This study deals with the potential of Pichia pastoris X-33 for the production of penicillin G acylase (PGAA) from Achromobacter sp. CCM 4824. Synthetic gene matching the codon usage of P. pastoris was designed for intracellular and secretion-based production strategies and cloned into vectors pPICZ and pPICZα under the control of AOX1 promoter. The simple method was developed to screen Pichia transformants with the intracellularly produced enzyme. The positive correlation between acylase production and pga gene dosage for both expression systems was demonstrated in small scale experiments. In fed-batch bioreactor cultures of X-33/PENS2, an extracellular expression system, total PGAA expressed from five copies reached 14,880 U/L of an active enzyme after 142 h; however, 60% of this amount retained in the cytosol. The maximum PGAA production of 31,000 U/L was achieved intracellularly from nine integrated gene copies of X-33/PINS2 after 90 h under methanol induction. The results indicate that in both expression systems the production level of PGAA is similar but there is a limitation in secretion efficiency.
- MeSH
- Achromobacter genetika metabolismus MeSH
- bioreaktory mikrobiologie MeSH
- exprese genu MeSH
- genetické vektory MeSH
- genová dávka MeSH
- klonování DNA MeSH
- kodon genetika MeSH
- penicilinamidasa genetika metabolismus MeSH
- Pichia genetika metabolismus MeSH
- promotorové oblasti (genetika) MeSH
- průmyslová mikrobiologie metody MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- transformace genetická MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Penicillin G acylase of Escherichia coli (PGAEc) is a commercially valuable enzyme for which efficient bacterial expression systems have been developed. The enzyme is used as a catalyst for the hydrolytic production of beta-lactam nuclei or for the synthesis of semi-synthetic penicillins such as ampicillin, amoxicillin and cephalexin. To become a mature, periplasmic enzyme, the inactive prepropeptide of PGA has to undergo complex processing that begins in the cytoplasm (autocatalytic cleavage), continues at crossing the cytoplasmic membrane (signal sequence removing), and it is completed in the periplasm. Since there are reports on impressive cytosolic expression of bacterial proteins in Pichia, we have cloned the leader-less gene encoding PGAEc in this host and studied yeast production capacity and enzyme authenticity. RESULTS: Leader-less pga gene encoding PGAEcunder the control of AOX1 promoter was cloned in Pichia pastoris X-33. The intracellular overproduction of heterologous PGAEc(hPGAEc) was evaluated in a stirred 10 litre bioreactor in high-cell density, fed batch cultures using different profiles of transient phases. Under optimal conditions, the average volumetric activity of 25900 U l-1 was reached. The hPGAEc was purified, characterized and compared with the wild-type PGAEc. The alpha-subunit of the hPGAEc formed in the cytosol was processed aberrantly resulting in two forms with C- terminuses extended to the spacer peptide. The enzyme exhibited modified traits: the activity of the purified enzyme was reduced to 49%, the ratios of hydrolytic activities with cephalexin, phenylacetamide or 6-nitro-3-phenylacetylamidobenzoic acid (NIPAB) to penicillin G increased and the enzyme showed a better synthesis/hydrolysis ratio for the synthesis of cephalexin. CONCLUSIONS: Presented results provide useful data regarding fermentation strategy, intracellular biosynthetic potential, and consequences of the heterologous expression of PGAEc in P. pastoris X-33. Aberrant processing of the precursor of PGAEc in the cytosol yielded the mature enzyme with modified traits.
- MeSH
- bioreaktory MeSH
- Escherichia coli enzymologie genetika MeSH
- fermentace MeSH
- klonování DNA MeSH
- penicilinamidasa biosyntéza genetika izolace a purifikace MeSH
- Pichia genetika metabolismus MeSH
- promotorové oblasti (genetika) MeSH
- proteiny z Escherichia coli biosyntéza genetika izolace a purifikace MeSH
- průmyslová mikrobiologie MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Penicilin G acylasa (PGA) patří k velmi důležitým průmyslovým enzymům, které jsou používány při produkci polosyntetických (3-laktámových antibiotik. Tento enzym katalyzuje hydrolýzu amidické vazby penicilinu G za vzniku 6-aminopenicilánové kyseliny, která slouží jako výchozí substance pro výrobu polosyntetických penicilinů. U kmene Escherichia coli W ATCC 11105 a ATCC 9637 je PGA kódována pga genem na chromozómu a syntetizována jako pre-pro-PGA (pp PGA) prekurzor, který je za pravděpodobné účasti chaperonového systému transportován do periplazmatického prostoru buňky. Zde po řadě proteolytických reakcí vzniká aktivní enzym PGA tvořený dvěma podjednotkami a a R. Exprese pga genu podléhá několika regulačním mechanizmům: teplotní represi, katabolické represi glukosou, represi kyslíkem a indukci fenyloctovou kyselinou (FOK). Tvorba aktivní PGA je také ovlivněna na post-translační úrovni, kde důležitou roli hrají intracelulární proteolytické reakce a transportní systém pre-pro PGA cytoplazmatickou membránou. Chromozomální oblast pga genu kmene E. coli W byla použita pro konstrukci mnoha rekombinantních plazmidy. Těmito plazmidy byly transformovány vhodné hostitelské kmeny, z nichž některé se dnes používají v průmyslu jako vysoko produkční mikroorganizmy.
Pencilin G acylase (PGA) is one of very important industrial enzymes used in the production of polysynthetic R-lactam antibbottks. This enzyme catalyzes the hydrolysis of the amidic bond of penicillin G with the development of 6-aminopenicillanic acid which serves as the initial substance for the production of semisynthetic penicillins. In the strain Escherichia coli W ATCC 11105 and ATCC 9637, PGA is coded by the pga gene on the chromozóme and synthesized as the pre-pro-PGA (pp PGA) precursor, which is transportnd, with probable participation of the chaperon system, to the periplasmatic space of the cell. Here after a series of proteolytic reactions the active enzyme PGA develops, consisting of two subunits a and R. Expression of the pga gene is subject to several regulatory mechanisms: temperature repression, catabolic repression by glucose, repression by oxygen, and induction by phenylacetic acid (FOK). The formation of active PGA is also influenced at the post-translation level, where an important role is played by intracellular proteolytic reactions and the transport system of pre-pro-PGA across the cytoplasmatic membrane. The chromozomal area of the pga gene of the E. coli W strain was employed for the construction of many recombinant plasmids. These plasmids served to transprm suitable host strains, some of which are now used in industry as highly productioe microorganisms.
