J L García

Nucleotide sequence and expression of the pneumococcal autolysin gene from its own promoter in Escherichia coli

Autolysins are enzymes that have several important biological functions and also seem to be responsible for the irreversible effects induced by the beta-lactam antibiotics. The pneumococcal autolysin gene (lyt) has been subcloned from the plasmid pGL30 [García et al., Mol. Gen. Genet. 201 (1985) 225-230] and we have found that the E form of the autolysin is synthesized in Escherichia coli using its own promoter. The high amount of autolysin obtained in the heterologous system when the lyt gene is present in different orientations in the recombinant plasmids studied supports the idea that the autolysin promoter could be a strong one. The nucleotide sequence of the HindIII fragment of pGL80 (1213 bp) containing the autolysin structural gene has been determined. A unique open reading frame (ORF) has been found, a consensus ribosome-binding site and -10 and -35 promoter-like sequences as well as A + T-rich regions farther upstream were also identified. The lyt ORF encodes a protein of 318 amino acid residues having a calculated Mr of 36,532, which agrees with previous size estimates based on electrophoretic migration [Höltje and Tomasz, J. Biol. Chem. 251 (1976) 4199-4207; Briese and Hakenbeck, Eur. J. Biochem. 146 (1985) 417-427]. Our results also demonstrate that the lyt-4 marker represents the first example of a mutation in a structural gene of a bacterial autolysin. The polarity profile of the pneumococcal autolysin supports previous suggestions about the localization of this enzyme in the normal cell.

Complete nucleotide sequence of the penicillin acylase gene from Kluyvera citrophila

The penicillin acylase (PAC) from Kluyvera citrophila ATCC21285 has been purified to homogeneity and found to be composed of two non-identical subunits of 23 and 62 kDa, in contrast with the previous findings [Shimizu et al., Agr. Biol. Chem. 39 (1975) 1655-1661]. The nucleotide (nt) sequence of the K. citrophila pac gene contained in the 3-kb PvuI-HindIII fragment of pKAP1 [García and Buesa, J. Biotechnol. 3 (1986) 187-195] has been determined, showing that it encodes a protein of 844 amino acid (aa) residues. The aa analysis of the N-terminal and C-terminal sequences of the purified subunits showed that they were derived from a common precursor protein of 93.5 kDa, from which a signal peptide of 26 aa, responsible for the periplasmic translocation of the protein, and an internal connecting polypeptide of 54 aa, have been removed in the maturation of the PAC. The comparison of the nt sequences of the pac genes from K. citrophila and Escherichia coli ATCC11105 [Schumacher et al., Nucl. Acids Res. 14 (1986) 5713-5727] revealed 80% homology, suggesting a common ancestral pac gene origin. The results reported here should allow investigation of the unusual mechanism of maturation of this prokaryotic protein, as well as manipulation, using DNA recombinant techniques, of the catalytic properties of this industrially important enzyme.

Overproduction and rapid purification of the amidase of Streptococcus pneumoniae.

Oligonucleotide-directed mutagenesis of a plasmid containing the lytA gene coding for the pneumococcal amidase has allowed the separation of the coding sequence of the gene. This sequence has been placed in plasmid pIN-III(lppP-5)-A3 downstream from both a modified lipoprotein promoter and the lactose promoter to construct the recombinant plasmid pGL100. When Escherichia coli RB 791 (pGL100) was grown in the presence of lactose, the pneumococcal amidase accounted for 7% of the total protein present in this strain after 18 h incubation at 37°C. The overproduced amidase was purified in a single-step procedure using a choline-Sepharose 6B column taking advantage of the fact that this enzyme was the unique protein with affinity for choline present in extracts obtained from E. coli RB791 (pGL100). The development of the above design opens up the possibility of studying the mechanism that regulates the activity of this important autolysin by using physicochemical techniques that require the availability of high amounts of purified amidase.

Characterization of the transcription unit encoding the major pneumococcal autolysin

The pneumococcal lytA gene coding for the major autolysin (amidase) can be expressed in Streptococcus pneumoniae and Escherichia coli using unchanged promoter and termination signals. A region containing several -10, -35 and -44 promoter elements, identical to other previously described prokaryotic promoter sequences, has been found upstream from the transcription start point. A transcription terminator consisting of a hairpin structure (-20.8 kcal/mol) typical of Rho-independent prokaryotic terminators was also localized. The lytA gene has a rather long (240-bp) leader sequence with a high A + T content (70%) that contrasts with the very short (2-bp) untranslated region of the polA gene [López et al., J. Biol. Chem. 264 (1989) 4255-4263], the unique pneumococcal transcription unit that had been characterized so far. Although two open reading frames have been found in the leader region it seems unlikely that these sequences can be translated due to the absence of appropriate ribosome-binding sites.

Bioconversion of Phytosterols into Androstadienedione by Mycobacterium smegmatis CECT 8331

The C19 steroid 1,4-androstadiene-3,17-dione (androstadienedione, ADD) is an added value product used as a synthon in the pharmaceutical industry for the commercial production of corticosteroids, mineralocorticoids, oral contraceptives, and other pharmaceutical steroids. Phytosterol biotransformation catalyzed by microbial whole cells is actually a very well-established research area in white biotechnology. The protocol below provides detailed information on ADD production by the mutant CECT 8331 of Mycobacterium smegmatis mc2155 using phytosterols as raw material in a lab scale. This protocol describes the bioconversion of phytosterols into ADD in a single fermentation step.

Biocatalysis for Industrial Production of Active Pharmaceutical Ingredients (APIs)

From an industrial point of view, biocatalysis is particularly important in the production of active pharmaceutical ingredients (APIs), offering increasingly high demands for regio-, stereo,- and enantioselectivity of drugs. Biocatalytic processes are more ecofriendly, sustainable, and profitable, and hence biocatalysis is proving to be key for the development of the so-called bioeconomy. Thus, in the manufacturing of APIs, as either pharmaceutical products or intermediates, biocatalysis is considered as a “green” technology to efficiently discriminate between isomers in a racemic mixture under mild conditions, compared to chemical salt resolutions which may involve contaminants and are expensive. In addition to enantioselectivity, regioselectivity on complex molecules is another inherent feature of chemoenzymatic processes, which avoids the need of protecting groups, and reduces the number of synthetic steps. Finally, biocatalysis can also be used to produce achiral APIs where classical chemical methods are too complicated. Therefore, a huge number and quantity of enzymes are now available in the market (Reetz, 2013). Although isolated enzymes are mainly chosen for their simple implementation in biocatalyzed processes, whole-cell biotransformations are generally preferred for complex reactions involving more than one enzyme or cofactor, or for reactions where enzymes are not suitable for isolation. We herein provide examples of the application of isolated enzymes (wild-type or mutants) and whole cells, either in soluble or immobilized form, in the synthesis of some valuable APIs.