The Superiority of Bacillus megaterium over Escherichia coli as a Recombinant Bacterial Host for Hyaluronic Acid Production

Abstract

(1) Background: Hyaluronic acid (HA) is a polyanionic mucopolysaccharide extensively used in biomedical and cosmetic industries due to its unique rheological properties. Recombinant HA production using other microbial platforms has received increasing interest to avoid potential toxin contamination associated with its production by streptococcal fermentation. In this study, the Gram-negative strains Escherichia coli (pLysY/Iq), E. coli Rosetta2, E. coli Rosetta (DE3) pLysS, E. coli Rosetta2 (DE3), E. coli Rosetta gammiB(DE3)pLysS, and the Gram-positive Bacillus megaterium (MS941) were investigated as new platforms for the heterologous production of HA. (2) Results: The HA biosynthesis gene hasA, cloned from Streptococcus equi subsp. zoopedemicus, was ligated into plasmid pMM1522 (MoBiTec), resulting in pMM1522 hasA, which was introduced into E. coli Rosetta-2(DE3) and B. megaterium (MS941). The initial HA titer by the two hosts in the LB medium was 5 mg/L and 50 mg/L, respectively. Streptococcal hasABC and hasABCDE genes were ligated into plasmid pPT7 (MoBiTec) and different E. coli host strains were then transformed with the resulting plasmids pPT7hasABC and pPT7hasABCDE. For E. coli Rosetta-gamiB(DE3)pLysS transformed with pPT7hasABC, HA production was 500 ± 11.4 mg/L in terrific broth (TB) medium. Productivity was slightly higher (585 ± 2.9 mg/L) when the same host was transformed with pPT7 carrying the entire HA operon. We also transformed B. megaterium (MS941) protoplasts carrying T7-RNAP with pPT7hasABC and pPT7hasABCDE. In comparison, the former plasmid resulted in HA titers of 2116.7 ± 44 and 1988.3 ± 19.6 mg/L in LB media supplemented with 5% sucrose and A5 medium + MOPSO, respectively; the latter plasmid boosted the titer final concentration further to reach 2476.7 ± 14.5 mg/L and 2350 ± 28.8 mg/L in the two media, respectively. The molecular mass of representative HA samples ranged from 105 − 106 Daltons (Da), and the polydispersity index (PDI) was <2. Fourier transform infrared spectroscopy (FTIR) spectra of the HA product were identical to those obtained for commercially available standard polymers. Finally, scanning electron microscopic examination revealed the presence of extensive HA capsules in E. coli Rosetta-gamiB(DE3)pLysS, while no HA capsules were produced by B. megaterium. (3) Conclusions: Our results suggested that Gram-positive bacteria are probably superior host strains for recombinant HA production over their Gram-negative counters. The titers and the molecular weight (MW) of HA produced by B. megaterium were significantly higher than those obtained by different E. coli host strains used in this study.

Keywords: Bacillus megaterium; Escherichia coli; Hyaluronan; Streptococcus equi subsp. zooepidemicus; hasABCDE genes.

Figure 1

Biosynthetic pathway of HA in S. equi subsp. zooepidemicus.

Figure 2

Comparison of HA productivity between pP_T7hasABC E. coli lysY/I^q at (A) 37 °C and at (B) 30 °C, (***): p ˂ 0.0001.

Figure 3

Comparison of HA productivity presented by (Figure 3A); A) (pP_T7hasABC) E.colilysY/I^q, B) Rosetta DE3 pLysS, and C) Rosetta gamiB DE3 pLysS, (***): p ˂ 0.0001. (Figure 3B) Comparison of HA productivity by E. coli Rosetta gamiB DE3 pLysS transformed with (pP_T7hasABC) in A) LB medium + glucose (1%), B) SOC medium, and C) TB medium, (**): p < 0.001, (***): p ˂ 0.0001.

Figure 4

Comparison of HA productivity in TB medium between (A) pP_T7hasABC Rosetta gamiB (DE3) pLysS and (B) (pPT₇hasABCDE) Rosetta gamiB (DE3) pLysS, (*): p ˂ 0.05.

Figure 5

Comparison of HA production presented by (Figure 5A); pP_T7hasABC B. megaterium MS941 pre-transformed with T7RNAP in complex media of A) LB + glucose (1%), B) glucose (3%), C) glucose (5%), D) LB + sucrose (1%), E) LB + sucrose (3%), F) LB + sucrose (5%), and G) TB medium, (***): p < 0.0001. (Figure 5B): Comparison of HA production by Pp_T7hasABC B. megaterium MS941 pre-transformed with T7RNAP in semi minimal media A) A5, B) A5 + K, and C) A5 + MOPSO, (*): p ˂ 0.05.

Figure 5

Comparison of HA production presented by (Figure 5A); pP_T7hasABC B. megaterium MS941 pre-transformed with T7RNAP in complex media of A) LB + glucose (1%), B) glucose (3%), C) glucose (5%), D) LB + sucrose (1%), E) LB + sucrose (3%), F) LB + sucrose (5%), and G) TB medium, (***): p < 0.0001. (Figure 5B): Comparison of HA production by Pp_T7hasABC B. megaterium MS941 pre-transformed with T7RNAP in semi minimal media A) A5, B) A5 + K, and C) A5 + MOPSO, (*): p ˂ 0.05.

Figure 6

Graphical conclusion of the superiority of HA productivity in B. megaterium over E. coli. HA productivity using B. megaterium platform showed higher HA titer with lower polydispersity and no capsulated HA in the culturing media of A5 + MOPSO and LB + sucrose (5%) (up-arrow represents “increase”, down-arrow represents “decrease”).

Figure 7

(A): SEM photo of induced pP_T7hasABCDE E. coli Rosetta-gamiBpLysS. HA capsule was found intact in the cell, with white arrow. (B): SEM: Photo of induced pP_T7hasABCDE E. coli Rosetta-gamiBpLysS after incubation for 20 min with hyaluronidase enzyme dissolved in phosphate buffered saline.

Figure 7

(A): SEM photo of induced pP_T7hasABCDE E. coli Rosetta-gamiBpLysS. HA capsule was found intact in the cell, with white arrow. (B): SEM: Photo of induced pP_T7hasABCDE E. coli Rosetta-gamiBpLysS after incubation for 20 min with hyaluronidase enzyme dissolved in phosphate buffered saline.

Figure 8

(A): SEM photo of induced pP_T7hasABCDE B. megaterium MS941. It was clear that HA particles were outside the protoplasts and do not form a distinct capsule. They appeared as white particles (black arrow). (B): SEM photo of induced pP_T7hasABCDE B. megaterium MS941 after incubation for 20 min with hyaluronidase enzyme dissolved in phosphate-buffered saline.

Figure 8

(A): SEM photo of induced pP_T7hasABCDE B. megaterium MS941. It was clear that HA particles were outside the protoplasts and do not form a distinct capsule. They appeared as white particles (black arrow). (B): SEM photo of induced pP_T7hasABCDE B. megaterium MS941 after incubation for 20 min with hyaluronidase enzyme dissolved in phosphate-buffered saline.

Figure 9

(A): FTIR of HA standard versus HA extracted from pP_T7hasABCDE E. coli Rosetta-gamiBpLysS. (B): FTIR of HA standard versus HA extracted from pP_T7hasABCDE B. megaterium.