Full Text - Construction of recombinant Bacillus subtilis strains for efficient pimelic acid synthesis

Construction of recombinant Bacillus subtilis strains for efficient pimelic acid synthesis

¹ Northwest A&F University, College of Life Sciences, Yangling, People's Republic of China
² Northwest A&F University, College of Animal Sciences, Yangling, People's Republic of China

Financial support: The financial supplement of National New Productions Project from Science and Technology Ministry (P. R. China) is gratefully acknowledged.

Keywords: Bacillus subtilis, biosynthesis, integration, P_glv promoter, pimelic acid, single crossover event.

As a precursor, pimelic acid plays an important role in biotin biosynthesis pathway of Bacillus subtilis. Fermentations supplemented with pimelic acid could improve the production of biotin, however, with a disadvantage-high cost. So it is necessary to improve the biosynthesis of pimelic acid via genetic engineering in B. subtilis. In this study, we constructed a recombinant B. subtilis strain for improving the synthesis of pimelic acid, in which a maltose-inducible P_glv promoter was inserted into the upstream of the cistron bioI-orf2-orf3 and, meanwhile, flanked by the tandem cistrons via a single crossover event. The copy number of the integrant was amplified by high-concentration resistance screen and increased to 4-5 copies. The production of pimelic acid from multiple copies integrant was about 4 times higher than that from single copy (1017.13 μg/ml VS. 198.89 μg/ml). And when induced by maltose the production of pimelic acid was about 2 times of that under non-induction conditions (2360.73 μg/ml VS. 991.59 μg/ml). Thus, these results demonstrated that the production of pimelic acid was improved obviously through reconstructed B. subtilis. It also suggested that our expression system provided a convenient source of pimelic acid that would potentially lower the cost of production of biotin from engineered B. subtilis.

Biotin is a sulfur-containing vitamin as a cofactor involved in central pathways in prokaryotic and eukaryotic metabolism, i.e. as the CO₂-carring prosthetic group of carboxylases, decarboxylases and trans carboxylases (Bower et al. 1996; Streit and Entcheva, 2003; Pirner and Stolz, 2006). The biotin biosynthesis in many prokaryotic microorganisms has been discovered and the genes involved in the biosynthesis pathway had been identified and cloned (Otsuka and Abelson, 1978; Gloeckler et al. 1990; Kiyasu et al. 2001). The genes involved in the conversion of pimelic acid to biotin are arranged in a single operon in the order bioWAFDB in B. subtilis. The pimelic acid is the known precursor of biotin biosynthesis in B. subtilis (Streit and Entcheva, 2003). The amount of its synthesis in natural strains could not meet the demand for industrial production of biotin. In the absence of added pimelic acid feeding, levels of biotin and biotin vitamins were greatly reduced. Adding pimelic acid in the fermentations of biotin can remarkably improve the production of the direct precursor dethiobiotin (Berkovitch et al. 2004).

Intriguing world market of biotin, in which humans and animals require several hundred micrograms of biotin per day, has been mainly supported by the chemically synthesized biotin so far (Streit and Entcheva, 2003). Since the chemical synthesis usually caused environmental burden, the biosynthesis of biotin became an attracted strategy. The well-characterized biotin biosynthesis pathway of most microbes and the modern DNA recombinant techniques make it available to develop genetic engineering bacterium for biotin-overproduction (Streit and Entcheva, 2003; Lin et al. 2010; Cronan and Lin, 2011; Lin and Cronan, 2011). Thereby, much effort has been made and the recombinant strains over-producing biotin were obtained in B. subtilis, Serratia marcescens and E. coli (Sakurai et al. 1995; Van Arsdell et al. 2005). Although the biotin yields achieved are already close to an almost profitable amount, none of the genetic engineering recombinant strains really produces enough biotin to allow cost-effective production yet (Streit and Entcheva, 2003). Because the high cost raising by the addition of biotin precursors during the biotin fermentation processes limited the commercial application.

The pimelic acid was considered to be one of the preferred additives. The production of dethiobiotin in the feeding added the pimelic acid is about ten-fold higher than in the fermentation with no addition (Berkovitch et al. 2004). However, the addition of pimelic acid was linked with high cost and environmental burden. So constructing an efficient system to improve the biosynthesis of pimelic acid is one of the necessary strategies and pre conditions for biotin over-producing recombinant strains. Cytochrome P450BioI (CYP107H1) is believed to supply pimelic acid equivalents for biotin biosynthesis in Bacillus subtilis (Cryle and De Voss, 2004; Cryle and Schlichting, 2008). There are some reports showed that the pimelic acid was formed most likely by P450_BioI from acyl ACPs by oxidative cleavage (Stok and De Voss 2000; Green et al. 2001). Further research indicated that in-chain cleavage of fatty acids or fatty acyl ACPs is the likely in vivo role of P450_BioI (Cryle et al. 2003; Cryle and De Voss, 2004). Moreover, the crystal structure and some other details of the acyl-ACP BioI were investigated (Cryle and Schlichting, 2008; Cryle, 2010). Therefore, it is an economical and efficient method that improve the production of pimelic acid by high-expressed the BioI in vivo.

In this study, a resultant recombinant was obtained, in which the bioI, orf2 and orf3 were created a repeated copy and the P_gl_v was flanked by the three cistrons. The copy number of expression cassette inserted into the chromosome was amplified via selection stress of antibiotic, and the pimelic acid was produced in high level. Hence, we constructed an efficient system for improving the biosynthesis of pimelic acid in B. subtilis.

Bacterial strains, plasmids, oligonuclotides and DNA manipulation techniques

The bacterial strains and plasmids used in this study were listed in Table 1, and specific primers used in this study were listed in Table 2. The isolation and manipulation of recombinant DNA was performed with standard techniques (Sambrook and Russell, 2001). B. subtilis electrotransformation was performed as previously described (Yang et al. 2006).

Construction of integration vector and recombinant Bacillus subtilis strain

To construct the delivery vector, the chloramphenicol-resistance gene (Cm^R, 1.0 kb) and the cistrons bioI-orf2-orf3 (2.8 kb) were PCR amplified from the pGJ103 (Yang et al. 2006; Zhang et al. 2007) and B. subtilis 168 chromosome DNA, using the primer pairs Cm-up/Cm-down and Cobio1-up/ Cobio2-down, respectively. The amplified Cm^R gene and the cistrons were cloned into pGEM-T vector respectively, resulting in pLHX1 and pLHX2. And then, the kpnI-ApaI-treated Cm^R gene was cloned into the corresponding sites of pE3 vector, yielding pLHX5. The ApaI-XbaI-treated P_glvpromoter was cloned into corresponding sites of pLHX2, resulting in pYG57. The expression cassette of the cistrons, excised from pYG57 with ApaI and XbaI, was cloned into the corresponding sites of pLHX5, yielding pLHX8.

To facilitate single crossover event between the P_glv-bioI-orf2-orf3 cassette and chromosomal DNA, B. subtilis PY79 was electro-transformed with 0.5 μg pLHX8 and the recombinants were selected on TBAB agar plate with 5 μg/ml chloramphenicol. Since the delivery vector could not self-replicate in B. subtilis, the colonies appeared on the screen plate should be integrants (Figure 1). The obtained recombinant was designed as BpLHX8.

To examine the single crossover event between pLHX8 and chromosomal DNA, the BpLHX8 chromosomal DNA digested with EcoRI and KpnI was subjected to southern blot analysis by using the DIG-labeled Cm^R gene as probe. For further confirming that the single crossover event occurred as expected in BpLHX8, PCR assays were carried out by using of four primer pairs.

Because the repetition of bioI-orf2-orf3 was created by the single crossover event in the recombinant B. subtilis BpLHX8, the copy-number amplification of the region flanked by the bioI-orf2-orf3 in the BpLHX8 chromosome can be performed. For amplifying the P_glv-bioI-orf2-orf3 cassette, BpLHX8 was incubated and screened in the culture medium with high concentration of chloramphenicol. The B. subtilis BpLHX8 was streaked repeatedly in TBAB medium with Cm^R concentrations up to 50 μg/ml, and the isolated resistance colon was, designed as BpLHX8-Cm₅₀ (Van Arsdell et al. 2005). As a result, we obtained recombinant strain BpLHX8-Cm₅₀.

To examine the efficiency of pimelic acid synthesis in the reconstructed strains, the production of pimelic acid from BpLHX8 and BpLHX8-Cm₅₀ were determined, respectively. The two strains were cultured in 2 x MSR medium without antibiotics respectively, while B. subtilis PY79 was used as negative control.

B. subtilis PY79, BpLHX8 and BpLHX8-Cm₅₀ were cultured in 5 ml of TBAB (Sambrook and Russell, 2001) at 37ºC, respectively, with the appropriate concentration of Cm^R or not. Then 600 μl overnight culture was inoculated in 30 ml fresh 2 x MSR medium (5.0% yeast extract, 3.0% bactotrypton, 0.6% K₂HPO₄, 1.0% glucose) without antibiotics (Ye et al. 1999). For maltose induction, cells were grown to the cell density of 100 klett units and maltose was added to a final concentration of 5% (Yang et al. 2006). For different culture time after induction, the cells were harvested by centrifugation at 10000 g for 30 min, and the supernatants were filtrated with 0.45 μm MF-Millipore^TM Filters (Whatwan) for chromatography analysis.

The chromosomal DNA was digested with EcoRI-XbaI and separated on a 0.8% BIOWEST agarose gel. The DNA was transferred with 0.4 M NaOH to a nylon filter (Hybond-N⁺, Amersham) and fixed with UV light (Stratalinker UV cross-linker, Stratagene). Southern blot was performed with DIG DNA labeling and Detection Kit (Roche Diagnostics Corporation) according to the instructions provided by the suppliers. The copy number of cistrons of BpLHX8-Cm₅₀ was determined using a DIG-labeled Cm resistance gene probe. In this experiment, 5x, 3x, 1x samples (the EcoRI-XbaI-treated BpLHX8 chromosomal DNA) were designed, respectively.

Reverse phase-high performance liquid chromatography (RP-HPLC) was used to determinate the production of pimelic acid in recombinant strains. RP-HPLC system was comprised of a LC-10A^VP pump (SHIMADZU), a SPD-10A^VP UV/VIS detector (SHIMADZU), a ValueChrom data acquisition system (Bio-Rad). The Zorbax SB-C18 column (5 μm, 4.6 x 250 mm, Agilent) was maintained at 45ºC with an injection volume of 10 μl, wavelength 210 nm. The mobile phase for determination of pimelic acid consisted of a mixture of 0.02M K₂HPO₄ (pH 3.0)-methanol (90:10, v/v) at a flow-rate of 1.3 ml/min.

To introduce the strong promoter P_glv into the upstream of bioI and create a repeat of bioI-orf2-orf3 in B. subtilis chromosome DNA, the delivery vector pLHX8 was constructed, in which the bioI-orf2-orf3 was under the control of P_glv. The chloromycetin resistance gene was employed as a selection marker in the delivery vector for the recombinant selection and copy number of gene amplification.

When the single crossover event between the delivery vector constructed in this study and the B. subtilis chromosome occurred using the bioI-orf2-orf3 as homogenous arms, the entire delivery vector would be introduced into the B. subtilis chromosome and the repeated bioI-orf2-orf3 would be generated.

To reconstruct B. subtilis strains, the resultant delivery vector pLHX8 was used to transform the B. subtilis PY79 through a single crossover event, resulting in the Cm^Rrecombinant strain BpLHX8 (Figure 1). Then BpLHX8 chromosomal DNA was subjected to southern blot analysis. The results indicated that a 1 kb-size band generated from the B. subtilis BpLHX8 chromosomal DNA, whereas no band generated from B. subtilis PY79 chromosomal DNA (Figure 2). This confirmed that the pLHX8 was inserted into the B. subtilis PY79 chromosome. Further PCR assay showed that the single crossover event between the delivery vector and B. subtilis PY79 chromosomal DNA occurred in accordance with our strategy and the P_glv promoter located upstream the bioI-orf2-orf3. As expected, the reconstructed strain was obtained (Figure 3).

The gradient of Cm^R (50 μg/ml) was used to increase the copy-number of cistrons in BpLHX8. To estimate the copy number of P_glv-bioI-orf2-orf3 cassette, southern blot was preformed and the result (Figure 4a) suggested approximate 4-5 copies of P_glv-bioI-orf2-orf3 cassette in BpLHX8-Cm₅₀. Additionally, the PCR determination also confirmed that the estimation of copy-number of target cassette in BpLHX8-Cm₅₀chromosome.

And the production of pimelic acid from BpLHX8-Cm₅₀ was maximal (1017.13 μg/ml) at 24 hrs that was about 5 times higher than that from BpLHX8 (Figure 4b). Again, this result demonstrated the amplification of copy number of P_glv-bioI-orf2-orf3 cassette contributed to the accumulation of pimelic acid. Whereas, no detectable-levels pimelic acid was shown in B. subtilis PY79. This probably resulted from the production of pimelic acid in B. subtilis PY79 is too low to detect for it beyond the sensibility of HPLC. The results mentioned above demonstrated that the production of pimelic acid from the reconstructed strains was increased significantly compared to the wild type.

In the recombined strains, BpLHX8 and BpLHX8-Cm₅₀, the bioI-orf2-orf3 was under the control of maltose utilization operon promoter P_glv, of which the P_glv is an inducible promoter and is positive regulated by maltose. Thereby, to improve the production pimelic acid production from the BpLHX8-Cm₅₀, the effect of the maltose on pimelic acid production was investigated. The production of pimelic acid from BpLHX8- Cm₅₀in medium supplemented with maltose was higher than without it. The production of pimelic acid from BpLHX8-Cm₅₀ with maltose-induction reached maximal at 24 hrs (Figure 4c) and was about 2.4 times higher than no induction (2360.73 μg/ml VS. 991.59 μg/ml). This suggested that maltose induction obviously improve the pimelic acid biosynthesis in the reconstructed strains.

Increasing the expression level of genes that involved in metabolite pathway is common approach to improve the accumulation of secondary metabolites. The usual strategy to achieve this aim is employing the strong promoter driven the target genes or increasing the copy-number of gene. The pimelic acid is an important precursor in biosynthesis pathway of biotin. Its accumulation can remarkably increase the production of the biotin. The bioI, orf2 and orf3 genes of B. subtilis were involved in pimelic acid biosynthesis pathway. Thus, we employed the strong promoter P_glv driven the BioI-orf2-orf3 to enhance the gene expression level, and meanwhile, created a repetition of BioI-orf2-orf3 via a single crossover event in B. subtilis chromosome that make the copy-number amplification of BioI-orf2-orf3 available. The resultant recombinant demonstrated the accumulation of pimelic acid and again, increasing copy number enhanced the pimelic acid production significantly. Furthermore, maltose induction doubled the production of pimelic acid. Thus, we reconstructed B. subtilis strains in this study for efficient production of pimelic acid through integration approach and high level production of pimelic acid was obtained. By using of the reconstructed B. subtilis strains, the pimelic acid will not need to be added in the biotin ferment. Moreover, the inducer of this system, maltose, is cheap and economical. Therefore, this method can reduce the industrial cost and environmental pollution significantly. Hence, this work supplies a potential and practical system which will play an important role in the biotin ferment.

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