enterobacteraceae 89. B. subtilis offers the advantage of lacking LPS and excreting proteins at a high rate into the medium 61. It thus stands a better chance as PHA producer for biomedical applications 90.

The use of CO₂ as a potential inexpensive renewable C source can help in reducing PHA production cost 5. Synechococcus sp. MA19 was observed to produce 55% w/w PHA from CO₂ 91. A. eutrophus was shown to accumulate PHA at the rate of 1.55 g/L/h, which was higher than that recorded with cyanobacteria or photosynthetic bacteria 92. The strategy being proposed is that if hydrogen (H₂) production becomes cheap then R. eutropha can produce PHB from CO₂ and oxidation of (H₂), under dark fermentative conditions 93. Bacillus seems to meet the requirements of this proposal. Bacillus spp. such as B. coagulans, B. licheniformis and B. subtilis have been shown to evolve 1.5 to 2.36 mol H₂/mol glucose 36. Biowastes rich in starch such as damaged wheat grains have been employed as feed for generating H₂ (45 to 64 L/kg Total solids) by B. licheniformis and B. subtilis 94. More recently, B. cereus strain EGU43 and B. thuringiensis strain EGU45 have been reported to generate 0.63 mol of H₂/mol of glucose and upto 500 mg PHB/L 37.