Potential Probiotics Bacillus subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895 Co-Aggregate with Clinical Isolates of Proteus mirabilis and Prevent Biofilm Formation

A urinary tract infection (UTI) is a multi-factorial disease including cystitis, pyelonephritis, and pyelitis. After Escherichia coli, Proteus mirabilis is the most common UTI-associated opportunistic pathogen. Antibiotic resistance of bacteria and infection recurrence can be connected to biofilm formation by P. mirabilis. In this study, human and sheep isolates of P. mirabilis were investigated for antibiotic sensitivity using an antibiotic disk test. Co-aggregation of the tested potential probiotic bacilli, Bacillus amyloliquefaciens B-1895 and Bacillus subtilis KATMIRA1933, with the isolated pathogen was also evaluated. Then, the anti-biofilm activity of naturally derived metabolites, such as subtilin and subtilosin, in the bacilli-free supernatants was assessed against biofilms of P. mirabilis isolates. The isolated pathogens were sensitive to 30 μg of amikacin and 5 μg of ciprofloxacin but resistant to other tested antibiotics. After 24 h, auto-aggregation of B. amyloliquefaciens B-1895 was at 89.5% and higher than auto-aggregation of B. subtilis KATMIRA1933 (59.5%). B. amyloliquefaciens B-1895 strongly co-aggregated with P. mirabilis isolates from human UTIs. Cell-free supernatants of B. amyloliquefaciens B-1895 and B. subtilis KATMIRA1933 showed higher antimicrobial activity against biofilms of P. mirabilis isolated from humans as compared with biofilms of sheep isolates. According to our knowledge, this is the first report evaluating the anti-biofilm activity of probiotic spore-forming bacilli against clinical and animal UTI isolates of P. mirabilis. Further studies are recommended to investigate the anti-biofilm activity and the mode of action for the antimicrobial substances produced by these bacilli, subtilosin and subtilin.

     

Engineering Bacillus subtilis ATCC 6633 for improved production of the lantibiotic subtilin

To improve the production of the lantibiotic subtilin in Bacillus subtilis ATCC 6633, two genetic engineering strategies were followed. Firstly, additional copies of subtilin self-protection (immunity) genes spaIFEG have been integrated into the genome of the producer strain. Their expression significantly enhanced the subtilin tolerance level, and concomitantly, the subtilin yield 1.7-fold. Secondly, a repressor of subtilin gene expression, the B. subtilis general transition state regulator protein AbrB, was deleted. A sixfold enhancement of the subtilin yield could be achieved with the abrB deletion mutant; however, the produced subtilin fraction predominantly consists of succinylated subtilin species with less antimicrobial activity compared to unmodified subtilin.     

Enhanced production of 2,3-butanediol by engineered <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Production of 2,3-butanediol by Bacillus subtilis takes place in late-log or stationary phase, depending on the expression of bdhA gene encoding acetoin reductase, which converts acetoin to 2,3-butanediol. The present work focuses on the development of a strain of B. subtilis for enhanced production of 2,3-butanediol in early log phase of growth cycle. For this, the bdhA gene was expressed under the control of P _alsSD promoter of AlsSD operon for acetoin fermentation which served the substrate for 2,3-butanediol production. Addition of acetic acid in the medium induced the production of 2,3-butanediol by 2-fold. Two-step aerobic–anaerobic fermentation further enhanced 2,3-butanediol production by 4-fold in comparison to the control parental strain. Thus, addition of acetic acid and low dissolved oxygen in the medium are involved in activation of bdhA gene expression from P _alsSD promoter in early log phase. Under the conditions tested in this work, the maximum production of 2,3-butanediol, 2.1 g/l from 10 g/l glucose, was obtained at 24 h. Furthermore, under the optimized microaerophilic condition, the production of 2,3-butanediol improved up to 6.1 g/l and overall productivity increased by 6.7-fold to 0.4 g/l h in the engineered strain compared to that in the parental control.     

Paclitaxel production using co-culture of <Emphasis Type="Italic">Taxus</Emphasis> suspension cells and paclitaxel-producing endophytic fungi in a co-bioreactor

The co-culture of the suspension cells of Taxus chinensis var. mairei and its endophytic fungi, Fusarium mairei, in a 20-L co-bioreactor was successfully established for paclitaxel production. The co-bioreactor consists of two-unit tanks (10 L each) with a repairable separate membrane in the center, culturing Taxus suspension cells in one tank and growing fungi in another. By optimizing the co-culture conditions, there was a desirable yield of paclitaxel in Taxus cell cultures. The Taxus cell cultures by co-culture produced 25.63 mg/L of paclitaxel within 15 days; it was equivalent to a productivity of 1.71 mg/L per day and 38-fold higher than that by uncoupled culture (0.68 mg/L within 15 days). The optimum conditions for co-culture in the co-bioreactor were: B5 medium, inoculating fungi when Taxus cells had grown for 5 days in the co-bioreactor, hydrophilic separate membrane in the center of the co-bioreactor, and air flow rate of 1:0.85 v/v/m in fungus cultures.     

The presence of sboA and spaS genes and antimicrobial peptides subtilosin A and subtilin among Bacillus strains of the Amazon basin

This report demonstrates the usefulness of PCR for the genes spaS and sboA as a means of identifying Bacillus strains with a potential to produce subtilin and subtilosin A. One collection strain and five Bacillus spp. isolated from aquatic environments in the Amazon basin were screened by PCR using primers for sboA and spaS designed specifically for this study. The sequences of the PCR products showed elevated homology with previously described spaS and sboA genes. Antimicrobial peptides were isolated from culture supernatants and analyzed by mass spectrometry. For all samples, the mass spectra revealed clusters with peaks at m/z 3300–3500 Da, corresponding to subtilosin A, subtilin and isoforms of these peptides. These results suggest that the antimicrobial activity of these strains may be associated with the production of subtilosin A and/or subtilin. The PCR used here was efficient in identifying novel Bacillus strains with the essential genes for producing subtilosin A and subtilin.     

Systems metabolic engineering of Bacillus subtilis for efficient biosynthesis of 5-methyltetrahydrofolate

5-Methyltetrahydrofolate (5-MTHF) is the major form of folate in human plasma and is the only folate form that can penetrate the blood–brain barrier. It has been widely used for the prevention and treatment of various diseases. It is mainly produced by chemical synthesis. However, the low production rate cannot meet the increasing demand. In addition, chemical synthesis is potentially detrimental to the environment. Despite various microorganisms synthetizing 5-MTHF, an efficient 5-MTHF bioproduction approach is lacking because of the tight regulation of the 5-MTHF pathway and limited metabolic flux toward the folic acid pathway. In this study, the 5-MTHF synthetic pathway in Bacillus subtilis was systematically engineered to realize 5-MTHF accumulation and further improve 5-MTHF production. Specifically, the 5-MTHF synthesis pathway with dihydrofolate (DHF) as the precursor was strengthened to shift the metabolic flux to 5-MTHF biosynthesis by replacing the native yitJ gene with Escherichia coli metF, knockout of purU, and overexpressing dfrA. The intracellular level of 5-MTHF increased 26.4-fold, reaching 271.64 µg/L. Next, the 5-MTHF precursor supply pathway was strengthened by co-overexpression of folC, pabB, folE, and yciA. This resulted in a 93.2-fold improvement of the 5-MTHF titer, which reached 960.27 µg/L. Finally, the clustered regularly interspaced short palindromic repeats interference system was used to identify key genes in the competitive and catabolic pathways for repression to further shift the metabolic flux toward 5-MTHF biosynthesis. The repression of genes thyA (existing in the purine metabolic pathway), pheA (existing in the competitive metabolic pathway), trpE (existing in the competitive metabolic pathway), and panB (existing in the pantoate synthesis pathway) significantly increased the titer of 5-MTHF. By repressing the pheA gene, the 5-MTHF titer reached 1.58 mg/L, which was 153.8-fold that of the wild-type strain of B. subtilis 168. Through medium optimization, the 5-MTHF titer reached 1.78 mg/L, which was currently the highest titer of 5-MTHF in B. subtilis. Apart from the highest titer of 5-MTHF, the highest titer of total folates including 5-MTHF, 5-FTHF, folic acid, and THF could reach 3.31 mg/L, which was 8.5-fold that in B. subtilis. To the best of our knowledge, the 5-MTHF and total folate titers reported here are the highest using a Generally regarded as safe (GRAS) bacterium as the production host. Overall, this study provides a good starting point for further metabolic engineering to achieve efficient biosynthesis of 5-MTHF by GRAS bacteria.     

Engineering <Emphasis Type="Italic">Bacillus subtilis</Emphasis> for isobutanol production by heterologous Ehrlich pathway construction and the biosynthetic 2-ketoisovalerate precursor pathway overexpression

In the present work, Bacillus subtilis was engineered as the cell factory for isobutanol production due to its high tolerance to isobutanol. Initially, an efficient heterologous Ehrlich pathway controlled by the promoter P₄₃ was introduced into B. subtilis for the isobutanol biosynthesis. Further, investigation of acetolactate synthase of B. subtilis, ketol-acid reductoisomerase, and dihydroxy-acid dehydratase of Corynebacterium glutamicum responsible for 2-ketoisovalerate precursor biosynthesis showed that acetolactate synthase played an important role in isobutanol biosynthesis. The overexpression of acetolactate synthase led to a 2.8-fold isobutanol production compared with the control. Apart from isobutanol, alcoholic profile analysis also confirmed the existence of 1.21 g/L ethanol, 1.06 g/L 2-phenylethanol, as well as traces of 2-methyl-1-butanol and 3-methyl-1-butanol in the fermentation broth. Under microaerobic condition, the engineered B. subtilis produced up to 2.62 g/L isobutanol in shake-flask fed-batch fermentation, which was 21.3% higher than that in batch fermentation.     

A modified electro-transformation method for Bacillus subtilis and its application in the production of antimicrobial lipopeptides

A modified electroporation method using trehalose is presented for the transformation of Bacillus subtilis. The new method improved the transformation efficiency of B. subtilis nearly 2,000-fold compared with the usual method, giving 4 × 10⁵ transformants/μg DNA. Using this method, B. subtilis was engineered to improve production of antimicrobial lipopeptides and produced 1.8-fold more surfactin and 2.9-fold more fengycin.     

Identification of unstructured model for subtilin production through Bacillus subtilis using hybrid genetic algorithm

Subtilin production is favorable when Bacillus subtilis 168 is subjected to stress condition such as nutrient scarcity. A mathematical model underlying such growth process has immense applicability in determining the optimal operating conditions at industrial scale. We present this work with multiple objectives of a) selection of a substrate for creating the minimal nutrient media for B. subtilis thereby enhancing subtilin production, b) experimental study of the growth along with morphological characteristics of B. subtilis and product profile in nutrient scarcity condition and c) identification of an optimal unstructured model for subtilin production using a computational framework. First, we show that subtilin can be produced while B. subtilis is grown using galactose and B. subtilis undergoes morphological changes and takes filamentous shape. We then constructed a series of plausible models and used a hybrid method combining Genetic Algorithm and gradient based search methodologies, for model selection. The estimated kinetic parameters and the stoichiometric analysis indicate that the B. subtilis growth/death, product profile and respiratory mechanism undergo specific modifications in galactose as an adaptive response. Current study provides an inexpensive platform to produce subtilin and the predictive framework presented here has potential applications for large scale production of subtilin.     

High-yield production of L-valine in engineered Escherichia coli by a novel two-stage fermentation

L-valine is an essential amino acid and an important amino acid in the food and feed industry. The relatively low titer and low fermentation yield currently limit the large-scale application of L-valine. Here, we constructed a chromosomally engineered Escherichia coli to efficiently produce L-valine. First, the synthetic pathway of L-valine was enhanced by heterologous introduction of a feedback-resistant acetolactate acid synthase from Bacillus subtilis and overexpression of other two enzymes in the L-valine synthetic pathway. For efficient efflux of L-valine, an exporter from Corynebacterium glutamicum was subsequently introduced. Next, the precursor pyruvate pool was increased by knockout of GTP pyrophosphokinase and introduction of a ppGpp 3′-pyrophosphohydrolase mutant to facilitate the glucose uptake process. Finally, in order to improve the redox cofactor balance, acetohydroxy acid isomeroreductase was replaced by a NADH-preferring mutant, and branched-chain amino acid aminotransferase was replaced by leucine dehydrogenase from Bacillus subtilis. Redox cofactor balance enabled the strain to synthesize L-valine under oxygen-limiting condition, significantly increasing the yield in the presence of glucose. Two-stage fed-batch fermentation of the final strain in a 5 L bioreactor produced 84 g/L L-valine with a yield and productivity of 0.41 g/g glucose and 2.33 g/L/h, respectively. To the best of our knowledge, this is the highest L-valine titer and yield ever reported in E. coli. The systems metabolic engineering strategy described here will be useful for future engineering of E. coli strains for the industrial production of L-valine and related products.     

Production of phloroglucinol by Escherichia coli using a stationary-phase promoter

Escherichia coli was metabolically engineered using a new host-vector system to produce phloroglucinol. The key biosynthetic gene phlD (encoding a type III polyketide synthase) from Pseudomonas fluorescens was expressed in E. coli using the stationary-phase promoter of the fic gene and a high-copy plasmid. In shake-flasks, the engineered strain produced phloroglucinol up to 0.28 g/l with a productivity of 0.014 g/l h. About 9.2% of the glucose consumed was converted to phloroglucinol after 20 h. Compared with the widely used inducible T7 promoter system, this strain did not require IPTG induction and the final titer of phloroglucinol was 22% higher.     

Production of β-carotene by recombinant Escherichia coli with engineered whole mevalonate pathway in batch and fed-batch cultures

Recombinant Escherichia coli engineered to contain the whole mevalonate pathway and foreign genes for β-carotene biosynthesis, was utilized for production of β-carotene in bioreactor cultures. Optimum culture conditions were established in batch and pH-stat fed-batch cultures to determine the optimal feeding strategy thereby improving production yield. The specific growth rate and volumetric productivity in batch cultures at 37°C were 1.7-fold and 2-fold higher, respectively, than those at 28°C. Glycerol was superior to glucose as a carbon source. Maximum β-carotene production (titer of 663 mg/L and overall volumetric productivity of 24.6 mg/L × h) resulted from the simultaneous addition of 500 g/L glycerol and 50 g/L yeast extract in pH-stat fed-batch culture.     

Enhanced extracellular production of L-asparaginase from Bacillus subtilis 168 by B. subtilis WB600 through a combined strategy

L-asparaginase (EC 3.5.1.1, ASN) exhibits great commercial value due to its uses in the food and medicine industry. In this study, we reported the enhanced expression of type II ASN from Bacillus subtilis 168 in B. subtilis WB600 through a combined strategy. First, eight signal peptides (the signal peptide of the ASN, ywbN, yvgO, amyE, oppA, vpr, lipA, and wapA) were used for ASN secretion in B. subtilis by using Hpa II promoter, respectively. The signal peptide wapA achieved the highest extracellular ASN activity (28.91 U/mL). Second, Hpa II promoter was replaced by a strong promoter, P43 promoter, resulting in 38.1 % enhanced ASN activity. By two rounds of error-prone PCR mutation, the P43 promoter variants with remarkably enhanced strength (D7, E2, H6, B2, and F3) were identified. B2 (−28: A → G, −13: A → G) achieved ASN activity up to 51.13 U/mL. Third, after deletion of the N-terminal 25-residues, ASN activity reached 102.41 U/mL, which was 100 % higher than that of the intact ASN. At last, the extracellular ASN of the B. subtilis arrived at 407.6 U/mL (2.5 g/L of ASN protein) in a 3-L bioreactor by using a fed-batch strategy. The purified ASN showed maximal activity at 65 °C and its half-life at 65 °C was 61 min. The K _m and k _cat of the ASN were 5.29 mM and 54.4 s⁻¹, respectively. To the best of our knowledge, we obtained the highest yield of ASN in a food-grade host ever reported, which may benefit the industrial production and application of ASN.

     

Efficient constitutive expression of thermostable 4-α-glucanotransferase in Bacillus subtilis using dual promoters

4-α-Glucanotransferases possess strong transglycosylation activity which has been used in various carbohydrate chemistry fields. Due to safety issues of the recombinant enzymes we chose Bacillus subtilis as an expression host to produce a thermostable 4-α-glucanotransferase from Thermus scotoductus (TSαGT). The HpaII promoter in the Gram-positive bacterial vector pUB110 was used first to express TSαGT gene in B. subtilis. However, the activity of TSαGT in B. subtilis was only 4% of that in our previous Escherichia coli system. Two expression systems constructed by sequential alignment of another constitutive promoter for either α-amylase from B. subtilis NA64 or maltogenic amylase from Bacillus licheniformis downstream of the HpaII promoter elevated the TSαGT productivity by 11- and 12-fold, respectively, compared to the single HpaII promoter system. In conclusion, the dual promoter systems in this study were much better than the single promoter system to express the TSαGT gene in B. subtilis.     

Production of cyclomaltodextrin glucanotransferase of Bacillus circulans var. alkalophilus ATCC21783 in B. subtilis

Summary The cyclomaltodextrin glucanotransferase (CGTase, E.C. 2.4.1.19) gene from an alkalophilic Bacillus circulans var. alkalophilus ATCC21783 was cloned into Escherichia coli and B. subtilis. When cloned from E. coli to B. subtilis, the entire insert containing the CGTase gene was, depending on the plasmid construction, either unstable or the recombinant B. subtilis did not secrete the enzyme in significant amounts. To achieve efficient enzyme production in B. subtilis, the gene was placed under the control of the B. amyloliquefaciens -amylase promoter. In one of the constructions, both the promoter and the signal sequence of the gene were replaced with those of B. amyloliquefaciens, whereas in another construction only the promoter area was exchanged. The recombinant B. subtilis clones transformed with these plasmid constructions secreted CGTase into the culture medium 14 times as much as did the parental strain in shake flask cultures. In fermentor cultures in an industrially feasible medium the enzyme production was substantially higher, yielding 1.2 g/l of CGTase, which is about 33 times the amount of the enzyme produced by the parental strain in corresponding fermentations. Both of the plasmid constructions were stable when grown over 50 generations without antibiotic selection.     

Engineering alternative butanol production platforms in heterologous bacteria

Alternative microbial hosts have been engineered as biocatalysts for butanol biosynthesis. The butanol synthetic pathway of Clostridium acetobutylicum was first re-constructed in Escherichia coli to establish a baseline for comparison to other hosts. Whereas polycistronic expression of the pathway genes resulted in the production of 34 mg/L butanol, individual expression of pathway genes elevated titers to 200 mg/L. Improved titers were achieved by co-expression of Saccharomyces cerevisiae formate dehydrogenase while overexpression of E. coli glyceraldehyde 3-phosphate dehydrogenase to elevate glycolytic flux improved titers to 580 mg/L. Pseudomonas putida and Bacillus subtilis were also explored as alternative production hosts. Polycistronic expression of butanol biosynthetic genes yielded butanol titers of 120 and 24 mg/L from P. putida and B. subtilis, respectively. Production in the obligate aerobe P. putida was dependent upon expression of bcd-etfAB. These results demonstrate the potential of engineering butanol biosynthesis in a variety of heterologous microorganisms, including those cultivated aerobically.     

NMR resonance assignment of the autoimmunity protein SpaI from Bacillus subtilis ATCC 6633

Bacillus subtilis ATCC 6633 produces the lipid II targeting lantibiotic subtilin. For self-protection these gram-positive bacteria express a cluster of four self-immunity proteins named SpaIFEG. SpaI is a 16.8 kDa lipoprotein which is attached to the outside of the cytoplasmic membrane via a covalently linked diacylglycerol anchor. Together with the ABC-transporter SpaFEG, SpaI protects the membrane from subtilin insertion and there is evidence for a direct interaction of SpaI with subtilin. As a prerequisite for further structural studies of SpaI and the SpaI/subtilin complex we report here the full ¹H, ¹⁵N, ¹³C chemical shift assignment for a stable 14.9 kDa C-terminal fragment of SpaI.     

Detection and identification of antibiotic biosynthesis genes in Bacillus subtilis strains

Two Bacillus subtilis strains L10 and G1, previously isolated from fermented pickles, were tested for antibacterial activity against Vibrio harveyi using a well-diffusion agar assay. Antibiotic biosynthesis genes were then detected in both bacterial strains and included the following genes: bacA, bacB and bacD genes for bacilysin production, ppsE/fenB gene for plipastatin/fengycin production, albF and albA genes for subtilosin production, and srfAB and sfP genes for surfactin production. Based on these results, two B. subtilis strains could be considered as potential biological control agents in aquaculture.     

Expression and fermentation optimization of oxidized polyvinyl alcohol hydrolase in E. coli

Oxidized polyvinyl alcohol (PVA) hydrolase (OPH) is a key enzyme in the degradation of PVA, suggesting that OPH has a great potential for application in textile desizing processes. In this study, the OPH gene from Sphingopyxis sp. 113P3 was modified, by artificial synthesis, for overexpression in Escherichia coli. The OPH gene, lacking the sequence encoding the original signal peptide, was inserted into pET-20b (+) expression vector, which was then used to transform E. coli BL21 (DE3). OPH expression was detected in culture medium in which the transformed E. coli BL21 (DE3) was grown. Nutritional and environmental conditions were investigated for improved production of OPH protein by the recombinant strain. The highest OPH activity measured was 47.54 U/mL and was reached after 84 h under optimal fermentation conditions; this level is 2.64-fold higher that obtained under sub-optimal conditions. The productivity of recombinant OPH reached 565.95 U/L/h. The effect of glycine on the secretion of recombinant OPH was examined by adding glycine to the culture medium to a final concentration of 200 mM. This concentration of glycine reduced the fermentation time by 24 h and increased the productivity of recombinant OPH to 733.17 U/L/h. Our results suggest that the recombinant strain reported here has great potential for use in industrial applications.