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Design and synthesis of hyaluronic acid coding has2 gene

The initial step of our project was the artificial synthesis of the has2 gene sequence coding for the hyaluronic acid synthase of the naked mole rat. The sequence was taken from the freely accessible genome database “UCSC genome browser”, whereby the sequence data was gained by shot-gun-sequencing (Kim et al., 2011). The gene sequence was adapted to the altered codon usage in Bacillus megaterium with JCat (Grote et al., 2005). Additionally the open reading frame (ORF) of the has2 gene was modified so that prokaryotic ribosomal binding site (RBS), rho-independent transcription termination as well as restriction enzymes that are also part of the multiple cloning site (MCS) of the target plasmid pSMF2.1 were excluded. For the upcoming cloning process of the has2 gene the nucleotide sequence was terminally modified with the restriction sites MluⅠ (5`end) und SacⅠ(3`end). Moreover a </i>B. megaterium</i> specific RBS was added downstream the MluⅠrestriction site for efficient protein expression. We paid attention to the fact that amino acid sequence did not change. Subsequently the 1693 bp long modified gene sequence has2 (fig. 1) was handed over to Eurofins who performed the gene synthesis and the cloning into the standard delivery plasmid pexK4 plasmid (Eurofins).

Genomic amplification of genes that may optimise the hyaluronic acid production

The hyaluronan synthase catalyses the synthesis of hyaluronic acid by consumption of the endogenous synthesised precursor molecules UDP-GlcUA and UDP-GlcNAc. The biochemical pathways for their production were already identified in streptococci of group A and C. Hence it was possible to identify genes that could have a beneficial effect on the concentration of these precursor molecules for high yield hyaluronic acid production in B. megaterium Eventually eight relevant pathway genes, including UDP-GlcDH, glk, pgmB, gtaB, pgi, glmS, glmM and gcaD, were chosen for the approach of pathway engineering. The sequences of these genes originate from MegaBac v9 database and were used for primer design and amplification of genomic DNA of B. megaterium. Restriction sites not only at the 5` end but also at the 3` end were added for subsequent cloning into the pSB1C3 plasmid (table 1).

To verify the correct amplification of the genes the PCR products were loaded onto an agarose gel. As it is evident in figure 2 the bands showed the expected lengths for the genes glk (969 bp), pgmB (707 bp), </i>gta</i>B (888 bp), pgi (1350 bp), glmS (1800 bp), glmM (1340 bp) and gcaD (1380 bp). Since UDP-GlcDH was planned for cloning into pSMF2.1-has2 plasmid it was amplified in a separate step, described below.

[[File:|thumb|center||Figure 2: PCR amplification of genes coding for the enzymes that are involved in synthesis the hyaluronic acid production. All amplified gene sequences show the expected lengths.
Lane 1: glk (969 bp)
Lane 2: pgmB (707 bp)
Lane 3: gtaB (888 bp)
Lane 4: pgi (1350 bp)
Lane 5: glmS (1800 bp)
Lane 6: glmM (1340 bp)
Lane 7: gcaD (1380 bp)
]] -->

Test of restriction enzymes

Before starting with the cloning procedures it was necessary to check if the ordered restriction enzymes work properly. Therefore the pSMF2.1 plasmid was digested separately with each restriction enzyme (MluⅠ, SacⅠ, SphⅠ) that should be used for the following cloning of has2 and UDP-GlcDH into the expression plasmid pSMF2.1. Displayed in Figure 3 all restriction enzymes (MluⅠ, SacⅠ and SphⅠ) linearised the plasmid, indicating proper functioning.

Cloning of the hyaluronan synthase gene (has2) into pSMF2.1

A digestion with the restriction enzymes SacⅠ und MluⅠwas used to extract the has2 gene (1693 bp) from the pexK4 plasmid (2507 bp) for the following cloning into the final pSMF2.1 plasmid (6510 bp). Agarose electrophoresis was used to detect the digestion products which showed the expected lengths (fig.4). The band of has2 gene was clearly visible at 1693 bp. For cloning of has2 into pSMF2.1 plasmid it first needed to be digested with the same restriction enzymes SacⅠ und MluⅠ. Subsequently the restricted plasmid (6510 bp) was separated by agarose gel electrophoresis (fig. 4). A restriction control without the restriction enzymes was carried out as well.

After reisolation the pSMF2.1 plasmid was treated with alkaline phosphatase. The treatment causes an elimination of the phosphate group at the 5` terminus of the restricted plasmid and thereby prevents the restricted plasmid from relegation.

Afterwards the digested insert has2 was ligated with the restricted and phosphatase treated pSMF2.1 plasmid. Figure 5 displays the control of ligation. The approach with ligase showed a high molecular band at 8203 bp indicating that insert and plasmid were successfully ligated. As expected the negative control showed only the two original bands representing digested has2 insert DNA and restricted pSMF2.1 plasmid.

After transformation of competent E. coli cells with the resulting pSMF2.1-has2.construct and selection on ampicillin agar plates, plasmid DNA of transformants was isolated and presence of has2 insert DNA was checked by control digestion. Figure 6 shows the result for one representative E. coli transformant.

Obviously the pSMF2.1-has2 plasmid was successfully absorbed by the E. coli transformant as both pSMF2.1 plasmid DNA at 6510 bp and has2 insert DNA at 1693 bp are visible after digestion with SacⅠ und MluⅠ. The isolated plasmid DNA was used in a following step for transformation of B. megaterium.

Cloning of the UDP-GlcDH gene into pSMF2.1-has2 plasmid

In the same way the UDP-glucose-6-dehdyrogenase (UDP-GlcDH) gene was cloned into the pSMF2.1-has2 plasmid. However, instead of an outsourced gene synthesis like in the case of has2, the UDP-GlcDH gene was amplified from the genomic DNA of B. megaterium. In addition modified primers were used to add the SacⅠ restriction site at the 5` terminus and the SphⅠ at the 3´ terminus of the PCR product. The presence and size of the PCR product was checked by agarose gel electrophoresis. pSMF2.1-has2 plasmid was digested with SacⅠ and SphⅠ for cloning UDP-GlcDH and also checked by agarose gel electrophoresis (fig.7). A specific band could be detected at a size of 1382 bp indicating a proper PCR amplification of UDP-GlcDH gene (fig.7). The restriction of pSMF2.1-has2 plasmid with SacⅠ and SphⅠ resulted in a linearised plasmid.

The next step was the ligation of the restricted pSMF2.1-has2 plasmid with the amplified and digested UDP-GlcDH gene. As demonstrated in figure 8 the ligation was efficient because a shift in pSMF2.1-has2 plasmid size and a weaker insert band could be detected. However, the ratio of UDP-GlcDH insert and pSMF2.1-has2 plasmid appeared not to be optimal due to a visible band at about 2750 bp resulting from a ligation of the insert UDP-GlcDH (1382 bp) with itself.

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